Protocol Dna Illinois

FOREWORD
This manual is the property of the Illinois State Police with all rights reserved. No portion of this manual can
be reproduced without written permission of the Illinois State Police.
The body of knowledge which comprises forensic science is a compilation of procedures adapted from
other disciplines that encompass many of the physical and natural sciences. During the history of forensic
science, a multitude of scientists have greatly contributed to the protocols, methods and procedures that
have become a routine part of analysis. Every effort has been made in this manual to give proper recognition
to the authors of specific procedures; however, in some instances, the original source of forensic procedures
has been lost in antiquity. For others, the general procedures belong to the public domain and are recorded
in many basic references concerning forensic science. In addition, many of the procedures described in this
manual have been adapted from standard laboratory practices, and the citation of thousands of references
which deserve credit for aiding in the development of these procedures is neither practical nor possible. To
all those scientists who have contributed to the knowledge of forensic science contained herein, we do
extend collective recognition and gratitude.
Procedures manuals which offer reliable information that is then combined with corresponding training
manuals serve as the foundation for effective quality management of analyses. Extensive effort has been
made to ensure that the routine procedures described herein will produce accurate and valid analytical
results. However, not all possible analyses that may be encountered in casework can be appropriately
covered in a procedures manual, nor can all possible variations to a described procedure be included.
Therefore, this manual is written with the understanding that minor variations that do not significantly alter the
described procedure may be used. An analyst may use a non-routine procedure not specifically stated in
this manual, provided all the following conditions are met:
1. The procedure used is based upon documented and scientifically accepted practice.
2. A notation is made on the worksheet indicating the procedure followed is not specified in the
procedures manual.
3. The analyst also indicates on the work sheet why the particular procedure was selected over
a procedure contained in this manual. Rationale must be detailed sufficiently to withstand
close scrutiny by independent examiners.
4. The analyst provides documentation showing that the non-routine procedure had been tested
prior to application with evidence. Test criteria shall include test samples that approximate
the characteristics of the evidence, the results obtained with the routine procedure, and the
results obtained with the non-routine procedure. Documentation will also include related
data concerning the non-routine procedures sensitivity, precision and possible sources of
error.
5. The non-routine procedure used will be recorded to a standard such that another scientist of
similar skills and experience can understand fully the procedure used and the results
obtained.
Additionally, there may be procedures which pertain to all sections. Such is the case with laboratory
reagents. In order to standardize the testing and monitor the shelf life of reagents used by analytical sections,
the Forensic Sciences Command has developed protocols which are universal for all sections. These
protocols regarding reagent expiration and testing are found in the Command Quality Manual.
Accepted Date: March 8, 2002
Next Review Date: July 31, 2006
Forensic Biology/DNA Procedures Manual
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Procedure: Clean Technique
ILLINOIS STATE POLICE
FORENSIC BIOLOGY/DNA
PROCEDURES MANUAL
PROCEDURE: CLEAN TECHNIQUE
Prepared by:

Barbara Llewellyn Date
Statewide DNA Technical Leader
Approved by:

Sandra N. Brown Date
R&D Laboratory Director
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INTRODUCTION
All Forensic Biologists and DNA analysts must follow clean technique. The purpose of clean technique
is to prevent unwanted DNA from entering a sample. The first line supervisor through the laboratory
director are responsible for ensuring this policy is followed. There are two main sources of unwanted
DNA. The first is extraneous DNA. This is a DNA profile in a case sample or blank that is consistent with
a laboratory employees DNA profile. The second is cross contamination. This is DNA that is transferred
between case samples. There are many sources of cross contamination and extraneous DNA such as:
aerosols, liquids or dry flakes/dust, unclean tools, unclean gloves, and contaminating materials on lab coats.
There are also laboratory areas that are more at risk for the introduction of unwanted DNA such as
evidence examination areas and extraction areas. As a result, proper precautions must be taken to ensure
that cross contamination and the introduction of extraneous DNA does not occur. These precautions must
include putting on a clean lab coat with sleeve covers or a disposable lab coat, gloves and masks to
examine all evidence and for DNA extractions up to the phenol stage. Please refer to Appendix VI DNA
Quality Assurance, Sample Handling and Facility Requirements for additional information.
SAFETY CONSIDERATION
Observe Standard Laboratory Practices
Warning: Treat all reagents/samples as potential biohazards.
Refer to safety considerations under the DNA Isolation/Methods section
PREPARATION
10% Bleach Solution
Sterile Cotton cloth or swab
INSTRUMENTATION
Standard Laboratory Instrumentation
MINIMUM STANDARDS AND CONTROLS
Refer to the minimum standards and controls under the DNA isolation section.
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PROCEDURE OR ANALYSIS
Initial Steps Prior to Starting Analysis:
1. Gloves must be worn whenever an individual is in the laboratory. The gloves must either be
sterile gloves or the gloves must be bleached and then dried with a paper towel after the gloves
are put on. Gloves must be changed between exhibits. Gloves must also be changed after
handling non-evidence items prior to returning to casework. These non-evidence items may
include but are not limited to, refrigerators/freezers, biohazard waste bins, equipment, computers,
and telephones. Gloves should be changed following common sense and clean technique.
2. Messages should be taken for all phone calls (except in an emergency) received while the analysts
are working in the laboratory. Time can be set aside each day to return phone calls.
Conversations between laboratory personnel should be kept at a minimum when an analyst is
extracting or setting up samples.
3. The temperature of the DNA Laboratory must be monitored and kept at a comfortable level to
prevent the DNA analysts from becoming too hot.
4. First decontaminate the surface on which samples are to be processed with a 10% bleach
solution. Wet the surface (counter top, lab bench etc.) that will be utilized to examine evidence
thoroughly with 10% bleach solution. Spread the 10% bleach across entire surface with a paper
towel. Ensure surface is dry before examining evidence. Dont store bleach solutions in open
containers. Replace the bleach solution daily with a fresh bleach solution.
5. All tubes utilized for extraction or amplification must be stored in closed containers.
6. All instruments which will be used to process forensic samples (e.g., forceps, scissors,
scalpel/razor blades, bone cutting equipment, pipetters and metal probes) must be
decontaminated by autoclaving or rinsing with a 10% bleach solution. Caution: some surfaces
may resist wetting and will require addition of a detergent. In addition, these items may also be
placed under an ultraviolet light source for at least 15 minutes. Note: UV light will not destroy
DNA on surfaces that are not directly exposed to the light.
7. Place evidence samples in clean containers or on clean surfaces for processing. Large glassine
weighing papers are suggested.
8. Use a 10% bleach solution to rinse or wipe instruments between samples. Instruments may be
rinsed with distilled water. After rinsing with a 10% bleach solution, use kimwipes to wipe the
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instrument. Use a new Kimwipe each time.
9. Do not process DNA samples in a scrape down area.
10. For each extraction protocol followed, a sterile swab or piece of cotton material must be
processed as a manipulation blank.
11. When preparing reagents, one large stock solution may be prepared and then divided. Each
analyst must have individual mini-stocks of each reagent. No sharing of mini-stocks. Mini-stocks
may be replenished from the main stock. Individual analysts must use disposable beakers to
portion out solutions when appropriate.
12. Clean centrifuge rotors and work area with 10% bleach solution before use and if noticeable
sample spill occurs.
Sample Processing Through DNA Extractions:
1. Forensic Biology and DNA Analysts must wear a mask, gloves and disposable sleeve covers or
disposable lab coats while examining all items of evidence.
2. The process of examining DNA evidence and cutting samples for DNA extraction and adding
reagents must take place in a biohood with the analyst wearing a lab coat, gloves, mask and
disposable sleeve covers or a disposable lab coat. The biohood must have a hepa filter and a
UV light. The UV light will be used for 30 minutes prior to extracting samples in hood and 30
minutes upon completion. Any tubes that will be utilized during the extraction process must be
exposed to the UV for 30 minutes. This hood will be used to extract DNA only. Until a biohood
is available, the analysts may process samples on a freshly bleached bench top wearing a lab
coat, gloves, mask and disposable sleeve covers or a disposable lab coat.
3. Exhibits will be processed one at a time. Put away the previous exhibit before opening the next
exhibit.
4. Follow these criteria to determine the order to process the stains: process questioned stains
before opening standards; process small/dilute samples prior to large/concentrated samples.
Process the manipulation blanks last.
5. Clean instruments and fresh paper must be used for each exhibit.
6. Open only one tube at a time. Close the appropriately labeled tube immediately after a sample
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has been placed into it. (Do not process samples in a manner that would allow cuttings, flakes
or aerosols of biological material to fall into tubes destined to hold another sample).

7. Microfuge tube openers must be used for opening tubes at all times rather than fingers. Tube
openers must be cleaned in 10% bleach solution after each use.
8. Aerosol resistant tips (ART) must be used. Place the tip on the pipetter immediately before use.
If the pipetter has been set down do not use the tip.
9. During sample extraction, the approximate volume of all reagents required for a set of samples
must be poured from the stock into a disposable beaker. Never place a pipetter or pipette into
a stock reagent bottle. When adding aliquoted reagents and pre-aliquoted reagents to each
sample (such as proteinase K and DTT), a new ART tip must be used. When finished, discard
the beaker and aliquot of reagent .
All stock reagents must be closed when processing stains for extraction.
10. DO NOT use repeating pipetters (except for indexing samples).
11. Process the manipulation blank identical to all the other samples.
12. Use Spin-EASE" tubes to spin stain extraction buffer from cloth or cotton. The contents must
be transferred to regular microfuge tubes prior to phenol extraction.
13. Gloves must be changed between each exhibit, up to the extraction stage. Thereafter, if gloves
become contaminated with sample they must be discarded and replaced with new ones.
14. In order to prevent any liquid from getting on the lids of microfuge tubes, the tubes must be spun
briefly prior to opening.
Amplification Set-up
1. All amplifications must be set up in designated hoods. If the face shield on the hood does not
shield the analysts face, the analyst must wear a face mask during amplification set up.
2. Pipetters that are dedicated for amplification set-up located in the amplification set-up hood must
be used.
3. All hoods must be bleached before and after the samples are prepared for amplifications. Ultra-
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violet light must be used as an additional decontamination. Expose the hood to UV light for at
least 15 minutes before and after samples are prepared for amplifications.
4. Do not set the tube rack down in the post-amplification room while transferring samples to the
thermal cycler. If the rack is set down, it must be bleached before being utilized in the main
laboratory again.
5. After working with amplified DNA, an analyst must not work with any other biology or non-
amplified evidence.
Post-Amplification Room
1. All surfaces and applicable instruments must be thoroughly cleansed with 10% bleach solution.
Any non-disposable glassware or plastic ware must be thoroughly washed with laboratory soap
and a 10% bleach solution and rinsed with ddi H
2
O.
2. Designated lab coats must be worn in the post-PCR rooms and these lab coats must be worn
only in the post-PCR rooms. These coats must be removed from the laboratory for cleaning in
a closed container.
3. The sample preparation hood and pipetters must be bleached before and after the samples are
prepared.
REPORT WORDING
See Appendix I.
REFERENCES
Refer to the references under the DNA Isolation Methods Section.
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Method: DNA Isolation
PROCEDURES MANUAL
PROTOCOL: DNA Analysis
METHOD: DNA Isolation
Prepared by:

Approved by:

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INTRODUCTION
DNA profiles obtained from biological evidence provide information as to the source of the sample.
The goal of DNA isolation is to recover/extract high molecular weight DNA in sufficient quantities. Through
the use of standard procedures, DNA is isolated from nucleated cells from various biological specimens.
Cell lysis is achieved by the addition of proteinase K, SDS, EDTA, phenol, and chloroform allows for the
release of the DNA. This DNA is then further purified through microcon filtration.
SAFETY CONSIDERATIONS
Observe Standard Laboratory Practices.
Warning: The following are considered hazardous reagents:
Chloroform is toxic and is a suspected human carcinogen. It is harmful if inhaled, ingested or exposed
to the eyes or skin. Inhalation may lead to loss of consciousness, dizziness, gastrointestinal distress,
respiratory failure or death. It may cause skin irritation or destruction, stinging and/or temporary
injury to the cornea of the eye. If ingested seek medical assistance immediately. Treat eye and skin
exposure with routine laboratory safety procedures. Wear appropriate personal protective equipment
to prevent exposure.
Hydrochloric Acid is a corrosive chemical that causes severe burns if inhaled, ingested or exposed
to the eyes or skin and may cause permanent tissue damage. Accidental skin or eye contact will be
treated according to routine laboratory safety procedures. Do not induce vomiting if ingested. Get
medical assistance immediately. Wear appropriate personal protective equipment and use the fume
hood when using this chemical.
Isoamyl alcohol is harmful if inhaled, ingested or absorbed through the skin. Its vapor is irritating
to the eyes, mucous membranes and upper respiratory tract. It may cause nervous system
disturbances. Accidental skin or eye contact will be treated according to routine laboratory safety
procedures. If ingested, wash out mouth with water if victim is conscious. Obtain medical assistance
immediately. Wear appropriate personal protective equipment to prevent exposure.
Phenol is a toxic and corrosive chemical that is harmful or fatal if ingested, inhaled, or absorbed
through the skin. Furthermore, this chemical can cause irritation and/ or damage to the eyes. It is also
a suspected carcinogen. Accidental skin or eye contact will be treated according to routine laboratory
safety procedures. If ingested, immediately seek medical attention and DO NOT induce vomiting.
Special protection: use of a local exhaust hood is required; use chemical resistant protective gloves
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(LATEX gloves are NOT a sufficient barrier of protection) such as neoprene or rubber.
Phenol-Chloroform-Isoamyl Alcohol is a toxic and corrosive solution that is harmful or fatal if
ingested, inhaled, or absorbed through the skin. Furthermore, this solution can cause irritation and/
or damage to the eyes and is a suspected carcinogen. Accidental skin or eye contact will be treated
according to routine laboratory safety procedures. If ingested, immediately seek medical attention and
DO NOT induce vomiting. Special protection: use of a local exhaust hood is required.
Sodium Dodecyl Sulfate (SDS) potentially causes burns when in contact with the skin or eyes. It
causes irritation if inhaled and may cause severe digestive tract irritation if ingested. Accidental skin
or eye contact will be treated according to routine laboratory safety procedures (give milk or water
if ingested and seek medical assistance). When preparing solutions using this chemical, proper
personnel protective equipment, including a dusk mask or respirator, should be employed.
Sodium Hydroxide is a corrosive chemical which may be fatal if absorbed through the skin. It
causes severe eye and skin burns, gastrointestinal burns if ingested and severe irritation if inhaled.
Accidental skin or eye contact will be treated with routine laboratory safety practices. Do NOT
induce vomiting if ingested, seek immediate medical aid. Wear appropriate personal protective
equipment and use the fume hood when using this chemical. It generates a lot of heat when put into
solution so add this chemical slowly and carefully.
8-Quinolinol is harmful if ingested, inhaled or absorbed through the skin. It is irritating to the eyes,
skin, mucous membranes and upper respiratory tract. Laboratory experiments have shown mutagenic
effects. Accidental skin or eye contact will be treated with routine laboratory safety practices. If
ingested, rinse mouth out with water and seek medical attention. Wear appropriate personal
protective equipment and use the fume hood when using this chemical.
Xylene (Note: A Xylene Substitute may be used which does not have the following toxic properties).
This substance has caused adverse reproductive and fetal affects in animals. Long term exposure may
be associated with brain and nervous system damage. It is harmful if ingested, inhaled, or exposed
to the eyes or skin. Accidental skin or eye contact will be treated with routine laboratory safety
practices. If ingested, do not induce vomiting; and get immediate medical aid. If inhaled, get medical
aid immediately. Wear appropriate personal protective equipment when using this chemical.
Xylene substitute. This chemical may be harmful if high vapor concentrations are inhaled. Dizziness,
headaches or unconsciousness may result. Accidental skin or eye contact will be treated with routine
laboratory safety practices. If ingested, contact poison center and obtain medical attention
immediately. Wear appropriate personal protective equipment and use the fume hood when using this
chemical.
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PREPARATIONS
DNA Isolation Reagents:
7M Ammonium Acetate
Anhydrous ammonium acetate 53.96 g
Bring to 100 ml with ddi H
2
O.
Store the raw chemical at 4C (very hygroscopic).
___________________________________________________
Chloroform/Isoamyl Alcohol (24:1)
Chloroform 96 ml
Isoamyl alcohol 4 ml
Prepare under fume hood.
___________________________________________________
390 mM DTT
Dithiothreitol 620 mg
ddi H
2
O 10 ml
Aliquot into convenient size volumes and freeze at -20/C. Remix after thawing before use.
___________________________________________________
500 mM EDTA pH 8.0
EDTA-2H
2
O@Na
2
930.5 g
ddi H
2
O 4.0 l
NaOH pellets 75-100 g
When fully dissolved, add additional NaOH to bring the pH to 8.0 (EDTA does not go into solution
until the pH nears 8.0.) Adjust the volume to 5.0 liter. Autoclave.
___________________________________________________
Phenol/Chloroform/Isoamyl Alcohol
Buffer-Saturated Phenol 100 ml
(Gibco Life Technologies catalogue #15513-021)
Chloroform 96 ml
Isoamyl alcohol 4 ml
8-Quinolinol (optional) 200 mg
Use caution! Phenol is very caustic!
Prepare under fume hood. Store in glass bottles.
___________________________________________________
Proteinase K (20g/l)
Proteinase K 500 mg
ddi H
2
O 25 ml
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Aliquot immediately into convenient size volumes and freeze at -20C.
Remix after thawing before use.
___________________________________________________
2.0 M Na Acetate
Na Acetate-anhydrous 164.1 g
ddi H
2
O 1.0 l
___________________________________________________
200 mM Na Acetate
2.0 M Na Acetate 10 ml
ddi H
2
O 90 ml
___________________________________________________
20% SDS
SDS 1000 g
ddi H
2
O to 5 l
Heat gently to about 65/C and stir to dissolve. Use a nuisance mask.
Prepare reagent in the fume hood.
___________________________________________________
20X SSC
NaCl 175.3 g
Na
3
Citrate-2H
2
O 88.2 g
ddi H
2
O 800 ml
Adjust to pH 7.0 with HCl. Bring to 1.0 liter with ddi H
2
O.
___________________________________________________
1X SSC
20X SSC 5 ml
ddi H
2
O 95 ml
___________________________________________________
Stain Extraction Buffer (SEB)
1.0 M Tris, pH 7.5 10 ml
500 mM EDTA, pH 8.0 20 ml
NaCl 5.84 g
20% SDS 100 ml
Add ddi H
2
O to 1.0 liter. Store at room temperature.
___________________________________________________
TE
1.0 M Tris pH 8.0 10 ml
500 mM EDTA, pH 8.0 2 ml
Add ddi H
2
O to 1.0 liter. Autoclave.
___________________________________________________
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TNE
1.0 M Tris, pH 7.5 1.0 ml
NaCl 584 mg
500 mM EDTA, pH 8.0 400 :l
Add ddi H
2
O to 100 ml.
__________________________________________________
1.0 M Tris, pH 7.5
Tris base 121.1 g
ddi H
2
O 800 ml
Adjust to pH 7.5 with approximately 65 ml concentrated HCl. Add ddi H
2
INSTRUMENTATION
MINIMUM STANDARDS & CONTROLS
A manipulation blank consisting of a sterile swab or piece of cotton material must be processed after
handling unknowns and standards for each extraction protocol followed. The purpose of this control is to
ensure that contamination has not occurred due to the manipulation of the sample or the reagents used in
the procedure. Whenever an additional manipulation is done on a sample (i.e., 10 second injections or
reconcentration of the DNA) the same manipulation must be done to the manipulation blank.
Refer to the appropriate procedure for the type of body fluid DNA is being extracted from.
REPORT WORDING
See Appendix I.
REFERENCES
1. Farley, M.; J. Harrington. Forensic DNA Technology; Lewis Publishers: Michigan, 1991.
2. Giusti, A.; Baird, M.; Pasquale, S.; Balazs. I.; and J. Glassberg. Application of
Deoxyribonucleic Acid (DNA) Polymorphism to the Analysis of DNA Recovered from
Sperm. Journal of Forensic Sciences. 1986. Vol 31, No 2, pp 409-417.
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3. Hochmeister, M.; Budowle, B.; Borer, V.; Eggman, V.; Comey, C.; Dirnhofer, R. Typing of
Deoxyribonucleic Acid (DNA) Extracted from Compact Bone from Human Remains.
Journal of Forensic Sciences. Nov 1991. Vol 36, No 6, pp 1649-1661.
4. Labor, T.; OConnor, J.M.; Iverson, J.T.; Liberty, J.A.; Bergman, D.L. Evaluation of Four
Deoxyribonucleic Acid (DNA) Extraction Protocols for DNA Yield and Variation in
Restriction Fragment Length Polymorphism (RFLP) Sizes under Varying Gel Conditions.
Journal of Forensic Sciences. March 1992, Vol 37, No 2, 404-424.
5. Lee, H.C.; Gaensslen, R.E.; Pagliaro, E.M.; Guman, M.B.; Berka, K.M.; Keith, T.P.; Fops,
P. The Effect of Presumptive Test, Latent Fingerprint and Some Other Reagents and
Materials on Subsequent Serological Identification, Genetic Marker and DNA Testing in
Bloodstains. Journal of Forensic Identification. 1989, Vol 36,
pp 339-358.
Also see the following ISP validation studies (listed by number):
9: Extraction study and the use of DTT for bloodstain extractions.
20, 21, 22, & 23:
Effect of various environmental insults on DNA recovery.
24: Effect of two hour resolubilization vs. overnight in TE buffer.
30: Efficiency of the differential extraction for spermatozoa recovery.
41: Evaluation of the use of micro-concentrator tubes as a replacement for ethanol precipitation of
DNA.
42: Evaluation of the efficiency of stain extraction buffer in the differential extraction of sperm-cell-
containing mixed samples.
Accepted Date: April 11, 2002
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Appendix VI: Forensic Biology/
DNA Quality Assurance
PROCEDURES MANUAL

APPENDIX VI: FORENSIC BIOLOGY/DNA
QUALITY ASSURANCE

Prepared by:

Approved by:

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APPENDIX VI
Forensic Biology/DNA Quality Assurance
Table of Contents
I. Goals and Objectives
II. Organization and Management
III. DNA Personnel Qualifications and Training
IV. Sample Handling and Facility Requirements
V. Evidence Control
VI. Validations
VII. Analytical Procedures
VIII. Equipment Calibration and Maintenance
IX. Proficiency Testing
X. Audits
XI. Reports
XII. Reviews
XIII. Safety
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Goals and Objectives
Overall goals:
To provide comprehensive, uniformly accessible, high quality, state of the art forensic biology
services to the citizens of the State of Illinois; and,
To ensure the quality of this Forensic Biology/DNA testing.
Objectives:
To have documented Forensic Biology/DNA procedures which ensure the output of a quality
product;
To routinely monitor Forensic Biology/DNA testing; and,
To document the identification and correction of problems with Forensic Biology/DNA testing.
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Forensic Biology/DNA Quality Assurance Program
Organization and Management
The Forensic Biology/DNA Quality Assurance Program is part of the Command's Quality Assurance
Program. The following topics are addressed in the Command QA Manual:
Command Quality Assurance Program
Competency Testing
Proficiency Testing
Administrative Reviews
Command Quality Assurance Reviews
Mock Trial/Court Appearance Rating
External Proficiency Testing
Blind Proficiency Testing
Corrective Action
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Forensic Biology/DNA Personnel Qualifications and Training
I. Personnel conducting DNA casework
A. Prerequisites for DNA training/casework
Prior to assuming casework responsibilities, each analyst must have a bachelor's degree
in a natural science or its equivalent. Each analyst must have successfully completed
college course work in the following areas prior to beginning DNA supervised casework.
1. Molecular Biology
2. Genetics
3. Biochemistry
To qualify, courses do not have to have these titles, but must cover equivalent material.
The training coordinator will review the prerequisites to determine if the course
work/equivalents meet the prerequisite requirements.
B. Training/Qualifying
1. Each individual will complete a formal period of training or evaluation prior to
assuming casework responsibilities.
a. New analysts will complete the documented DNA training program.
b. Experienced analysts will have their technical knowledge reviewed and
evaluated.
2. The training/qualifying program will be documented in a training file.
a. The training coordinator will document the successful completion of the
training/qualifying program in a training file. A check list will be maintained
summarizing the training.
b. Upon the completion of the training program, the training file will be sent
to the Director of Training or designate.
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c. Upon completion of the training, the trainer will provide a copy of the
training checklist and a letter confirming the completion of training to the
trainees Laboratory Director.
C. Experience
All individuals will have worked in a DNA laboratory for a minimum of 6 months prior to
assuming DNA casework responsibilities.
D. Certification
Final approval for conducting casework rests with the Command Administration.
Initially, each analyst will be certified based on a recommendation by a training coordinator
prior to beginning casework within the Command.
1. To be certified in this manner, an individual will complete and pass the following:
a. Demonstrate the ability to analyze blood and body fluid stains using the
appropriate DNA technology;
b. Demonstrate the ability to reproduce accurate and precise results;
c. Demonstrate the ability to conduct analysis on non-probative cases;
d. Demonstrate theoretical knowledge of DNA analysis;
e. Successfully complete competency tests;
f. Successfully complete a mock trial; and
g. Successfully complete supervised casework.
2. Upon completion of training when an analyst returns to his/her laboratory, or when
a certified analyst transfers to a new laboratory, a competency test will be given
for STR analysis. Five samples must be analyzed. These samples will be
requested by the Laboratory Director through the Assistant Technical Leader or
the Statewide Technical Leader.
E. Continuing Education
1. Each analyst must be responsible for keeping abreast of current developments
within the field. Each DNA analyst must complete annual continuing education as
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required by DAB standard 5.1.3.1. Annual is defined as per calendar year.
Examples of how this may be accomplished include:
a. Professional organizations and their meetings;
b. In-service training;
c. Attendance at formal training courses;
d. Participation at in-house technical meetings/courses/seminars;
e. Review of current literature; and,
f. College coursework.
2. The Laboratory Director will provide the opportunity to participate in these
activities as outlined in the following directives:
a. Tuition Reimbursement
b. Society Memberships
c. Section Advisory Committees (SAC)
d. Attendance at Professional Meetings
e. Out-of-State Travel Requests
II. Statewide DNA Technical Leader/Assistant DNA Technical Leader/Assistant DNA Coordinators
Technical leadership of the DNA section will be provided and conducted in accordance with
Command programs and Quality Assurance Standards for Forensic DNA Testing Laboratories:
the Quality Assurance Program;
the Training Program; and
the Research and Development Program.
The Statewide Technical Leaders duties are as follows:
1. Performs technical review of casework; notified of and assists on difficult or non-routine
cases.
2. Reviews proficiency testing performance and all quality assessments of analysts made by
quality review coordinators.
3. Reviews day to day quality control checks.
4. Trouble shoots analytical procedures.
5. Oversees training of laboratory staff.
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6. Monitors expert testimony; notified of outside experts to lend assistance in court
preparation.
7. Is accountable for the laboratorys quality assurance program to the extent that he or she
has the authority to terminate the laboratorys DNA testing in the event of a technical
problem until the problem is solved.
8. If serving as an off-site Technical Leader, minimally makes and provides written reports
of quarterly review visits to the laboratory.
9. Provides input on technical matters which arise from audits.
The Assistant DNA Technical Leaders duties are as follows:
1. Review all required mixture cases for analysts in their laboratory.
2. Aid with analytical questions and advise on disagreements between analysts on
interpretation.
3. Identify cases to be directed to the Statewide Technical Leader, as noted in the FB/DNA
Procedures Manual, and work with the Statewide Technical Leader to resolve case
interpretation issues.
4. Assist, when appropriate, in troubleshooting any issues which might question the reliability
of the analytical work of the laboratory.
5. Be responsible for addressing report wording questions for bench level analysts.
6. Review proficiency testing performance and all quality review assessments of analysts in
their laboratory.
7. Be responsible for reporting to the Statewide Technical Leader and laboratory
management any contamination or extraneous DNA issues identified in a case or
laboratory.
8. Ensure all quality control requirements are up to date and prepare the laboratory for annual
quality review audits and biannual external audits.
9. Oversee the training of laboratory staff by reviewing current training and determining
additional needs.
10. Monitor expert testimony either by actual viewing of testimony or by review of court cards,
transcripts, or supervisor evaluations to ensure proper content and accuracy.
11. Lend assistance in court preparation when notified of outside experts.
12. Give input on technical matters which arise from audits and ensure compliance with DAB
standards.
13. Provide accurate and timely communications to the DNA analysts regarding Command
decisions affecting DNA.
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The Assistant DNA Coordinators duties are as follows:
Procedures Manual.
2. Assist, when appropriate, the Statewide Technical Leader in troubleshooting any issues
which might question the reliability of the analytical work of the laboratory.
4. Be responsible for reporting any contamination or extraneous DNA issues identified in a
case to the Statewide Technical Leader and laboratory management.
5. Ensure all quality control requirements are up to date according to DAB standards and
prepare the laboratory for annual quality review audits and biannual external audits.
III. CODIS Manager - will meet all DNA analysts requirements if they will be performing casework.
If they will not be performing casework they must meet the requirements for a databasing analyst.
IV. Databasing analysts
Databasing analysts will meet all DNA analysts requirements except for non-probative casework,
supervised casework and mock trials.
V. Laboratory technicians
A. Will have documented training, education and experience commensurate with their
responsibilities as outlined in job description;
B. Will not conduct laboratory tests with evidence samples unless properly trained and
proficiency tested.
C. Will perform tests such as, hybridizations, reagent preparation, film developing, monitoring
and performing QA/QC testing, and, be proficiency tested in each of these areas.
D. Will receive evidence for DNA analysis in accordance with the Evidence Control Policy
and the DNA case acceptance policy outlined in the Command Directives; and,
E. Will return DNA evidence in accordance with the Evidence Control Policy.
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Sample Handling and Facility Requirements
I. All forensic biology and DNA analysts will follow clean technique as documented in the Forensic
Biology/DNA procedures manual.
II. Laboratories conducting DNA analyses will conduct the following activities either in a separate
space or at a separate time:
Evidence examination;
DNA extractions; and,
PCR set up.
III. Aqueous amplified products will be contained in a room separate from non-amplified product.
Amplified product may be removed from the amplification area for disposal only. If amplified
product is removed from the amplification area it will be sealed in a closed container. Product gels
will be photographed in the amplification area.
IV. The laboratory will follow the decontamination procedures outlined in clean technique found in the
Forensic Biology/DNA procedures manual.
V. The laboratory administration will monitor clean technique practices. This monitoring will include,
but not be limited to, a review of the bleach logs. This monitoring must be documented by
laboratory administration initialing the bleach log. This is to ensure that all surfaces and equipment
are being bleached properly. These surfaces must include all counter spaces and floors,
centrifuges, pipettes, holders, pipette tip boxes, refrigerators, freezers, doors, chairs, light switches,
etc. All reagents must be kept in a closed cabinet or drawer. In addition, clean technique practices
will be evaluated during laboratory inspections, QA visits, and Statewide Technical Leader visits.
VI. Cleaning and Sterilization Procedures
A. Glassware and plastic containers will be cleaned with detergent and completely rinsed with
tap water by hand or using a dishwasher. Before laboratory use, these items will be rinsed
with distilled water. Items that come in contact with DNA samples will be cleaned with
detergent and 10% bleach solution. Then rinsed with distilled water.
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B. The reagent preparation section details those reagents which require sterilization by
autoclaving. The operations manual for the autoclave will be reviewed by the analyst prior
to operation.
C. All waste from the PCR room will be sealed in a closed container before being removed
from the PCR room.
D. The laboratory will follow the decontamination procedures outlined in Clean Technique
found in the Forensic Biology/DNA Procedures Manual.
1. The Forensic Biology/DNA laboratory floor will be mopped using a freshly
prepared 10% bleach solution once a week. The PCR room must be mopped
last. This must be documented in a log located in the Forensic Biology/DNA
laboratory.
2. The entire Forensic Biology/DNA laboratory (computer tops, equipment, etc.),
including the PCR room, must be bleached once a week with a freshly prepared
10% bleach solution. This must be documented in a log located in the Forensic
Biology/DNA laboratory.
3. The bleach log is used to monitor decontamination of facilities and equipment
according to the Quality Assurance Standards for Forensic DNA Testing
Laboratories (Standard 6.1.4).
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Evidence Control
I. The laboratory will have a documented evidence control system to ensure the integrity of physical
evidence. This is outlined in the following Command directives:
Evidence Receipt Forms
Submission of Physical Evidence by Mail
Submission of Forensic Biology Evidence
Blood Evidence
Submission of Evidence to the FBI
Collection of Biological Standards
Access to Physical Evidence
Evidence Packaging
Transferring Cases Between Laboratories
Case Tracking
Destruction of Physical Evidence
Documentation of Case Related Phone Calls or Conversations
Signature Requirements for Case Reports
Minimum Standards for Evidence Marking
Internal Evidence Chain
Case Acceptance Policy for DNA Analysis
Clean Technique
Uniform Guidelines for Mailing Evidence
II. Retention of materials generated during DNA analysis:
A. Materials generated by RFLP analysis:
1. Extracted DNA will be maintained on all probative samples.
2. Extracted DNA from standards may be discarded if the original sample is
preserved and available.
3. Membranes used to obtain RFLP profiles are considered documents and will be
retained frozen indefinitely.
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B. Materials generated by PCR analysis:
1. Extracted DNA remaining after analysis must be dried down in a Speedvac and
returned to the agency.
3. Amplified DNA may be destroyed if extracted DNA and/or the evidence is
4. Amplified DNA must be double packaged in plastic, one inside the other, and
returned to the agency if no other samples are available. The package must be
labeled Amplified DNA, Do Not Open.
5. Amplified DNA that is retained will be kept in a locked, dedicated freezer in the
post-amplification area.
C. Records maintained on materials generated during DNA analysis:
1. A record will be maintained of all stain cards and probative evidence retained after
DNA analysis prior to January 1, 2001. The record will include case number,
exhibit number and location.
2. A record will be maintained on all extracted DNA retained after DNA analysis
prior to January 1, 2001. The record will include case number, exhibit number
and location.
3. A record will be maintained on all amplified DNA retained after DNA analysis
and location.
III. Evidence Return Policy
A. All items of evidence including stains and extracted DNA must be returned after analysis
is completed. A sticker that states Biological Evidence - store at room temperature must
be placed on the outside of each item of evidence being returned.
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B. After 3 years, the case file will be returned to the originating laboratory.
IV. Notations To Be Made in The Case File
A. Any known history of the evidence prior to arrival in the laboratory.
B. Packaging at time of arrival.
C. Description, sketch, and/or photograph of evidence item showing location of stains and
where stain material was removed. A drawing or photograph or physical description of
the evidence item is mandatory when any potentially probative stains are removed. This
should reflect stain location and size of samples.
D. Size, shape, pattern and the appearance of stains.
E. Which samples were extracted for DNA and date extraction started.
F. Storage condition of evidence from time of arrival in the laboratory.
G. Disposition of evidence items and/or remaining stain specimen.
H. If assistance is received from an evidence technician for yield gels or slot blots, the
technician must initial the worksheet, picture or lumirad. If the technician prepares samples
for injections, it must be documented at the bottom of the amplification sheet and the
technician must initial it.
V. Stain Storage
A. Blood Standards
1. Standards from cases with no probative material and a live victim will be
2. Standards from deceased victims received by the laboratory will be retained
regardless of the presence of probative material.
B. Probative Material
After all work is completed on a case, all samples, including extracted DNA, are to be
returned to the submitting agency, along with any specific storage instructions.
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Validations
I. The laboratory will use validated methods and procedures as outlined in the Command Directives
and meet Quality Assurance Standards for Forensic DNA Testing Laboratories.
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References for STR Validation
1. Perkin-Elmer Applied Biosystems AmpFlSTR Profiler Plus PCR Amplification Kit Users Manual,
1997.
2. Weber JL. Abundant class of human DNA polymorphisms which can be typed using the
polymerase chain reaction. AM J Hum Genet 1989; 44:388-396.
3. Edwards A, Civitello A, Hammond HA, Caskey CT. DNA typing and genetic mapping with
trimeric and tetrameric tandem repeats. Am J Hum Genet 1991; 49:746-756.
4. Edwards A, Hammond HA, Jin L, Caskey CT, Chakraborty R. Genetic variation at five trimeric
and tetrameric tandem repeat loci in four human population groups. Genomics 1992; 12:241-253.
5. Hammond HA, Jin L, Zhong Y, Caskey CT, Chakraborty R. Evaluation of 13 short tandem
repeat loci for use in personal identification applications. Am J Hum Genet 1994;55:175-189.
6. Baylor College of Medicine, inventors. Perkin-Elmer Applied Biosystems Corporation, assignee.
AmpFlSTR Profiler Plus PCR Amplification Kit.
US Patent 5,364,759.
7. Yamamoto T, Uchihi R, Nozawa H, Huang X, Leong Y, Tanaka M, Mizutani M, Tamaki K and
Katsumata Y. Allele distribution at nine STR loci-D3S1358, vWA, FGA, TH01, TPOX,
CSF1PO, D5S818, D13S317 and D7S820-in the Japanese population by multiplex PCR and
Capillary Electrophoresis. J Forensic Sci 1999;44:167-170.
8. Fregeau CJ, Bowen KL and Fourney RM. Validation of highly polymorphic fluorescent multiplex
short tandem repeat systems using two generations of DNA sequences. J Forensic Sci
1999;44:133-166.
9. Crouse CA, Rogers S, Amiott E, Gibson S and Masibay A. Analysis and interpretation of short
tandem repeat microvariants and three banded allele patterns using multiple allele detection
systems. J Forensic Sci 1999;44:87-94.
AmpFlSTR Profiler PCR Amplification Kit.
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11. Perkin-Elmer Applied Biosystems. AmpFlSTR Profiler PCR Amplification Kit Users Manual.
1997.
AmpFlSTR Blue PCR Amplification Kit.
13. Baylor College of Medicine, inventors. Promega Corporation, assignee. GenePrint PowerPlex
Fluorescent STR System.
14. GeneScan and GenoTyper [computer programs] Macintosh version, 1997.
15. Perkin-Elmer Applied Biosystems. Performance Optimized Polymer 4 (POP-4) US Patent
5,552,028.
5,552,028.
17. Gentra Systems Inc. Puregene DNA Isolation Kits.
18. Comey CT, Koons BW, Presley KW, Smerick JB, Sobieralski CA, Stanley DM, Baechtel FS,
DNA extraction strategies for amplified fragment polymorphism analysis. J. Forensic Sci.
39:1254-1269.
19. Perkin-Elmer Applied Biosystems QuantiBlot
TM
Human DNA Quantitation Kit Users Manual,
1996.
20. Walsh PS, Fildes NJ, Reynolds R. Sequence analysis and characterization of stutter products at
the tetranucleotide repeat locus vWA. Nucleic Acids Res. 1996;24:2807-2812.
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Analytical Procedures
I. Procedures.
The laboratory will have approved, written analytical procedures.
A. Procedures used in DNA and Forensic Biology Analysis will be approved
according to the Command Directives.
B. Procedures being developed as part of the R&D Program may be used in
casework with Command approval.
II. Reagents.
The laboratory will use reagents that are suitable for the methods employed.
A. The laboratory will maintain a log for documenting commercial biological reagents
utilized in the laboratory.
1. The log will be maintained on all chemicals received in the laboratory
2. Information contained in the log will include the manufacturer, the date a
chemical was received, the lot numbers received, the quantity received,
the storage conditions and the expiration date if appropriate.
3. An annual inventory of these reagents will be conducted by a person
designated in each laboratory.
B. The laboratory will maintain a log for documenting the formulation of all reagents
1. The formulations for all reagents are found in the Procedures Manual and
the Reagent Log.
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2. Information kept in the log must include the date the reagent was
prepared, the lot numbers of chemicals used to prepare the reagent, the
concentration and quantity of the reagent prepared, the identity of the
analyst preparing the reagent, the storage conditions of the reagent.
C. Reagents will be labeled with the identity of the reagent, the concentration of the
reagent, the date of preparation and date of expiration (if necessary), the identity
of the individual preparing the reagent and the storage conditions. Deionized water
must be autoclaved for use in dilutions for extractions and amplifications.
D. Expiration Dates
Manufacturer suggestion for expiration dates of reagents must be followed. If
there is no manufacturer suggested expiration date for a reagent then the expiration
date will be set at one year for purchased and prepared reagents. In addition,
when frozen reagents are thawed for use, they will have an expiration date of one
year from the date they are thawed and put into use. Autoclaved water will have
an expiration date of six months.
E. Critical Reagents.
The following reagents have been defined as critical reagents:
1. Species testing antisera
2. ABAcard kits
3. Nylon membrane
4. DNA quantitation kits (Expiration date: listed on kit)
5. DNA amplification kits (Expiration date: listed on kit)
6. DNA Internal Lane standard - ROX 500 (Expiration date: Six
months after receipt)
7. Formamide
8. Yield Gel Standards
F. These critical reagents must be quality controlled in house.
1. Procedures for quality control of these reagents are found in this
manual.
2. A critical reagent log will be maintained documenting all quality
control procedures performed on a particular lot of a reagent.
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3. If a particular supply, chemical, reagent or material does not meet
the required quality control standard(s), the manufacturer will be
notified and the entire lot rejected.
4. The procedures for critical reagents do not have to be run
individually but may be combined with other procedures as
appropriate.
5. Quality control records will be maintained indefinitely.
G. In procedures specifying using previously quality controlled lot, appropriate controls and
known standards will be used if an old lot is available.
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III. Basic Forensic Biology Procedures
A. Blood Standards
Dry stain cards will be produced from all whole blood specimens as soon after receipt as
possible.
B. Dry Blood
The minimum work on stains will be to indicate blood. Classifying the stain as human
material may be conducted when appropriate.
C. Semen
Test all kit swabs using the acid phosphatase test. Semen will be identified by the presence
of spermatozoa or by the P30 ABAcard test. Examination for the presence of
spermatozoa includes observation of either intact or identifiable heads where acrosomal
cap, point of attachment, size and shape are clearly visible. If the examination for
spermatozoa is negative or inconclusive, then the P30 ABAcard test will be conducted on
the sample. Examination of clothing items, including underwear, will be conducted if
determined to be of probative value. Otherwise clothing will not be routinely examined.
Once semen is identified in a case, the analyst will routinely defer all other semen testing
pending DNA results.
D. Vaginal secretion will be indicated by the identification of glycogen-containing squamous
epithelial cells (positive Lugols stain test). This test is not confirmatory for vaginal
secretion.
E. Saliva
Saliva will be indicated by a positive amylase test. (Phadebas or radial gel diffusion
technique.) This is not confirmatory for saliva.
F. Urine
Urine will be indicated by a positive urea nitrogen test or a positive creatinine test.
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IV. Standards & Controls
The laboratory will monitor the analytical procedures using appropriate controls and standards.
A. The following standards and controls will be used in Forensic Biology casework and are
included in the Forensic Biology\DNA Procedures Manual:
1. Dry Blood - Stain Identification
a. Kastle-Meyer- a positive and negative control each day the test is used.
b. Ouchterlony Immunological Tests - a positive and negative control each
run.

Test all antisera (human and animal) against the following series of known
standards when a new lot is received:
Human, swine, bovine, deer, goat, cat, dog, sheep, chicken or duck
(bird), rat or hamster or rabbit (rodent).
Antisera testing will be documented in a logbook. Logbook information
will include antisera lot numbers and brand names. Documentation of
manufacturers recommended expiration dates will also be included in the
logbook.
2. Semen ID
a. Acid phosphatase - two-step procedure. Run known semen and
negative control with each set of tubes opened each day.
b. P30 by ABAcard - run 10ng and 4ng known semen control and negative
control with each new lot number of cards. Results to be recorded in the
critical reagent log. Run a control blank with each daily batch of sample.
3. Lugols Stain
Run known vaginal squamous epithelial cells as a positive control. Run known
buccal squamous epithelial cells as a negative control. Run controls each day the
test is used.
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4. Amylase (Phadebas tablets)
Run known dry saliva extract as positive control and a control blank each day the
test is used.
5. Amylase (radial gel diffusion method)
Run a positive control consisting of an aqueous dilution of 1/500 of fresh liquid
saliva and a control blank, each day the test is used.
6. Urea Nitrogen
Run known dry urine extract, a control blank, and a portion of the suspected urine
stain with the phenol, hypochlorite and nitroprusside without the urease added
each day the test is run.
7. Creatinine Test
Known urine stain and a control blank.
B. The following standards and controls will be used in PCR casework and are included in
the Forensic Biology/DNA Procedures Manual. These standards and controls must work
properly.
1. Quantitation standards for estimating the amount of DNA recovered by extraction.
See Yield Gel and Slot Blot sections of the Forensic Biology/DNA Procedures
Manual.
2. Positive and negative amplification controls.
3. Allelic ladder for variable number tandem repeat sequence PCR based systems.
4. Manipulation blanks will be used to monitor clean technique. If during quantitation
DNA is visible in the manipulation blank, it must be reported to the Assistant
Technical Leader and the Statewide Technical Leader. The sample will be re-
extracted. If no case material remains, it may be necessary to utilize the extracted
sample upon approval by a the Assistant Technical Leader and the Statewide
Technical leader. Twenty percent of the manipulation blank must be amplified in
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at least Profiler Plus. If extraneous DNA is detected in any manipulation blank,
negative control or any sample, the case must be brought to the attention of the
Assistant Technical Leader and the Statewide Technical Leader. When an
extraneous DNA profile is obtained in a manipulation blank but does not appear
in any of the probative samples, the entire DNA analysis will be repeated, if
possible. However, if sufficient probative sample is not available for retesting, the
incident will simply be documented in the analysts case notes and the extraneous
DNA profile will not be reported. An interpretation may be made depending on
the specific case situation. The Assistant Technical Leader or DNA Supervisor
must inform the Statewide Technical Leader and the Quality Assurance Program
Administrator. An incident report must also be made by the DNA supervisor and
placed in the Incident notebook located in the analysts laboratory. A copy of the
report must be given to the Quality Assurance Program Administrator. The
Quality Assurance Program Administrator may file a Quality Issue Tracking form
on the incident. A copy of this form will be maintained by the Quality Assurance
Program Administrator. These incident reports will be monitored by the Assistant
Technical Leader and the Statewide Technical Leader. If there is more than one
occurrence of contamination by an analyst within a six month period, laboratory
management must notify the Quality Assurance Program Administrator and the
Statewide Technical Leader. In addition, if the extraneous DNA compromises the
analysts ability to determine genotypes of the probative profile, a situation report
must be filled out by the laboratory management..
V. The laboratory will have written general guidelines for interpretation of data.
A. The laboratory will verify that all control results are typed correctly.
B. For a given population(s) and/or hypothesis of relatedness, the probability of observing a
DNA profile will be estimated using a standard population genetic method(s) and/or
directed method (as described in Forensic Biology/DNA Procedures Manual). These
calculations will be derived from a documented population database appropriate for the
calculation.
VI. All cases with extraneous DNA will be reviewed by the Statewide Technical Leader.
A. When an extraneous DNA profile is obtained in a probative sample which cannot be
reanalyzed and the extraneous DNA profile is identified in at least four loci as having come
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from a laboratory employee, the extraneous DNA profile will be reported in the laboratory
report.
B. When an extraneous DNA profile is obtained in a probative sample which can be
reanalyzed, it will be. If the extraneous DNA profile does not reappear in the probative
sample, the extraneous profile will not be reported but will be documented in the case
notes. If the extraneous profile does reappear and is identified in at least four loci as having
come from a laboratory employee, the extraneous DNA profile will be reported in the
laboratory report.
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Quality Control of P30 Analysis by ABACard
1. Purpose: To compare new lot numbers of ABACard for P30 analysis against known
positive and negative (blank) controls.
2. Procedure:
A. Extract a positive 10ng and 4ng standards and a negative (blank) sample according to the
guidelines of the Forensic Biology/DNA Procedures Manual.
B. Incubate at room temperature for 2 hours with gentle agitation on a shaker.
C. Pipette 200ul of extract to the sample well S of the test strip.
D. Record results at 10 minutes.
3. Assessment of Results:
A positive is indicated by the presence of two pink lines: one line in the control of C area and one
in the test or T area.
A negative is indicated by the presence of one pink line in the control or C area.
An error is indicated if no C line appears.
Results must be obtained for both the 10ng and 4ng standards.
If results are not what is expected, please repeat.
If, after repeating the test, the results still do not coincide with the expectations of the test, notify
the manufacturer.
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Quality Control of P30 by ABACard
DATE LOG# NEGATIVE
10NG
POSITIVE
4NG
POSITIVE ANALYST
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Quality Control of Species Antisera
1. Purpose: To compare new lot numbers of antisera (human and animal) to the appropriate series of
known standards which are human, swine, bovine, deer, goat, cat, dog, sheep, chicken or
duck (bird), rat or hamster or rabbit (rodent) as outlined in the Forensic Biology/DNA
Procedures Manual.
2. Procedure:
A. Punch the gel with a series of 7 wells to form a hexagon with a central well.
B. Place the antiserum to be QCd in the central well and bloodstains for 6 different species in the
surrounding wells.
C. Cover the petri dish and leave undisturbed at room temperature overnight. The petri dish can be
placed in a 37/ oven to decrease the incubation period or in the refrigerator for a longer
incubation period.
D. Any known stain which forms a precipitin band with the antiserum must be checked using the
following triangular 3-well Ouchterlony pattern:
Set up a positive control: known blood sample in both left and right wells of the triad.
Set up a negative control: known blood sample in one well and a negative (blank) in the other.
Place the antisera being QCd in the third well.
E. Cover the petri dish and leave undisturbed overnight at room temperature. The petri dish can be
incubation period.
F. Record the results on an Ouchterlony worksheet.
Precipitin bands which form a continuous arc of convergence (identity) between the antiserum and the
2 extract wells are considered positive results.
If no or partial precipitin bands form and a positive test result is expected, repeat test. If a positive
result is noted for a species other than what antisera it is directed against, repeat test.
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If, after repeating the test, the results do not coincide with the expectations of the test, notify the
manufacturer.
Quality Control of Ouchterlony Plates
Species being tested __________________________
Anti-Serum Lot Number __________________________
Anti-Serum Brand __________________________
Expiration Date __________________________
Date QCd __________________________
Analyst __________________________
ANTI____________________
TESTED AGAINST OBSERVED RESULT
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Verification of yield gel standards
1. Purpose: To compare newly prepared standards against previously QCd standards or
commercially prepared standards to ensure consistency
2. Procedure:
A. Prepare new yield gel standards as outlined in the Forensic Biology/DNA Procedures Manual.
B. Prepare a 1% minigel as outlined in the Forensic Biology/DNA Procedures Manual.
C. Load 6ul of each new standard and previously QCd standards into wells.

D. Run at 200 volts for 8 minutes
E. Remove gel from tank and examine on an ultraviolet light transilluminator
F. Photograph gel

-Compare intensity and width of previously QCd standards against new standards.
-There should be no appreciable difference in fragment width and intensity.
-If there is a discernible difference, remake standards and repeat procedure.
This procedure must be run for each new quantitation standard set.
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Verification of Yield Gel Standards
Cathodal Origin
Lane Exhibit Volume DNA(ng)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Middle Origin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Include Photograph Along
With This Sheet
Analyst_________ Date_________
Lot# ____________
Date Received__________________
Amount Received_______________
Date of Dilution ________________
Previously QCd Lot # or
D a t e o f D i l u t i o n i f S a m e
Lot#_________________________
Notes:
Are the intensities and widths
of the new standards comparable
t o t h o s e p r e v i o u s l y
QCd?________________
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Verification of Slot Blot Standards New Lot# (Slot Blot Kit)
1. Purpose:
To compare newly prepared standards against calibrators present in kit.
2. Procedure:
Conduct slot blot analysis as outlined in the Forensic Biology/DNA Procedures Manual with newly
prepared standards from the new lot number kit. Also run calibrators present in kit.
-Compare intensity of calibrators to newly prepared standards. The calibrators must fall within the
nanogram range specified by the manufacturer.
-If the calibrators fall out of range, reject new lot number and notify manufacturer.
4. Expiration Dates:
Once a critical reagent has exceeded its expiration date, discontinue its use in casework.
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Verification of Slot Blot Standards (Slot Blot Kit)
Analyst__________ Date__________ Kit Lot #__________
Date Received__________ # of Kits Received_____ Kit Expiration Date__________
1 2 3 4 5 6
A
B
C
D
E
F
G
H
Notes: Upon comparison to the newly prepared standards, do the
Calibrators fall within the nanogram range specified by the
manufacturer?__________________________________
SAMPLE ID
ng CALLED Include Photograph Along With This Sheet
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Verification of Charged Membrane for Slot Blot
1. Purpose: To evaluate a new lot of charged membrane for binding ability and band intensity.
2. Procedure:
A. Conduct slot blot analysis as outlined in the Forensic Biology/DNA procedures manual with new
lot number of charged membrane. Run slot blot standards and calibrators 1 and 2.
-Compare intensity of signal of standards to calibrators. If band intensity is weak, reject lot number
and notify manufacturer.
-Compare results to the results of previously QCd membrane. If there is a weaker signal, reject the
new lot of membrane.
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CHARGED MEMBRANE
(For Slot Blot)
Manufacturer______________________ Date Received_________________
Lot #s Received___________________ Quantity Received_______________
Expiration Date________________
Date QCd________________________ QCd by______________________
Standards
(ng)
Calibrators
NOTES:
Is band intensity adequate when compared to calibrators?_________________________
Is band intensity consistent with that of previously quality controlled membrane?____________________
ATTACH PICTURE OF SLOT BLOT
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Verification of STR Typing Kits for the ABI Prism 310 Genetic Analyzer
1. Purpose: To demonstrate that all amplification components contained in the kit can produce
accurate typing results.
2. Procedures:
A. Prepare amplification reaction mixture using components from new lot number kit, as prescribed
in the Forensic Biology/DNA Procedures Manual.
B. Controls consist of the Control DNA 9947A (present in kit) and a negative control consisting
of 20 :L ddi H
2
O.
C. Amplify for appropriate loci as outlined in the Forensic Biology/DNA Procedures Manual.
D. Type the amplified products using the 310 Genetic Analyzer as outlined in the Forensic
Biology/DNA Procedures Manual.
3. Assessment of Results
A. All control samples must type correctly.
B. If incorrect or incomplete typing results are obtained reject the lot number and notify
manufacturer.
The expiration date is located on the box.
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Verification of COfiler Kit
Analyst__________________________ QC Date ___________________
Kit Lot #___________________ Kit Expiration Date________________
# of Kits Received__________ Date Received_______________
Individual Kit Components
Please check and Note if Individual Component Lot numbers Vary From Kit to Kit
Primer Set#____________ AmpliTaq Gold #____________
Reaction Mix#___________ Mineral Oil #_____________
Allelic Ladder ___________ 9947A#_________________

9947A Control DNA Negative Control
D3S1358
D16S539
TH01
TPOX
CSF1PO
Amelogenin
D7S820
NOTES:
Do all Controls exhibit expected alleles? ____________________________
Include positive control electropherogram and negative control electropherogram along with this sheet.
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Verification of Profiler Plus Kit
Analyst__________________________ QC Date ___________________
Kit Lot # ________________________ Kit Expiration Date __________
# of Kits Received_________________ Date Received_______________
Allelic Ladder ___________ 9947A#_________________

D3S1358
vWA
FGA
Amelogenin
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
NOTES:
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Verification of GeneScan 500 In-lane Sizing Standard
1. Purpose: To test a new lot of GeneScan 500 in-lane sizing standard for calculation of fragments
separated during capillary electrophoresis.
2. Procedure:
A. Mix 1 :L of new lot of GeneScan 500 in-lane sizing standard with 24 :L deionized formamide
according to the Forensic Biology/DNA Procedures Manual.
B. Type the GeneScan 500 in-lane sizing standard using the 310 Genetic Analyzer according to the
Forensic Biology/DNA Procedures Manual.
A. The peaks corresponding to the 75, 100, 139, 150, 160, 200, 300, 340, 350, and 400 bp
fragments must be sharp and well defined with a fluorescence intensity of 150 or higher.
B. A recently amplified but never typed set of samples can be used to verify the new lot of 500 in-
lane sizing standard as long as the positive and negative controls and the 500 in-lane sizing
standard type correctly.
C. If the above conditions cannot be successfully demonstrated, reject lot number and notify
manufacturer.
The expiration date per manufacturer suggestions is six months after date of receipt.
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Analyst__________________________ QC Date ___________________
GS 500 in-lane Sizing Standard Lot #___________________
# Received__________ Date Received_______________
Expiration Date______________________
Peak present, sharp, and
well defined
Fluorescence Intensity
75
100
139
150
160
200
300
340
350
400
450
490
500
NOTES:
Are all expected peaks present, sharp, and well defined? _______________________
Include Gene Scan and/or GenoTyper data for sample ROX applied along with this sheet.
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Annual System Verification with Standard Reference Material 2391
Purpose: To annually verify that the entire PCR system is functioning within accepted criteria by the use of
SRM 2391 from the National Institute of Standards and Technology.
Procedure:
A. Extract as per ISP protocol
B. Amplify components #3-12, 19-20, using ISP protocol for appropriate loci.
C. Analyze samples using the ABI 310 Genetic Analyzer Capillary Electrophoresis Instrument.
Assess Results: Compare the results of each test to the NIST results for each component. Any
discrepancies will be reanalyzed and resolved.
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Evaluation of a New ABI 310 Genetic Analyzer
1. When a new Applied Biosystems 310 Capillary Electrophoresis unit is received into the laboratory,
it must be evaluated before it can be used for case work. The following studies must be conducted.
2. When major equipment changes are necessary for the Applied Biosystems 310 Capillary
Electrophoresis unit, one or more of the following studies may be required. The type of studies
required will be dependent on the type of equipment replaced.
A. Matrix
Prepare a matrix file using the 5-FAM, JOE, NED, and ROX matrix standard samples and the
Filter Set F module files according to Forensic Biology/DNA Procedures Manual.
B. Instrument Run Time
Before proceeding with the following studies the instrument electrophoresis run time must be
determined. Run five allelic ladders containing the ROX-500 in-lane sizing standard. Ensure that
the 400 base pair peak of the ROX-500 in-lane sizing standard is consistently being observed.
If the 400 base pair peak is consistently observed then leave the run time at 24 minutes. If the
400 base pair peak is not consistently being observed then increase the run time by one minute
and run five more allelic ladders to determine if 400 base pair peak is now being seen.
C. Precision and Reproducibility
Choose five samples that have been previously characterized using the AmpFlSTR system. Each
sample is to be run 20 times on the ABI 310 CE and analyzed using the GeneScan and
GenoTyper software according to ISP protocol. Determine the average, standard deviation,
minimum, and maximum values for the alleles at each locus for each sample. This part of the
precision study will also serve as a reproducibility study.
D. Sensitivity
Choose three samples previously characterized using the AmpFlSTR Profiler Plus system and
make a dilution series of each sample from 5 ng to 0.03 ng. These three samples should contain
alleles that represent both high and low alleles across all loci.
Note: The data from each of these studies must be maintained in a notebook that is kept near each
instrument along with a routine maintenance log. The routine maintenance log must include documentation
of cleaning the block, changing the polymer, changing the capillary, changing the buffer, and defragmenting
the Macintosh hard drive.
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Quarterly Sensitivity Check for the ABI 310 Genetic Analyzer
1. Purpose:
To monitor the peak height of one locus of the Profiler Plus positive control to allow the analyst to
observe a failing laser. This must be done quarterly.
2. Procedure:
A. Inject a Profiler Plus positive control ten times. Record the RFUS for the 23 allele at the FGA
locus.
B. Monitor the RFUs over time and note any significant decreases in RFUs. This may be an
indication that the laser is failing and a service engineer needs to be called.
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ABI 310 _____________________________________ Date_________________________
Analyst ______________________________________
Profiler Plus Positive Control Lot# _________________________________
FGA - 23 allele Fluorescence Intensity
1
2
3
4
5
6
7
8
9
10
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Annual Verification for the ABI 310 Genetic Analyzer
A. Matrix:
Prepare a matrix file using the 5-FAM, JOE, NED, and ROX matrix samples and the filter set F
module files according to ISP DNA protocol.
B. Sensitivity:
Choose three samples previously characterized using the AmpFlSTR Profiler Plus system and make
a dilution series of each sample from 5ng to 0.03ng. These three samples should contain alleles that
represent both high and low alleles across all loci.
Verification Study Date Completed Initials of Accepting
Supervisor
Matrix Installation
Sensitivity
**PLEASE ATTACH COPY OF MATRIX AND SENSITIVITY RUN**
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Quality Assurance
Equipment Calibration and Maintenance
I. Calibrations:
A. For each piece of equipment, the following calibration information will be listed:
1. Procedure
2. Frequency
3. Results
4. Course of action
B. Records will be maintained for calibrations.
II. Maintenance
A. Where appropriate, maintenance procedures and schedules will be listed for equipment.
B. Records will be maintained for maintenance.
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Balance Calibrations
I. Each balance is cleaned of any excess debris and leveled before checking calibration.
II. Standard weights are placed on the balance using cotton gloves or forceps.
III. Use the manufacturer's recommendation for which weight will be used as a minimum. If there is no
recommendation, use a minimum of 3 weights (high, low and medium) that represent the normal
weights measured on the balance.
IV. Frequency:
A. Check calibration monthly, or if used less frequently, prior to use.
V. Results:
A. Record the results in a log book.
B. Computerized records will be backed up.
VI. Course of action:
A. Compare the recorded results to the tolerance window as specified by the manufacturer.
B. If the weights are not within the specified tolerance window, then contact the manufacturer for
repairs.
VII. Maintenance
A. Procedure - The balance will be maintained by a company qualified to provide certification for
the balances' accuracy.
B The frequency of certification will be annually.
C. The results of certification (certificate) will be maintained in the laboratory.
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Conductivity Meter and Testing the Conductivity of Formamide
I. Calibration Procedure
A. Calibrate the conductivity meter according to the instructions for manual calibration using the
manufacturers instruction manual for calibration with a 100 siemens conductivity calibration
standard.
B. Calibrate before each use.
C. Acceptance of the lot of formamide signifies the calibration of conductivity meter.
II. Course of Action
A. Retry procedure if conductivity meter will not calibrate.
B. Reject Formamide if conductivity is greater than 100 siemens.
C. Call repair company for repairs if conductivity meter will not calibrate on second attempt.
D. No recorded documentation is necessary.
III. Maintenance
No maintenance is recommended by manufacturer.
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Oven Calibrations
I. Calibration
A. Procedure
1. Set temperature of oven at desired setting for laboratory procedures.
2. Place a NIST traceable thermometer in oven.
3. Read temperature after thirty minutes.
B. Frequency
1. Calibrate annually, but observe temperature before each use.
C. Results
1. Record the results on the appropriate form.
D. Course of Action
1. Adjust the temperature dial if necessary and recalibrate.
2. Call the repair company for any needed repairs if the oven is out of calibration.
II. Maintenance
A. No routine maintenance is recommended by the manufacturer.
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Freezer/Refrigerator Calibrations
A. Place a NIST traceable thermometer into the freezer or refrigerator.
B. Take temperature after 5 minutes.
C. Calibrate annually, but monitor at least monthly.
D. Record the results of calibration and monitoring in a log book.
A. Compare the recorded results with the ranges as specified by the manufacturer if provided. If
there is no specification, establish an appropriate range based on the use of the
refrigerator/freezer.
B. If the temperatures are not within the specified tolerance window, contact the manufacturer or
repair company for repairs.
III. Maintenance
A. No regular maintenance is required. Defrost/clean as necessary. Contact the repair company
for repairs.
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pH Meter
A. Calibrate the pH meter according to the instructions for manual calibration using the
manufacturers instruction manual for calibration with two buffers (one above and one below
desired pH).
C. For reagents with the pH noted in the procedure, the analysts initials signifies calibration of the
pH meter. If the pH meter does not calibrate, note in the maintenance log.
A. Retry procedure if pH meter will not calibrate.
B. Call repair company for repairs if pH meter will not calibrate on second attempt.
C. No recorded documentation is necessary.
III. Maintenance
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Pipette Calibrations
A. Pipettes can be calibrated according to in-house procedures or,
B. Pipettes can be contracted to either the manufacturer or a certified agency for calibration.
C. Calibrate annually.
D. Record results in a log book. If contracted out, modify the form to match the procedure.
II.. Course of Action
A. Compare the recorded results to the tolerance window, +/- 2.5%.
B. Remove pipette from casework if the recorded tolerance window is greater than +/- 2.5%.
C. Resubmit for repair and subsequent recalibration.
III. Maintenance
The only required maintenance is annual cleaning and calibration.
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Microscopes
1. Cleaning and Maintenance
Please refer to Appendix IV of the Microscopy Procedures Manual (excluding Ocular Scale
Calibration) for proper cleaning and maintenance.
Note: Due to the hazardous nature of xylene, a generic lens cleaner (usually isopropyl alcohol) or
a xylene substitute may be used for cleaning.
2. Microscopes will be checked quarterly (cleaning, general maintenance, and optimal illumination) and
documented in an appropriate logbook.
3. Microscopes will be covered at the end of the day.
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Alternate Light Source
1. Calibrations:
A. Check monthly. If used less frequently, check before each use.
B. A 1:50 dilution of semen will be visually examined. Record results in log book. New stains will
be prepared by the QA Coordinator every 3 years.
2. Course of Action:
A. If 1:50 dilution is visualized, the alternate light source is working properly.
B. If 1:50 dilution is not visible make a new stain. If still not visible, have alternate light source
serviced.
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Thermal Cycler Calibrations
A. Calibrate thermal cyclers according to the methods in the Perkin Elmer Cetus users manual for
the DNA thermal cycler.
B. Conduct temperature verification test monthly.
C. Conduct temperature uniformity test every 6 months.
(These test schedules are based on the recommendations of Perkin Elmer.)
D. Record the results in a log book.
Call Perkin Elmer if thermal cycler falls out of the documented ranged (found in the compatible
manual) and discontinue use.
III. Maintenance
Return the temperature verification system to Perkin Elmer annually for calibration.
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NIST Thermometer Calibration
A. Prepare an ice bath and place the NIST thermometer at an appropriate depth.
B. Take reading of NIST thermometer. Thermometer should read 0 degrees Celsius.
II. Record results in a log book.
III. Course of Action
A. If the thermometer reading is +/- 2 degrees from 0 degrees, the NIST thermometer will not be
used for any further calibrations.
B. The original certification should be maintained in the laboratory.
IV. Maintenance
None
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NIST Traceable Thermometer Calibrations
B. Place the thermometer being tested at the same depth as the NIST thermometer and read the
temperature.
C. Follow the same procedure for a boiling water bath, if applicable.
(It is assumed an ice bath will be 0 degrees Celsius and a boiling water bath will be 100 degrees
Celsius.)
D. Calibrate annually.
If the thermometer is greater than +/- 2 degrees from the NIST thermometer, it will not be used for
any further calibrations.
IV. Maintenance
None
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Water Bath Temperature Recording
I. Procedure
A. Place a NIST thermometer or NIST traceable thermometer in the bath for 5 minutes.
B. On the appropriate form, record the temperature results monthly.
C. Follow course of action if the temperature is not within the specified tolerance window (+/- 1
C).
A. Adjust the temperature setting and retake the bath temperature if reading is slightly off.
B. Call the manufacturer or repair company for repairs if the temperature will not adjust and do not
use the water bath.
III. Maintenance
Empty, clean and refill with fresh water as needed.
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Proficiency Testing
I. Proficiency testing is performed in accordance with Command QA Manual, which includes the
following:
Competency Tests
Internal Proficiency Tests
II. The results of proficiency test results will be checked and compared to the standards by the Quality
Assurance manager as outlined in the Command QA Manual.
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Audits
I. The Command's inspection program is covered in the Command QA Program:
Purpose of an Inspection Program
Vehicle Inspection Forms
General Inspection, Security, and Safety Guidelines
Laboratory Evidence Vault Inspection
II. In addition to the Command's inspection program, external auditors will review the DNA section in
the laboratory once every two years according to ASCLD/LAB criteria.
A. A record of the audit report will be maintained in the laboratory.
B. A copy of the external audit report will be sent to the Quality Assurance Program Administrator
with an action memo addressing issues identified by the auditor.
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Reports/Case Files
I. Report wording guidelines are found in the Procedures Manual.
II. DNA Case file preparation is provided in the attachment.
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CASEFILE ORGANIZATION
NUMBERED PAGES:
1. DNA COVER SHEET (Always numbered as page 1).
2. CONVERSATION RECORDS (All correspondence regarding case).
3. LABORATORY WORKSHEET(S) (Describing exhibits).
4. YIELD GEL WORKSHEET(S) WITH PHOTOGRAPH(S).
5. SLOT BLOT WORKSHEET(S) WITH FILM(S).
6. PROFILER PLUS AMPLIFICATION WORKSHEET(S).
7. COFILER AMPLIFICATION WORKSHEET(S).
8. ELECTROPHEROGRAMS:
a) Profiler Plus Positive Controls
b) COFILER Positive Controls
c) Profiler Plus Negative Controls
d) COfiler Negative Controls
e) MBs (Manipulation Blanks)
f) Blood Standard (Person 1) - Profiler Plus
g) Blood Standard (Person 1) - COfiler
h) Blood Standard (Person 2) - Profiler Plus
I) Blood Standard (Person 2) - COfiler
j) Case stain 1 - Profiler Plus
k) Case stain 1 - COfiler
l) Case stain 2 - Profiler Plus
m) Case stain 2 - COfiler
9. SUMMARY SHEET(S) - PROFILER PLUS/COFILER
10. STATISTIC PRINTOUTS/SHEETS
11. CODIS TRANSFER(S)/SEARCHES
12. CASE-RELATED MATERIAL (Xerox of biologists notes; medical reports; etc.).
NON-NUMBERED PAGES:
1. ORIGINAL DNA REPORT
2. EVIDENCE RECEIPTS
NOTE: All electronically generated data must be archived on a CD. This includes sample
sheets, injection lists, GeneScan and GenoTyper data.
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Reviews
I. Technical case reviews are outlined in Appendix V and the Command Directives. The technical
review form will be used only as a guide for the technical reviewer. Therefore, this form is not kept
as part of the case file. The review sheet is used to assist with communications between the technical
reviewer and the analyst. Approval of the case file contents by the technical proofer is indicated by
his/her initials on the cover sheet.
II. Each analyst will conduct an administrative review of his/her cases. When an analyst signs a case
report, he/she is indicating that an administrative review has been conducted.
III. Supervisory reviews are outlined in Command Directives. The supervisory review form is used as a
guide for the reviewer. This form is a suggested guide and may be retained by administrator as
documentation of the review but is not part of the case file (see Appendix B).
IV. Court monitoring is covered under:
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DNA Supervisory File Review Procedure
Purpose: To ensure that proper note taking and case file documentation procedures are followed.
Procedure: The reviewer must note the following:
1. General considerations
_____ Are all items in the file marked with the case number and additional information (such as the
item number, analysts initials and date) where appropriate?
_____ Are all case note pages numbered?
_____ Are the total number of pages of notes included?
2. Chain of evidence
_____ Check the evidence receipt for accuracy and completeness.
Has the chain of evidence been appropriately documented on the evidence receipt?
Are all items of evidence tracked adequately?
_____ Are all supporting documents (e.g., locker receipts) in the file?
_____ Check the report for accuracy and completeness of chain.
_____ Cross check the report and the evidence receipt.
_____ Check the notes.
Were additional exhibits generated and tracked appropriately?
3. Technical Documentation
_____ Check the case file cover sheet for completeness.
Did the technical proofer initial and date the cover sheet?
_____ Check for case notes.
These must include a description of what evidence was received and any additional exhibits
generated.
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Safety
I. The safety program is found in the Command Safety Manual.
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PROCEDURES MANUAL

QUALITY ASSURANCE

Prepared by:

Approved by:

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APPENDIX VI
Table of Contents
V. Evidence Control
VI. Validations
X. Audits
XI. Reports
XII. Reviews
XIII. Safety
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Overall goals:
Objectives:
product;
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Competency Testing
Proficiency Testing
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college course work in the following areas prior to participating in DNA training:
2. Genetics
3. Biochemistry
evaluated.
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C. Experience
D. Certification
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cases.
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preparation.
interpretation.
their laboratory.
laboratory.
additional needs.
standards.
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Procedures Manual.
proficiency tested.
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PCR set up.
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to operation.
from the PCR room.
laboratory.
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Evidence Control
Blood Evidence
Evidence Packaging
Case Tracking
Clean Technique
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and location.
and location.
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V. Stain Storage
A. Blood Standards
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Validations
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1997.
1999;44:133-166.
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1997.
5,552,028.
5,552,028.
39:1254-1269.
TM
1996.
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I. Procedures.
II. Reagents.
the Reagent Log.
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D. Expiration Dates
2. ABAcard kits
3. Nylon membrane
7. Formamide
manual.
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appropriate.
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A. Blood Standards
possible.
B. Dry Blood
C. Semen
secretion.
E. Saliva
F. Urine
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run.

logbook.
2. Semen ID
3. Lugols Stain
test is used.
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test is used.
6. Urea Nitrogen
7. Creatinine Test
properly.
Manual.
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calculation.
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report.
laboratory report.
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2. Procedure:
the manufacturer.
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DATE LOG# NEGATIVE
10NG
POSITIVE
4NG
POSITIVE ANALYST
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Procedures Manual.
2. Procedure:
surrounding wells.
incubation period.
incubation period.
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manufacturer.
Anti-Serum Brand __________________________
Expiration Date __________________________
Date QCd __________________________
Analyst __________________________
ANTI____________________
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2. Procedure:

F. Photograph gel

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Cathodal Origin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Middle Origin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Include Photograph Along
With This Sheet
Analyst_________ Date_________
Lot# ____________
Date Received__________________
D a t e o f D i l u t i o n i f S a m e
Lot#_________________________
Notes:
QCd?________________
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1. Purpose:
2. Procedure:
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1 2 3 4 5 6
A
B
C
D
E
F
G
H
manufacturer?__________________________________
SAMPLE ID
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2. Procedure:
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CHARGED MEMBRANE
(For Slot Blot)
Date QCd________________________ QCd by______________________
Standards
(ng)
Calibrators
NOTES:
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2. Procedures:
of 20 :L ddi H
2
O.
manufacturer.
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Analyst__________________________ QC Date ___________________
Allelic Ladder ___________ 9947A#_________________

D3S1358
D16S539
TH01
TPOX
CSF1PO
Amelogenin
D7S820
NOTES:
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Analyst__________________________ QC Date ___________________
Allelic Ladder ___________ 9947A#_________________

D3S1358
vWA
FGA
Amelogenin
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
NOTES:
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2. Procedure:
manufacturer.
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Analyst__________________________ QC Date ___________________
Expiration Date______________________
well defined
75
100
139
150
160
200
300
340
350
400
450
490
500
NOTES:
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Annual System Verification with Standard Reference Material 2391
SRM 2391 from the National Institute of Standards and Technology.
Procedure:
B. Amplify components #3-12, 19-20, using ISP protocol for appropriate loci.
Assess Results: Compare the results of each test to the NIST results for each component. Any
discrepancies will be reanalyzed and resolved.
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A. Matrix
D. Sensitivity
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1. Purpose:
2. Procedure:
locus.
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ABI 310 _____________________________________ Date_________________________
Analyst ______________________________________
1
2
3
4
5
6
7
8
9
10
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A. Matrix:
B. Sensitivity:
Supervisor
Matrix Installation
Sensitivity
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Quality Assurance
I. Calibrations:
1. Procedure
2. Frequency
3. Results
4. Course of action
II. Maintenance
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IV. Frequency:
V. Results:
repairs.
VII. Maintenance
FB-App VI (DNA)
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standard.
III. Maintenance
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Oven Calibrations
I. Calibration
A. Procedure
B. Frequency
C. Results
D. Course of Action
II. Maintenance
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III. Maintenance
for repairs.
FB-App VI (DNA)
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pH Meter
desired pH).
III. Maintenance
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III. Maintenance
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Microscopes
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1. Calibrations:
serviced.
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III. Maintenance
FB-App VI (DNA)
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NIST Thermometer Calibration
B. Take reading of NIST thermometer. Thermometer should read 0 degrees Celsius.
A. If the thermometer reading is +/- 2 degrees from 0 degrees, the NIST thermometer will not be
used for any further calibrations.
IV. Maintenance
None
FB-App VI (DNA)
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B. Place the thermometer being tested at the same depth as the NIST thermometer and read the
temperature.
Celsius.)
If the thermometer is greater than +/- 2 degrees from the NIST thermometer, it will not be used for
any further calibrations.
IV. Maintenance
None
FB-App VI (DNA)
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I. Procedure
C).
use the water bath.
III. Maintenance
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Proficiency Testing
following:
Competency Tests
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Audits
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Reports/Case Files
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NUMBERED PAGES:
NON-NUMBERED PAGES:
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Reviews
documentation of the review but is not part of the case file (see Appendix B).
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generated.
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Safety
Accepted Date: January 30, 2002
FB-App V (DNA)
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Appendix V: Technical
Proofing and Technical
Review
PROCEDURES MANUAL

APPENDIX V: TECHNICAL PROOFING AND
TECHNICAL REVIEW
Prepared by:

Approved by:

FB-App V (DNA)
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Review
APPENDIX V
Technical Proofing and Technical Review
All aspects of an analyst's case files will be proofed by another qualified analyst. The criteria to be
used are set forth on the Technical Proofing Checklist. The minimum scope of this review will be to
determine whether the analytical results support the interpretations and conclusions stated in the report.
FB-App V (DNA)
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Review
ILLINOIS STATE POLICE Case #
Division of Forensic Services
Forensic Sciences Command Initials

Date
TECHNICAL PROOFING CHECKLIST: STR
Reviewed by Page 1 of 2
General:
___ All file items marked with case/item number, initials, date (if appropriate).
___ DNA procedure consistent with procedures manual.
___ Adequate agency contact.
___ Adequate conversation record.
___ Notes paginated/total listed.
___ Case file organized according to policy.
Chain:
___ Evidence receipt/custody history report complete.
___ Supporting documents (e.g., locker receipts) in file.
___ Additional exhibits generated tracked appropriately.
Case notes:
___ Case number, date, initials.
___ Description of exhibit packaging.
___ The nature of the seals.
___ Brief description of exhibits including size.
___ Portion of exhibit consumed in analysis.
___ Date and description of repackaging.
Yield gel worksheet:
___ Worksheet filled out.
___ Photo attached and labeled.
___ Interpretation of quantity/quality of DNA.
___ Re-extractions and re-dilutions have own worksheet.
Slot blot worksheet:
___ Photo attached and labeled.
___ Quantitation of DNA correct and documented.
___ Re-dilutions have own worksheet.
Sample amplification worksheet: Profiler Plus
___ (+) and (-) controls and manipulation blanks.
Sample amplification worksheet: Cofiler
Genotyper electropherograms:
___ Positive control verifying ladder present for each sample tray.
___ (+) and (-) amplification controls present for each amplification.
___ Samples present.
___ Labeled with case number, sample number and date of electrophoresis run.
___ Protocol alterations clearly documented.
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ILLINOIS STATE POLICE Case #
Division of Forensic Services
Forensic Sciences Command Initials
Date
TECHNICAL PROOFING CHECKLIST: STR
Page 2 of 2
Allele summary sheet: Profiler Plus
___ Sheet filled out listing all samples.
___ (+) and (-) amplification control and manipulation blank checked.
Allele summary sheet: Cofiler
___ Sheet filled out listing all samples.
___ (+) and (-) amplification control checked.
Frequency calculations:
___ Calculation sheet filled out/ printout present.
___ Correct values entered.
___ Statistics documented for all probative samples.
Codis:
___ Documentation of appropriate CODIS uploads.
Other:
___ Interpretations correct and documented.
___ Gel or photo labeled.
Report:
___ Report references all items received.
___ Loci documented.
___ All matches/exclusions reported in results.
___ Frequency of probative profiles.
___ Conclusion statement identifies potential donors of probative profiles.
___ Conclusions supported by notes.
___ References to indexing searches if open profile.
___ Signature.
___ Coversheet/stats/reviewer initials.
Accepted Date: September 20, 2001
FB-App IV (DNA)
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Appendix IV: Worksheets
PROCEDURES MANUAL
APPENDIX IV: WORKSHEETS
Note: These are representations of the actual
worksheets.
Prepared by:

Approved by:

FB-App IV (DNA)
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ILLINOIS STATE POLICE Case
DIVISION OF FORENSIC SERVICES
FORENSIC SCIENCES COMMAND Initials
DNA COVERSHEET Date
Status: Case type: Examination type:
Analyst ID* (The number below represents (Check below.)
different people present.)
Date Submitted For
Technical Review Probative Match STR 1___________
Technical Review By Probative Exclusion STR 2 ___________
Date
Open Profile Examination #:
(Total below.**)
Technical Leader Review By No suspect
(as required)
Date_________ Parentage Extraction

Case Status* Remains ID _____________ Quantitation
Command TAT*
Section TAT* Total Number of Pages in File ____________________
*Optional for CALMS laboratories.
** Extraction- Each exhibit and manipulation blank
examined will be counted as 1. If a differential
extraction is conducted, count each fraction worked.
Quantitation- Every space for all yield gels/ slot blots
utilized for samples and manipulation blanks will be
counted as 1. The quantitation standards will not be counted.
FB-App IV (DNA)
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YIELD GEL WORKSHEET Date
Cathodal Origin
Lane Sample Resolub.
Volume
(L)
DNA
(ng / 4 L)
DNA
(ng / 1 L)
1
DNA Dilution Series
125
2 63
3 31
4 15
5 8
6 4
7
8
9
10
11
12
13
14
Middle Origin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
FB-App IV (DNA)
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SLOTBLOT WORKSHEET Date
Key: Sample
L loaded | ng reading
Standards (ng) 1 2 3 4 5 6
A 10
Calibrator 1
5 - 2.5 ng _____________
|
_____________
|
_____________
|
______________
|
B 5
Calibrator 2
0.6 - 0.31 ng _____________
|
_____________
|
_____________
|
_____________
|
C 2.5 ______________
|
_____________
|
_____________
|
_____________
|
______________
|
D 1.25 ______________
|
_____________
|
_____________
|
_____________
|
______________
|
E 0.6 ______________
|
_____________
|
_____________
|
_____________
|
______________
|
F 0.3 ______________
|
_____________
|
_____________
|
_____________
|
______________
|
G 0.15 ______________
|
_____________
|
_____________
|
_____________
|
______________
|
H Blank ______________
|
_____________
|
_____________
|
_____________
|
______________
|
FB-App IV (DNA)
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AMPLIFICATION WORKSHEET Date
Each amplification set (different date and/or time of set up) requires its own worksheet. Mark column(s) for Kit(s)
used.
Sample PP CO Slot Blot
(ng/L)
Diln
for
Amp.
Amount
of
DNA
(L)
Amount
of
H
2
O
(L)
Estimated
Amount
of DNA
(ng)

FB-App IV (DNA)
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STR SUMMARY WORKSHEET Date
SAMPLE
Locus
D3S1358
vWA
FGA
Ameloge
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
D16S539
TH01
TPOX
CSF1PO
Reviewed Date: November 7, 2001
FB-APP III (DNA)
Page 1 of 2
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Appendix III: Non-Routine
Procedures
PROCEDURES MANUAL

APPENDIX III: NON-ROUTINE PROCEDURES
Prepared by:

Approved by:

Reviewed Date: November 7, 2001
FB-APP III (DNA)
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Appendix III: Non-Routine
Procedures
APPENDIX III
NON-ROUTINE PROCEDURES
Evidence Return:
Since the initial amount of evidence may be limited and the entire sample used in analysis, evidence
materials subjected to DNA analysis will be preserved in the laboratory prior to trial whenever
possible. (All evidence remaining following an analysis should be preserved as described in the
evidence preservation section of the QA manual.)
After all work is completed on a case, all samples, including extracted DNA, is to be returned to the
submitting agency, along with any specific storage instructions. Evidence return procedures are found in
the Command Directives Manual.
Confirmation of a DNA Indexing Match:
1. Suspect's Standard
A. An additional blood sample from the offender will be submitted as a standard for
analysis.
B. Whenever possible the analyst who originally performed the DNA analysis will perform
the analysis using the additional blood standard.
2. Analysis
All probes used to analyze the evidence sample will be used to analyze the new suspect
sample. Additional well-characterized, polymorphic probes may also be used at the
discretion of the analyst. If a match is declared, a statistical evaluation of the frequency
of the evidentiary type will be made using as many probes as feasible.
Accepted Date: May 22, 1997 Appendix II: Minimum
Next Review Date: February 1, 2002 Standards & Controls
FB-App II (DNA)
Page 1 of 1
Version 2
PROCEDURES MANUAL

APPENDIX II: MINIMUM STANDARDS &
CONTROLS
Note: Minimum Standards & Controls, Critical
Reagents & Equipment Calibrations Are Included In
Appendix VI of the DNA Procedures Manual.
FB-App I (DNA)
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Appendix I: Report Wording
PROCEDURES MANUAL

APPENDIX I: REPORT WORDING
Prepared by:

Approved by:

FB-App I (DNA)
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Appendix I: Report Wording
APPENDIX I
REPORT WORDING
INTRODUCTION
The following suggestions are guidelines which will be followed whenever practical. Modifications
to this report format may be necessary based on the evidence in the case and may be used at the
discretion of the analyst.
PROCEDURE
I. Evidence Description
The first section of the report will consist of two columns with the following headings:
A. Exhibit
The exhibit number used will reflect the information of the original report and the evidence
receipt.
B. Item Submitted/Description
This column will contain a brief description of the exhibits/subexhibits submitted (e.g., blood
standard, vaginal swabs, stain from panties, etc.).
C. Examples
Examples for this section of the report are given below:
EXHIBIT DESCRIPTION
1 Blood standard: Name
2 Tissue: Name (Any type of standard other than blood)
3 Vaginal swabs (Questioned samples)
4 (Type of stain): Exhibit Description
II. Results
Note: Report Wording for STRs is included in the Amplification of STRs procedure (FB-
IIIE-3).
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Procedure: Amplification of
STRs
PROCEDURES MANUAL
METHOD: PCR
PROCEDURE: Amplification of STRs
Prepared by:
Approved by:
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INTRODUCTION
Short tandem repeat (STR) genetic markers are polymorphic DNA loci that contain a repeated nucleotide
sequence. The STR repeat unit can be from two to seven nucleotides in length. The number of repeat units
at an STR locus differs from individual to individual, so alleles of many different lengths are possible. This
makes them useful for human identification purposes.
STR loci can be amplified using the polymerase chain reaction (PCR) process. The AmpFlSTR Profiler
Plus PCR Amplification Kit co-amplifies the tetranucleotide repeat regions of the following nine short
tandem repeat loci: D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and
D7S820. A segment of the X-Y homologous gene amelogenin is also amplified. The AmpFlSTR Cofiler
PCR Amplification Kit co-amplifies the tetranucleotide repeat regions of the following six short tandem
repeat loci: D3S1358, D16S539, TH01, TPOX, CSF1PO and D7S820. A segment of the X-Y
homologous gene amelogenin is also amplified.
The alleles within each locus as well as the loci themselves are separated by size using capillary
electrophoresis. The use of multicolor dye-labeled primers allows loci that have alleles with overlapping
size ranges to be distinguished from one another when activated by a laser during the course of the capillary
electrophoresis run.
Standard Laboratory Practices.
Warning: Potential Biohazard.
Warning: Hazardous Reagents - Formamide is a suspected teratogen.
Electrical Shock Hazard: The ABI Prism 310 capillary electrophoresis unit contains a high
voltage power supply. Handle with caution. Under no circumstances
should any safety system be bypassed. Arcing may result from
incomplete drying of instrument components.
Laser Hazard: The ABI Prism 310 contains a laser. Operate only with doors closed.
Service by Perkin-Elmer personnel only.
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Formamide
An irritant and suspected teratogen. Causes irritation to the eyes, skin and mucous membranes. Do not
inhale or ingest. Wear protective eyewear, gloves and laboratory coat. Dispose of properly.
310 Genetic Analyzer Buffer with EDTA (10X)
Toxic properties have not been fully investigated. Handle with caution. Avoid contact with skin, eyes and
clothing. In case of exposure, wash affected area with large amounts of water for at least 15 minutes.
Wear protective eyewear, gloves and laboratory coat.
310 Genetic Analyzer Performance Optimizing Polymer 4 (POP 4)
May cause irritation to the respiratory tract, skin and eyes. Avoid contact with skin, eyes and clothing.
Do not inhale or ingest. Wear protective eyewear, gloves and laboratory coat.
PREPARATIONS
Deionized Formamide
Purchased deionized formamide does not need to be deionized again using resin. However, the
conductivity of all formamide must be verified using a conductivity meter. If the conductivity of the
deionized formamide does not meet the manufacturers specification then return it to the manufacturer for
a new lot.
Aliquot into convenient volumes and freeze at -15 to -20C with protection against defrosting.
Frozen aliquots can be stored for a maximum of 6 months. If the aliquot is not frozen discard immediately.
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Internal Lane Standard
Into a 1.5 ml Eppendorf tube, add the following reagents in the calculated volumes:
1. GeneScan-500 [ROX] Size Standard 1 ul x number of samples + one
2. Deionized formamide 24 ul x number of samples + one
Briefly vortex and spin.
Aliquot 25 ul into each sample tube.
Solution must be made fresh before each use.
Allelic Ladder
Profiler Plus Ladder
Allelic Ladder Mix 1.5 ul x number of ladders per tray
Ladder is premixed, no necessary preparation.
Cofiler Ladder
Allelic Ladder Mix 1.5 ul x number of ladders per tray
Ladder is premixed, no necessary preparation.
1X Genetic Analyzer Buffer with EDTA
Genetic Analyzer Buffer w/ EDTA (10X) 2 ml
ddi water 18 ml
Mix thoroughly.
Solution must be made fresh before each use.
60% Ethanol
EtOH 600 ml
ddi water 400 ml
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INSTRUMENTATION
Standard Laboratory Instrumentation:
ABI Prism 310 Genetic Analyzer.
Critical Laboratory Instrumentation:
Perkin-Elmer thermal cycler.
Standardization: see Quality Assurance Manual and PE thermal cycler manual for complete
instructions of the following tests:
1) Temperature Uniformity Test - Semi-annual
2) Temperature Verification Test - Monthly
SOFTWARE
ABI Prism 310 Genetic Analyzer Firmware, version 1.0.2 or higher.
ABI Prism 310 Collection Software, version 1.0.2 or higher.
ABI Prism 310 Module GS STR POP4 (1 mL)F.
GeneScan Analysis Software, version 2.1 or higher.
GenoTyper Analysis Software, version 2.0 or higher.
DatabankSTR Software
ISP Population Software Program
CODIS
Critical Reagents
Perform quality control on the following reagents (see Quality Assurance portion of Manual for
instructions):
1) GeneScan 500-ROX Internal Lane Sizing Standard
This is a series of 16 DNA fragments ranging in size from 35 to 500 base pairs, each
labeled with the fluorescent dye ROX. It is designed as an internal standard against which
each sample allele can be accurately sized after separation by capillary electrophoresis.
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2) AmpFlSTR Profile Plus PCR Amplification Kit
Contains all the amplification components needed for the co-amplification of 9 STR loci
and amelogenin. The kit also includes the allelic ladder for each locus and a positive
amplification control.
3) AmpFlSTR Cofiler PCR Amplification Kit
Contains all the amplification components needed for the co-amplification of 6 STR loci
and amelogenin. The kit also includes the allelic ladder for each locus and a positive
amplification control.
4) Formamide
Used to denature the amplified DNA for capillary electrophoresis.
Profiler Plus and Cofiler Controls:
Positive Amplification Control: 20 ul of Control DNA 9947A (supplied in kit)
Purpose: To ensure that amplification has occurred successfully. The following types must be obtained:
D3S1358 14, 15
vWA 17, 18
FGA 23, 24
AMELOGENIN X, X
D8S1179 13, 13
D21S11 30, 30
D18S51 15, 19
D5S818 11, 11
D13S317 11, 11
D7S820 10, 11
D16S539 11, 12
TH01 8, 9.3
TPOX 8, 8
CSF1PO 10, 12
Negative Amplification Control: 20 ul ddi water
Purpose: To ensure that extraneous DNA has not been introduced or that contamination has not
occurred due to the reagents used in the amplification.
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Manipulation Blank: sterile cotton swab processed through slot blot and amplified using the
AmpFlSTR Profiler Plus kit. Twenty percent of the total volume of the
manipulation blank must be amplified.
Purpose: To ensure that extraneous DNA has not been introduced or that contamination has not
occurred due to the reagents used during the extraction of the DNA or manipulation of the
sample.
PROCEDURE
All suspect standards must be typed in both AmpFlSTR Profile Plus and Cofiler for entry into the
suspect database.
All samples must be amplified and typed in Profiler Plus. Only probative samples and samples to be
entered into CODIS will be amplified and type in Cofiler. When an F2 fraction of a differentially
extracted sample is considered probative, both the F1 and F2 fraction of that sample must be
amplified and typed in Cofiler.
ROUTINE MAINTENANCE OF THE 310 GENETIC ANALYZER
Clean the instrument and supply fresh buffer and polymer approximately every 3 days.
Replace the capillary after approximately 100-150 electrophoresis runs.
The polymer must be allowed to reach room temperature before loading into syringe.
Visually inspect the polymer to ensure that no crystals are present.
Inspect block components and make sure they are dry before starting a run.
All maintenance to the 310 Genetic Analyzer must be recorded in a log book.
REPLACING CAPILLARY AND CLEANING INSTRUMENT
1. Carefully remove glass syringe from pump block and clean with warm tap water, followed
by ddi water.
2. Loosen capillary ferrule and remove end of capillary from pump block. (Temporarily place
end of capillary into a vial containing water to prevent it from drying out.)
3. Remove opposite end of capillary from thumbscrew by electrode, untape from heating
element and remove entire capillary from unit.
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4. Remove pump block from unit, detach buffer reservoir and fittings and flush thoroughly
with warm tap water, followed by ddi water, Dry with compressed air.
5. Replace buffer in both buffer reservoirs (pump block and autosampler tray) and replace ddi
water in both the wash vial and waster reservoir (autosampler tray).
6. Loosely re-attach fittings to pump block and re-attach pump block to unit.
7. Clean window of new capillary with 60% ethanol, then briefly wipe down with ddi water.
8. Attach end of capillary to pump block by tightening ferrule.
9. Align the opposite end of the capillary with tip of electrode, align window with laser and tape
capillary to heating element.
10. Prime syringe with approximately 20 ul of polymer. Expel polymer.
11. Fill syringe with new polymer and re-attach syringe to pump block. Generally, 100-300
ul of polymer is needed to prime the pump block and approximately 5 ul of polymer is utilized
for each sample injection. Calculate volume accordingly. Syringe should not be loaded with
more than 800 ul of polymer.
12. Prime the pump block with polymer.
13. Calibrate autosampler by aligning capillary with autosampler tray.
CLEANING INSTRUMENT ONLY
To clean the pump block without changing the capillary, omit steps 3, 7, 9, and 13.
AMPLIFICATION SET UP
Twenty percent of the manipulation blank must be amplified in at least Profiler Plus. If a DNA profile
is detected, the case must be brought to the attention of an Assistant Technical Leader and the
Statewide Technical Leader.
1. Dilute an appropriate quantity of sample DNA (0.6 - 2.5 ng) to 20 ul with autoclaved ddi
water. This quantity is dependent upon the quality of the DNA and the sensitivity of the 310
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instrument. Samples that contain less than 600 pg will not be amplified unless reviewed and
approved by an Assistant Technical Leader or the Statewide Technical leader in conjunction
with the Laboratory Director. Approval must be documented by the analyst on the
amplification sheet. Approval by an Assistant Technical Leader or the Statewide Technical
Leader and Laboratory Director for an exception will be documented in the case file. Samples
that do not appear on the Quanti-Blot will not be amplified.
Positive control: 20 ul of control DNA supplied with kit.
Negative control: 20 ul ddi water.
2. Label appropriate number of 0.5 ml reaction tubes (including positive and negative controls).
3. Prepare Amplification Master Mix*
4. Add 30 ul of Amplification Master Mix to labeled reaction tubes.
5. Gently add 1 or 2 drops of mineral oil to each reaction tube.
6. Add the 20 ul of DNA samples beneath the oil in each tube.
7. Place tubes in thermal cycler, select file for STR amplification parameters** and start
amplification.
After amplification, store tubes in either:
a) an amplified DNA dedicated refrigerator for up to 2 weeks.
b) an amplified DNA dedicated freezer indefinitely.
Tubes must be stored in a sealed container to protect them from light.
When shutting off thermal cycler, leave lid open to prevent condensation of moisture within wells.
* Amplification Master Mix Preparation:
Into a 1.5 ml Eppendorf tube, add the following reagents in the calculated volumes:
1. PCR Reaction Mix (in kit) . . . 21 ul x number of samples
2. Primer Set (in kit) . . . . . . . . 11 ul x number of samples
3. Taq (gold) Polymerase . . . . . . 1.0 ul x number of samples
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** STR Amplification Parameters:
1 cycle at 95C for 11 minutes.
28 cycles; each consisting of 1 minute at 94C, 1 minute at 59C and 1 minute at 72C.
1 cycle at 60C for 45 minutes.
Soak at 10
C PREPARATION OF AMPLIFIED DNA SAMPLES FOR 310 ANALYSIS
1. Preheat the block on the 310 to 60C.
2. Prepare fresh solution of internal lane standard by mixing appropriate volumes (see
preceding preparations section) of the 500 ROX size standard with deionized formamide.
3. Prepare fresh solution of allelic ladder (see preceding preparations section).
4. Label appropriate number of 0.5 ml sample tubes (including a system blank, a positive and
negative control and a minimum of 2 allelic ladders).
5. Aliquot 25 ul of deionized formamide containing internal lane standard into each sample
tube.
6. To each tube add 1.5 ul of one of the following:
a) amplified DNA
b) allelic ladder
c) positive control
d) negative control
7. Seal each tube with rubber septum, vortex lightly and spin down briefly.
8. Denature samples for 3-5 minutes at 95C.
9. Snap cool denatured samples for at least 5-10 minutes in an ice block or equivalent.
10. Place tubes in sample tray.
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a) first well (Position A1) contains system blank
b) second well (Position A3) and last well contain allelic ladders
c) wells between ladders contain the positive control, negative control, samples and
any additional ladders
11. Load sample tray into the 310.
12. Create sample sheet and injection list (as described in next section).
13. Start electrophoresis run.
CREATING A SAMPLE SHEET AND INJECTION LIST
1. Create a Sample Sheet.
2. Save the Sample Sheet File.
3. Open New File to make Injection List.
4. Import Sample Sheet onto Injection List.
5. Verify parameters:
a) Module: GS STR POP4 1ml F
b) Injection time: 5 seconds or 10 seconds at the analysts discretion.
c) Run temperature: 60C.
d) Run time: instrument dependent. See Quality Assurance Manual for analysis of
internal land standard.
6. Select appropriate Matrix.
7. Auto analyze: may be selected, but the run must be reanalyzed afterwards. This option
can be used to review results while the 310 is operating.
8. Save Injection List File.
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GENESCAN ANALYSIS
1. Retrieve Project File from 310 Results folder.
2. Open Project File.
3. Create New File for Size Standard. This must be done for each run.
4. Select internal lane standard from an allelic ladder and apply appropriate values to
peaks.
Profiler Plus: 75-400
COfiler: 75-350
5. Save.
6. Title internal size standard.
7. Select appropriate Analysis Parameters.
a) Analysis Range - select, This Range, where Start and Stop values will be dependent
on position of primer - dimer peaks.
b) Data Processing -
Baseline - checked
Multicomponent - checked
Smooth options - Light
c) Peak Detection -
Peak Amplitude Threshold - 150
Min. Peak Half width - 3
d) Size Call Range - Select, All Sizes or This Range, where Min. and Max. values will
cover at least 75-400bp.
e) Size Call Method - Local Southern Method.
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f) Split Peak Correction - None.
8. Select all 4 dyes.
9. Analyze.
10. Verify Internal Lane Standard for all samples.
11. Save.
12. Close Project File.
GENOTYPER ANALYSIS
1. Open GenoTyper Program (Profiler Plus or COfiler).
2. Import GeneScan Files.
3. Analyze project with Kazam Macro (Profiler Plus or COfiler).
4. Select sample for viewing, indication all 4 dyes.
5. Display Plot Window to generate electropherograms with allele calls, base pair sizes and
peakheights for all loci (Views). Include base pair sized for ROX electropherograms. Labels
from peaks must not be removed/changed under any circumstances.
6. Print Electropherograms displaying the internal lane standard peaks 75-400 bp for Profiler
Plus loci and 75-350 bp for COfiler loci.
7. Electronic Data will be retained and will be archived on a CD.
INTERPRETATION
These interpretation rules are to be followed as written. However, it is recognized that these rules cannot
cover all situations. On a case by case basis the analysts permitted to suspend the use of these rules
provided:
1. The analyst documents objective reasons for such suspension in his/her case notes, and:
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2. The Statewide Technical Leader has reviewed the case and agrees with the interpretation.
After such review the Statewide Technical Leader will sign off on the analysts notes to indicate
concurrence. If no agreement can be reached then the Quality Assurance Program Administrator will be
notified. The Statewide Technical Leader and Assistant Technical Leaders shall maintain a logbook of all
such requests to include a description of the requests, the justification(s) for suspending the interpretation
guidelines, and the final decision.
When an analyst confer with the Statewide Technical Leader or an Assistant Technical Leader about
questions, issues, or problems in casework, or to request a review of casework, or to discuss the results
of a review of casework, the conversation, to include the issues discussed and their resolution, will be
documented in the case file by the analyst.
General considerations:
1. The amount and condition of stain material available for DNA analysis is provided by the biologist.
2. The total yield, state of degradation of DNA, and presence of potential inhibitors needs to be taken
into consideration.
3. Interpretations will be based on the entire DNA profile for a sample.
4. GeneScan data may aid in interpretation. If GeneScan data is used for interpretation it must be
included in the case file.
5. It may be necessary to interpret data across more than one electropherogram. This will be
considered on a case by case situation and only when there is overlap of results at a minimum of one
locus. This situation must be reviewed by the Statewide Technical Leader.
Assessment of successful amplification and electrophoresis:
1. The GeneScan 500-ROX internal sizing standard will be examined for each sample and control to
ensure that all peaks in the analytical range (75-400 bp for Profiler Plus, 75-350 for COfiler) being
examined were sized correctly and that there are no extraneous peaks above 150 relative fluorescent
units (RFU). This will confirm that the base pair sizing of alleles was computed accurately.
2. The allelic ladder used for genotyping will be examined to determine that all allele designations have
been assigned correctly by the GenoTyper program. If the lot of ladder demonstrates low rfus, a 10
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second injection of the ladder can be conducted; however, the laboratorys Assistant Technical
Leader or Assistant DNA Coordinator must be notified whenever there are problems with the
Profiler Plus or COfiler ladders. The laboratorys Assistant Technical Leader or Assistant DNA
Coordinator is required to notify the Statewide Technical Leader and all other Assistant Technical
Leaders and Assistant DNA Coordinators with the lot number of the ladder that is exhibiting low
RFUs.
3. Each sample will be examined for fluorescent artifacts that may be present at a given base pair size
in two or more dyes. Such samples will require a second injection.
4. All peaks to be called should fall within the range of 150 to 4500 RFU. Interpretation of peaks
above 4500 RFU will be conducted with caution. Electropherograms demonstrating multiple such
loci will require the approval of the laboratorys Assistant Technical Leader or the Statewide
Technical Leader.
5. Each sample will be examined for fluorescent pull up. Fluorescent pull up is defined as any small
peaks present in one or more dyes which echo the presence of a relatively large peak at that same
base pair size in another one of the dyes. The base pair size of these peaks must be withing +/- 0.5
base pairs. Peaks determined to be fluorescent pull up must be noted and do not require a second
injection.
6. Negative amplification controls must not exhibit reproducible peaks greater than 150 RFU in any of
the dyes within the size ranges covered by any of the loci for each amplication.
7. For each amplification and electrophoresis run, the positive amplification control must type correctly
at all loci (see Profiler Plus and COfiler controls).
8. The loci D3S1358 and D7S820 shared by both Profiler Plus and COfiler Amplification Kits may be
used as a quality assurance measure.
9. Each analyst is required to search all open profiles against the Forensic Biology DNA
Analyst/Technician DNA Database. If a potential hit is made, the analyst will notify the Statewide
Technical Leader.
Allele definition:
1. For any given locus, minor peaks that are above 150 RFU and are located at a position one repeat
unit smaller than a major peak must be evaluated as to whether it represents stutter or a real allele.
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Such peaks may be considered as stutter in Profiler Plus if their peak height percentages to the larger
peaks are equal to or less than 12% at the D3S1179, D5S818, D13S217 and D7S820 loci; equal
to or less than 15% for the D3S1358, D21S11, vWA and FGA loci and equal to or less than 18%
at the D18S51 locus.
Such peaks may be considered at stutter COfiler if their peak height percentages to the larger peaks
are equal to or less than 10% at the CSF1PO locus, equal to or less than 12% at the D7S820 locus,
equal to or less than 15% for the D3S1358 and D16S539 loci, and equal to or less than 7% at the
TH01 and TPOX loci.
2. Peaks located at a position one base pair smaller than a major peak may represent incomplete A
nucleotide addition and will not be interpreted. If more DNA is available, the sample must be re-
amplified.
3. For any given locus, minor peaks above 150 RFU that are not attributed to stutter or fluorescent pull
up will be interpreted as a real allele.
4. Off ladder variant alleles will only need to be verified by reproducibility; either by reamplifying and
rerunning the sample or by comparing the sample to the corresponding standard within a given case.
Reproducible off ladder alleles greater than or less than the largest or smallest alleles in the allelic
ladder will be designated as > largest allele or < smallest allele for each given locus allelic ladder
range. Reproducible variants within the ladder will be evaluated against the allelic ladder and an
allelic call made. These calls will be made in the summary sheet, the table attached to the report, for
uploading into CODIS and for statistics. The minimum allele frequency specific for the locus will be
used as the minimum allelic frequency if no other frequency has been assigned to the variant.
5. Caution must be exercised when alleles are present at or below 214 RFU for the D3S1358, vWA,
D8S1179, D21S11, D5S818, D13S317, D7S820, D16S539, TH01, TPOX, and CSF1PO loci,
224 RFU for the D18S51 locus and 234 RFU for the FGA locus. In such cases, the sister allele can
be as low as the heterozygote model percentage for that locus and would fall below the threshold for
calling an allele. Thus, the sister allele would go undetected.
Single profile:
1. Single source profiels generally exhibit no more than two (2) alleles at a given locus. Deviations of
such profiles must be verified by reproducibility.
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2. The heterozygote model will not be applied when interpreting single source profiles.
3. If a single probative profile is identified in the F2 then the mixture which may be present in the F1
need not be differentiated so long as the alleles in the F1 are consistent with a mixture of the victim
and the probative profile.
Mixed profile:
1. Three alleles at any locus is indicative of a mixture.
2. The use of the heterozygote model will be used to differentiate major from minor genotypes. A 70%
heterozygote model will be used for interpretation of D3S1358, vWA, D8S1179, D21S11,
D5S818, D13S317, D7S820, D16S529, TH01, TPOX, and CSF1PO loci, a 67% heterozygote
model for the D18S51 locus and a 64% heterozygote model for the FGA locus.
3. If the appilcation of the heterozygote model or the assumption of the victims profile (such as in a
vaginal swab) does not differentiate contributors of a genotype (i.e. approximately equal
contributors), then all possible genotypes that do not violate the different levels of input DNA will be
used.
4. The heterozygote model will be applied to any locus in a DNA profile with caution which as a whole
exhibits any of the following anomalies and must be reviewed by an Assistant Technical Leader or
the Statewide Technical Leader:
A. Degradation: Diminished peak heights or allele dropout with increasing size of loci, generally
indicated by a two fold difference in peak heights between adjacent loci within a dye.
B. Inhibition: Loci specific peak height diminishment and/or allele (locus) dropout.
C. Preferential amplification: Uneven amplification of alleles within a locus attributable to large
differences in base pair size or copy number.
5. Mixtures involving two (2) contributors with no shared alleles.
A. The heterozygote model will be applied. Heterozygote pairs at a four allele locus may fall out
of the model at 300 RFU or less.
B. Minor components with alleles in a stutter position will be interpreted as follows:
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1. One of the minor alleles of a heterozygote pair falls in the stutter position and both are
called by GenoTyper but they fall out of the heterozygote model. Make a note that the
pair fall out of the heterozygote model because the peak height or one minor allele is
being raised by the stutter from the main allele.
2. One minor allele plus one peak in the stutter position that falls within the range of stutter.
Therefore, the peak is not being typed in GenoTyper. Review the GeneScan data and
obtain the peak height for the peak that is located in the stutter position. If the peak in
the stutter position and the minor allele falls out of the heterozygote model and the stutter
peak is less than the minor peak that was typed, the minor peak will be called. If,
however, the peak in the stutter position is equal to or greater than the minor allele that
was typed, the minor component will be considered inconclusive at that locus.
6. Mixtures involving two (2) contributors with shared alleles: three allele loci where the major genotype
is a heterozygote.
A. When both of the major alleles are 300 RFU or higher.
1. Determine if the major peaks are within the heterozygote model or the larger allele is in
a stutter position.
a. To determine the potential major genotype(s):
(1.) Examine the entire electropherogram and evaluate the different levels of input
DNA.
(2.) The major genotype will be reported as the observed heterozygote pair.
b. To determine the potential minor genotype(s):
(1.) If the sole minor allele is in a non-stutter position:
(a.) The heterozygote model will be applied if the peak height is 300 RFU or
above.
(b.) If the peak height is between 214 RFU (224 RFU for D18S51 or 34
RFU for FGA) and 299 RFU, the heterozygote model will nto be applied
to the minor profile and all possible combinations involving the minor
allele will be reported.
(c.) If the peak height is below 214 RFU (224 RFU for D18S51 or 234
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RFU for FGA), the minor allele will be reported and its sister allele
designated as inconclusive.
(2.) If the sole minor allele is in a stutter position:
(a.) If a minor component of the mixture is detected in a larger base pair
fragment, and that minor component is either a heterozygote pair or a
homozygote of 300 RFU or greater, then the minor genotype(s) can be
called without using the calculation for max stutter and the heterozygote
model will not be applied to the minor profile. All possible combinations
involving the minor allele will be reported.
(b.) If a minor component is not interpreted in a larger base pair fragment,
then maximum stutter will be subtracted from the minor allele and the
remaining peak height evaluated.
(i.) If the remaining peak height is 214 RFU or greater (224 RFU for
D18S51 and 234 RFU for FGA), then the heterozygote model will
not be applied and all possible combinations involving the minor
allele will be reported.
(ii.) If the remaining peak height is below 214 RFU (224 Rfu for
D18S51 and 234 RFU for FGA), the minor allele will be reported
and its sister allele designated as inconclusive.
(iii.) The heterozygote model will be applied if the peak height is 300
RFU or higher.
2. When the major alleles fall out of the heterozygote model and the larger peak of the
major heterozygote pair is not in the stutter position.
(1.) Examine the entire electropheragram and evaluate the different levels of input
DNA.
(2.) The major genotype will be reported as the observed heterozygote pair.
b. The minor genotype will be a combination of the minor peak with the taller of the
major heterozygote peaks.
B. When one or both alleles of the major heterozygote genotype are below 300 RFU.
1. The major genotype will be reported as all possible genotypes that do not violate the
different levels of input DNA.
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2. To determine the potential minor genotype(s):
a. If the sole minor is in a non stutter position:
(1.) If the peak height is greater than 214 RFU (224 RFU for D18S51 or 234 for
FGA) and 299 RFU, the heterozygote model will not be applied to the minor
profile and all possible combinations involving the minor allele will be reported.
(2.) If the peak height is below 214 RFU (224 RFU for D18S51 or 234 for
FGA), the minor allele will be reported and its sister allele designated as
inconclusive.
b. If the sole minor is in a stutter position:
(1.) Maximum stutter will be subtracted from the minor allele and the remaining
peak height evaluated:
(a.) If the remaining peak height is 214 RFU or greater (224 RFU for
D18S51 and 234 RFU for FGA), then the heterozygote model will not
be applied and all possible combinations involving the minor allele will be
reported.
(b.) If the peak height is below 214 RFU (224 RFU for D18S51 or 234
RFU for FGA), the minor allele will be reported and its sister allele
designated as inconclusive.
7. Mixtures involving two (2) contributors: three allele loci where major genotype is a homozygote. The
minor genotype will be reported as a heterozygote of the remaining two (2) alleles.
8. Mixtures involving two (2) contributors: two allele loci where the major genotype is a heterozygote.
A. When the major allele is 300 RFU or higher.
1. Determine if the major peaks are within the heterozygote model or if the larger allele of
the major heterozygote pair is in a stutter position.
DNA.
(2.) The major genotype will be reported as the heterozygote pair.
b. To determine the potential minor genotype(s):
(1.) If an interpretable minor genotype is detected in a larger base pair fragment,
the minor will be interpreted as all possible combinations of the two major
alleles.
(2.) If there is not an interpretable minor in a larger base pair fragment of the same
dye, the minor will be interpreted as not detected.
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2. Determine if the major peaks are not within the heterozygote model and the larger allele
is not in the stutter position.
DNA.
(2.) The major genotype will be reported as the heterozygote pair.
b. To determine the minor genotype(s):
(1.) If an interpretable minor genotype is detected in a larger base pair fragment
the minor will be interpreted as the homozygote genotype of the tallest allele
from the major heterozygote peaks.
(2.) If there is not an interpretable minor the minor will be interpreted as the taller
allele from the major heterozygote peaks and its sister will be designated as
inconclusive.
B. One or both alleles in the major heterozygote are <300.
1. The major will be interpreted as all possible combinations of the two observed alleles.
2. The minor will be interpreted as inconclusive.
9. Mixtures involving two (2) contributors: two allele loci where the major genotype is a homozygote.
A. To determine the potential minor genotype(s):
1. If a minor genotype is interpreted in a larger base pair fragment the minor genotype will
be reported as all possible combinations involving the minor allele that do not violate the
different levels of input DNA.
2. If there is no minor interpreted
a. If the minor allele is greater than 214 RFU (224 RFU at D18S51 or 235 RFU at
FGA).
(1.) Minor allele is in a stutter position. Maximum stutter will be subtracted from
the minor allele and the remaining peak height evaluated.
(a.) If the remaining peak height is 214 RFU or greater (224 RFU for
D18S51 or 234 RFU for FGA), the minor genotype(s) will be reported
as all possible combinations involving the minor allele that do not violate
the different levels of input DNA.
(b.) If the remaining peak height is below 214 RFU (224 RFU for D18S51
or 234 RFU for FGA), the minor allele will be reported and its sister
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allele designated as inconclusive.
(2.) Minor allele is in a non-stutter position.
The minor genotype(s) will be reported as all possible combinations
involving the minor allele that do not violate the different levels of input
DNA.
b. If the minor allele is less than 214 RFU (224 RFU for D18S51 or 234 RFU for
FGA), the minor allele will be reported and its sister allele designated as
inconclusive.
10. Mixtures involving two (2) contributors: one allele loci.
A. If a minor genotype is interpreted in a larger base pair fragment, the minor will be interpreted
as the homozygote genotype of the allele from the major.
B. If there is no interpreted minor in a larger base pair fragment, the minor will be interpreted as
not detected.
11. Mixtures involving greater than two (2) contributors.
A. Evaluate 5 second injection to determine if there is a possible three person mixture.
B. Do a 10 second injection if RFUs for the STR loci on the 5 second injection are low enough
that they will not result in alleles >4500 RFUs in the 10 second injection.
C. Determine if alleles in stutter position are being filtered or if they are below threshold by using
the GeneScan data.
D. Review the GeneScan data of the 10 second injection (or 5 second if only a 5 second injection
was done) to determine if there are peaks that could represent alleles below threshold.
Individual loci must be enlarged in the GeneScan software to better visualize these peaks. This
data must be included in the case file.
E. Alleles in the stutter position that are above threshold will be called and used for interpretation
and statistics.
F. Only loci in which all alleles present are above the threshold will be utilized for interpretation
and statistics.
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G. All GeneScan data for three person mixture samples must be included in the case file.
H. All mixtures of three or more people must be reviewed by the Statewide Technical Leader.
12. Any electropherogram exhibiting degradation, inhibition and/or preferential amplification will be
reviewed by the Statewide Technical Leader.
13. If a DNA profile exhibits an allele(s) whose peak height fall below 300 RFU, a 10 second injection
may be used to help clarify the profile. The additional minor alleles observed in the 10 second
electropherogram must be included in the Summary of the DNA Analytical Results Table. The
resultant 10 second electropherogram, however, must be mathematically interpreted using the RFUs
observed in the initial 5 second electropherogram. In addition, the manipulation blank(s) must be
injected for 10 seconds.
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Applying population frequency data to STR results
1. The allele frequency data for all STR loci for the Black, White and Hispanic populations are listed
in the next section. The Black, White and Hispanic populations were compiled by the FBI
Laboratory. The minimum allele frequency at a given locus for each population is calculated as 5/2N,
where N = the number of individuals in the database. The FBI Hispanic database is separated into
Southeastern and Southwestern Hispanics. The frequency of the probative samples will be calculated
for both databases and the most common overall frequency will be reported as the Hispanic profile
frequency.
2. Calculate frequency of probative samples.
Heterozygote: use 2pq.
Homozygote: use p
2
+ p(1-p)2; where 2 = 0.01.
3 Banded Patterns: use 2pq, where p and q are the two most common allele frequencies.
3. The combined frequency is calculated by multiplying together the individual frequencies determined
from independent loci. The frequency will be truncated to 2 significant digits, (no profile frequencies
will be rounded). Example: 1,490,000,000 = 1.4 billion.
4. The major and minor contributors of a mixture will be interpreted such that the genotype (profile as
a whole) will not violate the stutter and peak height ration (heterozygote) models. This will limit the
number of possible genotypes contributing to a mixture for which statistics need to be calculated.
5. If the probative contributor cannot be differentiated within a profile, then the frequency for each locus
will be the additive frequencies of all conceivable genotypes for that locus after application of the
70% heterozygote model.
6. If the probative contributor can be differentiated within a profile, then the frequency for those loci that
can be differentiated will be calculated separately for the major and minor contributors. For the
remaining loci where major and minor contributors cannot be differentiated the frequency for those
loci will be the additive frequencies of all possible genotypes for that locus after the application of the
heterozygote model.
7. Reproducible off ladder variants of a genotype will be given the minimum allele frequency of specific
for that locus.
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REPORT WORDING
The following report wording guidelines are designed to establish consistency in reporting results and to
accurately reflect the scientific weight given to the various situations encountered when interpreting and
calculating statistics. It is hoped that these guidelines will assist agencies in understanding the reports.
Modifications may be necessary and will be monitored by the Assistant Technical Leader and the
Statewide Technical Leader prior to issuing the report.
All cases in which single source stains that are being reported must be technically proofed prior to issuing
the report. All cases in which a mixture is reported must be technically proofed and further reviewed by
procedures previously described.
The first paragraph in the Results section will state which Exhibits were profiled and at which loci.
DNA from Exhibit(s) XXX was amplified using the Polymerase Chain Reaction (PCR) and profiled
at the following loci: D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317,
D7S820, D16S539, TH01, TPOX, CSF1PO and Amelogenin.
The following situations are those most commonly reported in forensic casework.
SITUATION 1. A single source stain when interpretable results are obtained for all loci:
Results
A human DNA profile was identified in Exhibit(s) XXX which matches the DNA profile of XXX and
does not match the DNA profile of XXX. This profile would be expected to occur in approximately
1 in XXX Black, 1 in XXX White or 1 in XXX Hispanic unrelated individuals.
For differential extractions the following statement should be used to identify a profile
detected in a non sperm fraction.
An additional human DNA profile was identified in Exhibit(s) XXX which matches the DNA profile
of XXX.
Conclusions
The (body fluid/DNA profile) identified/indicated in Exhibit(s) XXX is consistent with having
originated from XXX.
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The (body fluid/DNA profile) identified/indicated in Exhibit(s) XXX could not have originated from
XXX.
SITUATION 2. A single source stain when interpretable results are not obtained for all loci:
Results
A human DNA profile (use type instead of profile for a one locus match) was identified in Exhibit(s)
XXX at the D
1
, D
2
...and D
x
loci which matches the DNA profile of XXX and does not match the
DNA profile of XXX. This profile would be expected to occur in approximately 1 in XXX Black,
1 in XXX White or 1 in XXX Hispanic unrelated individuals.
Conclusions
The DNA profile identified in Exhibit(s) XXX is consistent with having originated from XXX.
The (body fluid/DNA profile) identified/indicated in Exhibit(s) XXX could not have originated from
XXX.
SITUATION 3. Mixtures:
a. Heterozygote Model
If the application of the heterozygote model does differentiate contributors of a genotype, then
separate statements will be reported.
Results
A mixture of human DNA profiles was identified in Exhibit(s) XXX that was interpreted as a mixture
of two people.
A mixture of human DNA profiles was identified in Exhibit(s) XXX that was interpreted as a mixture
of a least three people.
MAJOR (genotype differentiated at all loci)
One human DNA profile was identified in Exhibit(s) XXX which matches the DNA profile of XXX.
This profile would be expected to occur in approximately 1 in XXX Black, 1 in XXX White or 1 in
XXX Hispanic unrelated individuals.
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MINOR (genotype differentiated at all loci or complete profile of victim in F1)
A second human DNA profile was identified in Exhibit(s) XXX which matches the DNA profile of
XXX. This profile would be expected to occur in approximately 1 in XXX Black, 1 in XXX White
or 1 in XXX Hispanic unrelated individuals.
MINOR (no differentiation to a single genotype at all loci)
A second human DNA profile was identified in Exhibit(s) XXX and XXX cannot be excluded from
having contributed to this profile. This profile would be expected to occur in approximately 1 in
XXX Black, 1 in XXX White or 1 in XXX Hispanic unrelated individuals.
XXX can be excluded as having contributed to the mixed DNA profile identified in Exhibit(s) XXX.
If interpretable results are not obtained at all loci, then those loci for which results were
obtained must be listed.
Conclusions
The DNA profile identified in Exhibit(s) XXX could not have originated from XXX.
The mixture of DNA profiles identified in Exhibit(s) XXX is consistent with having originated from
XXX and XXX.
XXX could not have contributed to the mixed profile identified in Exhibit(s) XXX.
b. Assumption
If the application of the heterozygote model can only differentiate a probative genotype when a
non probative type is assumed, then a combined match statement will be reported to reflect the
assumption.
Common examples that one of the profiles present in the mixture is that of a know source,
-Known (victim/elimination) profile in swab of body orifice
-Known (victim/elimination) profile in underwear stain if warranted.
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-Consider all other items of clothing with caution when they belong to the victim or the
suspect.
Results
A mixture of human DNA profiles was identified in Exhibit(s) XXX which matches the combine
profiles of XXX and XXX. Assuming this profile is a mixture of XX (insert i.e. victims name) and
one other individual, approximately 1 in XXX Black, 1 in XXX White or 1 in XXX Hispanic
unrelated individuals cannot be excluded as the other contributor.
Conclusions
The DNA profile identified in Exhibit(s) XXX could not have originated from XXX.
The mixture of DNA profiles identified in Exhibit(s) XXX is consistent with having originated form
XXX and XXX.
c. No differentiation based on heterozygote model and assumption:
If the application of the heterozygote model and use of the assumption does not differentiate
contributors of a genotype, then a combined match statement will be reported (i.e. approximately
equal contributors).
Results
A mixture of human DNA profiles was identified in Exhibit(s) XXX which matches the combined
DNA profiles of XXX and XXX. approximately 1 in XXX Black, 1 in XXX White or 1 in XXX
Hispanic unrelated individuals cannot be excluded as having contributed to this mixed DNA profile.
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Conclusions
The mixture of DNA profiles identified in Exhibit(s) XXX is consistent with having originated from
XXX and XXX.
REPORT WORDING STATEMENTS/ OPTIONS SUMMARY
Results
The profile has been searched against the DNA Index. The search did not detect a profile consistent
with the profile in this case.
DNA from Exhibit(s) XXX was amplified using the Polymerase Chain Reaction (PCR) and profiled
at the following loci: D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317,
D7S820, D16S539, TH01, TPOX, CSF1PO and Amelogenin.
A human DNA profile was identified in Exhibit(s) XXX which matches the DNA profile of XXX and
does not match the DNA profile of XXX.
A second human DNA profile was identified in Exhibit(s) XXX and XX cannot be excluded from
having contributed to this profile.
A DNA profile (use type instead of profile for a one locus match) was identified at the D
1
, D
2
...and
D
x
loci in Exhibit(s) XXX which matches the DNA profile of XXX and does not match the DNA
profile of XXX.
A second DNA profile (use type instead of profile for a one locus match) was identified at the D
1
,
D
2
...and D
x
loci in Exhibit(s) XXX and XXX cannot be excluded from having contributed to this
profile.
This profile would be expected to occur in approximately 1 in XXX Black, 1 in XXX White or 1 in
XXX Hispanic unrelated individuals.
An additional DNA profile was identified in Exhibit(s) XXX which matches the DNA profile of
XXX.
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Low levels of an additional human DNA profile (use type instead of profile for a one locus match)
were detected in Exhibit(s) XXX that are not suitable for comparison to known standards.
Due to an insufficient amount of human DNA for PCR analysis, Exhibit(s) XXX was not profiled.
Exhibit(s) XXX was/were not profiled.
Exhibit(s) XXX was/were not examined.
No human DNA profile was identified in Exhibit(s) XXX.
A mixture of human DNA profiles was identified in Exhibit(s) XXX which matches the combined
profiles of XXX and XXX. Assuming this profile is a mixture of XXX (insert i.e. victims name) and
one other individual, approximately 1 in XXX Black, 1 in XXX White or 1 in XXX Hispanic
unrelated individuals cannot be excluded as the other contributor.
XXX can be excluded/cannot be excluded as having contributed to the mixed human DNA profile
identified in Exhibit XXX.
A mixture of human DNA profiles/A DNA profile was identified in Exhibit XXX in the non-
sperm/sperm fraction which...(reporting of fractions may be necessary on a case by cases basis).
A human DNA profile was identified in Exhibit XXX which is consistent with extraneous DNA from
a DNA analyst.
A human DNA profile was identified in Exhibit XXX. This profile has been searched against the
DNA Index. The search did not detect a profile consistent with the profile in this case.
The human DNA profile identified in Exhibit XXX was searched against the DNA Index. The search
detected a match with XXX, from (State), [SID#]. This individual demonstrates a DNA profile that
is consistent with the evidence profile and could be the donor of the biological material identified.
A human DNA profile was identified in Exhibit XXX. This profile has bee searched against the DNA
Index. The search detected a match to Laboratory Case XXX (Agency Name, Agency Case
Number), Exhibit XXX.
Statement to be included at the end of the results section of DNA reports when any sample is
entered into CODIS.
FB-IIIE-3
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Profile(s) from this case may be included in the DNA Index.
See attached table for summary of observed alleles.
Conclusions
The questioned profile from Exhibit XXX has been included in the DNA Index and will continue to
be compared to other profiles. You will be notified if a consistent profile is detected.
The blood indicated in Exhibit(s) XXX is consistent with having originated from XXX.
semen identified
saliva indicated
amylase identified (used for saliva inconclusive results)
DNA profile identified (used if no body fluid identified, etc.)
stain identified (for situations that above examples are not appropriate)
debris identified
etc.
The (...see above list...) Identified in Exhibit(s) XXX could not have originated from XXX.
No conclusions can be drawn as to the source of the semen identified/indicated in Exhibit(s) XXX
since no DNA profile other than that consistent with XXX (i.e. victim) could be identified.
No human DNA profile foreign to the victim was identified. (Used in situations when the above
statement is not appropriate.)
The mixture of human DNA profiles identified in Exhibit(s) XXX is consistent with having originated
from XXX and XXX.
The questions profile from Exhibit XXX has been included n the DNA Index and will continue to be
compared to other profiles. You will be notified if a consistent profile is detected.
Extraneous DNA Report Wording
A DNA profile was identified in Exhibit XXX which is consistent with a DNA analyst. Therefore,
FB-IIIE-3
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no interpretation can be made.
Requests
For results of previous biological examinations, please refer to my laboratory report and the
laboratory report by Forensic Scientist XXX from (...insert laboratory...).
For results of previous biological examinations, please refer to my laboratory report dated XXX.
DNA (PCR) analysis was not performed on Exhibit(s) XXX. If at a later date, it is determined that
the value of further analysis can significantly aid the case, please advise.
Upon submission of additional standards, further analysis can be conducted to resolve the source of
the open profile identified in Exhibit(s) XXX.
This information can be used for investigative purposes only. Please submit an additional blood
standard from XXX for confirmatory forensic analysis.
EVIDENCE DISPOSITION
DNA evidence will be maintained in the laboratory until such time as the case has been adjudicated or the
evidence is required by the agency.
Directions for completion of the Summary of DNA Analytical Results
1. Enter the exhibit number and description at the top of each column (1 A-Blood Standard Jane
Smith).
2. List all alleles in the profile that are above 150 RFUS in ascending numerical order not descending.
3. Complete each column for each exhibit profile.
4. The following abbreviations will be utilized:
ND - Not Detected (To be used when no alleles are called in GenoTyper.)
NT - Not Tested (To be used when the sample was not analyzed in a PCR system.)
5. The following statement must be placed on the last line of the results section after the CODIS
statement in the report: See attached table for summary of observed alleles.
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6. The following statement must be added to the bottom of each table: This is a summary of observed
alleles only. Interpretations are made according to established guidelines.
REFERENCES
References 1-6 written and compiled by Perkin Elmer Corporation.
1. GeneScan Analysis Software Users Manual (Version 2.1); 1996.
2. Fluorescent Genotyping Demonstration Kit Users Manual; 1997.
3. GeneScan Chemistry Guide; 1995.
4. AmpFlSTR Profiler Plus PCR Amplification Kit Users Manual; 1997.
5. AmpFlSTR COfiler PCR Amplification Kit User Bulletin; 1997.
6. Genetic Analyzer Instrument Users Manual; 1995.
7. ISP STR Validation Studies
8. Popstats 5.1 or higher frequency calculation program.
9. Thermal cycler manual
10. FBI Procedures Manual.
FB-IIIE-3
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ALLELE FREQUENCY DATA
D3S1358 Black
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<12-
12-
13-
14-
15-
15.2-
16-
17-
18-
19-
>19-
(alleles)
<12
12
13
14
15
15.2
16
17
18
19
>19
Count
2
1
5
51
122
0
129
84
23
2
1
Fraction
0.0048
0.0024
0.0119
0.1214
0.2905
0.0000
0.3071
0.2000
0.0548
0.0048
0.0024
Totals 420 1.0001
Minimum allele frequency = 0.0119
Null allele frequency = 0.0119
Min Allele Frequency and Null Allele Frequency calculated as 5/2N where N = 210
Note: single-allele patterns are entered twice in database
Provided by B. Budowle, FBI Academy.
D3S1358 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<12-
12-
13-
14-
15-
15.2-
16-
17-
18-
19-
>19-
(alleles)
<12
12
13
14
15
15.2
16
17
18
19
>19
Count
0
0
1
57
100
0
94
86
66
2
01
Fraction
0.0000
0.0000
0.0025
0.1404
0.2463
0.0000
0.2315
0.2118
0.1626
0.0049
0.0000
Totals 406 1.0000
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D3S1358 Southeastern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<12-
12-
13-
14-
15-
15.2-
16-
17-
18-
19-
>19-
(alleles)
<12
12
13
14
15
15.2
16
17
18
19
>19
Count
1
0
4
32
135
0
94
62
53
1
0
Fraction
0.0026
0.0000
0.0105
0.0838
0.3534
0.0000
0.2461
0.1623
0.1387
0.0026
0.0000
Totals 382 1.0000
D3S1358 Southwestern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<12-
12-
13-
14-
15-
15.2-
16-
17-
18-
19-
>19
(alleles)
<12
12
13
14
15
15.2
16
17
18
19
>19
Count
0
0
1
33
178
0
111
53
35
6
1
Fraction
0.0000
0.0000
0.0024
0.0790
0.4258
0.0000
0.2656
0.1268
0.0837
0.0144
0.0024
Totals 418 1.0001
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vWA Black
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<11-
11-
12-
13-
14-
15-
16-
17-
18-
19-
20-
21-
>21-
(alleles)
<11
11
12
13
14
15
16
17
18
19
20
21
>21
Count
0
1
0
2
24
85
97
66
49
26
10
0
0
Fraction
0.0000
0.0028
0.0000
0.0056
0.0667
0.2361
0.2694
0.1833
0.1361
0.0722
0.0278
0.0000
0.0000
Totals 360 1.0000
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vWA Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<11
11-
12-
13-
14-
15-
16-
17-
18-
19-
20-
21-
>21-
(alleles)
<11
11
12
13
14
15
16
17
18
19
20
21
>21
Count
0
0
0
2
40
44
79
103
87
33
4
0
0
Fraction
0.0000
0.0000
0.0000
0.0051
0.1020
0.1122
0.2015
0.2628
0.2219
0.0842
0.0102
0.0000
0.0000
Totals 392 0.9999
FB-IIIE-3
Page 39 of 74
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STRs
vWA Southeastern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<11-
11-
12-
13-
14-
15-
16-
17-
18-
19-
20-
21-
>21-
(alleles)
<11
11
12
13
14
15
16
17
18
19
20
21
>21
Count
0
0
0
2
33
48
129
146
90
24
8
0
0
Fraction
0.0000
0.0000
0.0000
0.0042
0.0688
0.1000
0.2688
0.3042
0.1875
0.0500
0.0167
0.0000
0.0000
Totals 480 1.0002
FB-IIIE-3
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STRs
vWA Southwestern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<11
11-
12-
13-
14-
15-
16-
17-
18-
19-
20-
21-
>21-
(alleles)
<11
11
12
13
14
15
16
17
18
19
20
21
>21
Count
0
1
0
0
25
31
146
90
79
29
5
0
0
Fraction
0.0000
0.0025
0.0000
0.0000
0.0616
0.0764
0.3596
0.2217
0.1976
0.0714
0.0123
0.0000
0.0000
Totals 406 1.0001
FB-IIIE-3
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FGA Black
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Range
<18-
18-
18.2-
19-
19.2
20-
20.2-
21-
21.2-
22-
22.2-
23.2-
23-
23.2-
24-
24.2-
24.3-
25-
26-
26.2-
27-
28-
29-
30-
>30-
(alleles)
<18
18
18.2
19
19.2
20
20.2
21
21.2
22
22.2
22.3
23
23.2
24
24.2
24.3
25
26
26.2
27
28
29
30
>30
Count
1
3
3
19
1
26
0
45
0
81
2
0
45
0
67
0
0
36
13
0
8
6
2
1
1
Fraction
0.0028
0.0083
0.0083
0.0528
0.0028
0.0722
0.0000
0.1250
1.0000
0.2250
0.0056
0.0000
0.1250
0.0000
0.1861
0.0000
0.0000
0.1000
0.0361
0.0000
0.0222
0.0167
0.0056
0.0028
0.0028
Totals 360 1.0001
FB-IIIE-3
Page 42 of 74
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STRs
FGA Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Range
<18-
18-
18.2-
19-
19.2
20-
20.2-
21-
21.2-
22-
22.2-
23.2-
23-
23.2-
24-
24.2-
24.3-
25-
26-
26.2-
27-
28-
29-
30-
>30-
(alleles)
<18
18
18.2
19
19.2
20
20.2
21
21.2
22
22.2
22.3
23
23.2
24
24.2
24.3
25
26
26.2
27
28
29
30
>30
Count
0
12
0
22
0
57
1
68
0
74
4
0
62
0
54
0
0
27
7
0
4
0
0
0
0
Fraction
0.0000
0.0306
0.0000
0.0561
0.0000
0.1454
0.0026
0.1735
0.0000
0.18888
0.0102
0.0000
0.1582
0.0000
0.1378
0.0000
0.0000
0.0689
0.0179
0.0000
0.0102
0.0000
0.0000
0.0000
0.0000
Totals 392 1.0002
FB-IIIE-3
Page 43 of 74
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STRs
FGA Southeastern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Range
<18-
18-
18.2-
19-
19.2
20-
20.2-
21-
21.2-
22-
22.2-
23.2-
23-
23.2-
24-
24.2-
24.3-
25-
26-
26.2-
27-
28-
29-
30-
>30-
(alleles)
<18
18
18.2
19
19.2
20
20.2
21
21.2
22
22.2
22.3
23
23.2
24
24.2
24.3
25
26
26.2
27
28
29
30
>30
Count
0
4
0
32
0
45
0
52
1
57
2
0
57
2
56
0
0
43
18
0
0
20
0
1
0
Fraction
0.0000
0.0105
0.0000
0.0838
0.0000
0.1178
0.0000
0.1361
0.0026
0.1492
0.0052
0.0000
0.1492
0.0052
0.1466
0.0000
0.0000
0.1126
0.0471
0.0000
0.0262
0.0052
0.0000
0.0026
0.0000
Totals 382 0.9999
FB-IIIE-3
Page 44 of 74
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STRs
FGA Southwestern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Range
<18-
18-
18.2-
19-
19.2
20-
20.2-
21-
21.2-
22-
22.2-
23.2-
23-
23.2-
24-
24.2-
24.3-
25-
26-
26.2-
27-
28-
29-
30-
>30-
(alleles)
<18
18
18.2
19
19.2
20
20.2
21
21.2
22
22.2
22.3
23
23.2
24
24.2
24.3
25
26
26.2
27
28
29
30
>30
Count
0
1
0
32
0
29
1
53
1
72
2
0
57
3
51
0
0
56
34
0
13
1
0
0
0
Fraction
0.0000
0.0025
0.0000
0.0788
0.0000
0.0714
0.0025
0.1305
0.0025
0.1773
0.0049
0.0000
0.1404
0.0074
0.1256
0.0000
0.0000
0.1379
0.0837
0.0000
0.0320
0.0025
0.0000
0.0000
0.0000
Totals 406 0.9999
FB-IIIE-3
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STRs
D8S1179 Black
Bin
1
2
3
4
5
6
7
8
9
10
11
12
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
16-
17-
>17-
(alleles)
<8
8
9
10
11
12
13
14
15
16
17
>17
Count
0
1
2
9
13
39
80
120
77
16
3
0
Fraction
0.0000
0.0028
0.0056
0.0250
0.0361
0.1083
0.2222
0.3333
0.2139
0.0444
0.0083
0.0000
Totals 360 0.9999
FB-IIIE-3
Page 46 of 74
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STRs
D8S1179 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
12
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
16-
17-
>17-
(alleles)
<8
8
9
10
11
12
13
14
15
16
17
>17
Count
0
7
4
40
23
57
133
79
43
5
1
0
Fraction
0.0000
0.0179
0.0102
0.1020
0.0587
0.1454
0.3393
0.2015
0.1097
0.0128
0.0026
0.0000
Totals 392 1.0001
FB-IIIE-3
Page 47 of 74
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STRs
Bin
1
2
3
4
5
6
7
8
9
10
11
12
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
16-
17-
>17-
(alleles)
<8
8
9
10
11
12
13
14
15
16
17
>17
Count
0
6
4
37
20
41
136
81
46
10
1
0
Fraction
0.0000
0.0157
0.0105
0.0969
0.0524
0.1073
0.3560
0.2120
0.1204
0.0262
0.0026
0.0000
Totals 382 1.0000
FB-IIIE-3
Page 48 of 74
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STRs
Bin
1
2
3
4
5
6
7
8
9
10
11
12
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
16-
17-
>17-
(alleles)
<8
8
9
10
11
12
13
14
15
16
17
>17
Count
0
1
1
38
25
49
132
100
47
10
3
0
Fraction
0.0000
0.0025
0.0025
0.0936
0.0616
0.1207
0.3251
0.2463
0.1158
0.0246
0.0074
0.0000
Totals 406 1.0001
FB-IIIE-3
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STRs
D21S11 Black
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Range
<24.2-
24.2-
24.3-
26-
27-
28-
29-
29.2-
30-
30.2-
30.3-
31-
31.2-
32
32.1-
32.2-
33-
33.2-
34-
34.2-
35-
35.2-
36-
>36-
(alleles)
<24.2
24.2
24.3
26
27
28
29
29.2
30
30.2
30.3
31
31.2
32
32.1
32.2
33
33.2
34
34.2
35
35.2
36
>36
Count
0
1
0
1
22
77
68
1
64
3
0
33
27
3
0
25
3
12
3
1
10
0
2
2
Fraction
0.0000
0.0028
0.0000
0.0028
0.0615
0.2151
0.1899
0.0028
0.1788
0.0084
0.0000
0.0922
0.0754
0.0084
0.0000
0.0698
0.0084
0.0335
0.0084
0.0028
0.0279
0.0000
0.0056
0.0056
Totals 358 1.0001
FB-IIIE-3
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STRs
D21S11 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Range
<24.2-
24.2-
24.3-
26-
27-
28-
29-
29.2-
30-
30.2-
30.3-
31-
31.2-
32
32.1-
32.2-
33-
33.2-
34-
34.2-
35-
35.2-
36-
>36-
(alleles)
<24.2
24.2
24.3
26
27
28
29
29.2
30
30.2
30.3
31
31.2
32
32.1
32.2
33
33.2
34
34.2
35
35.2
36
>36
Count
0
2
0
0
18
65
71
91
15
0
28
39
6
0
44
0
12
0
0
0
1
0
0
Fraction
0.0000
0.0051
0.0000
0.0000
0.0459
0.1658
0.1811
0.0000
0.2321
0.0383
0.0000
0.0714
0.0995
0.0153
0.0000
0.1122
0.000
0.0306
0.0000
0.0000
0.0000
0.0026
0.0000
0.0000
Totals 392 0.9999
FB-IIIE-3
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STRs
D21S11Southeastern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Range
<24.2-
24.2-
24.3-
26-
27-
28-
29-
29.2-
30-
30.2-
30.3-
31-
31.2-
32
32.1-
32.2-
33-
33.2-
34-
34.2-
35-
35.2-
36-
>36-
(alleles)
<24.2
24.2
24.3
26
27
28
29
29.2
30
30.2
30.3
31
31.2
32
32.1
32.2
33
33.2
34
34.2
35
35.2
36
>36
Count
0
0
0
1
7
48
92
1
91
13
0
29
32
4
0
44
0
14
0
4
0
0
1
1
Fraction
0.0000
0.0000
0.0000
0.0026
0.0183
0.1257
0.2408
0.0026
0.2382
0.0340
0.0000
0.0759
0.0838
0.0105
0.0000
0.1152
0.0000
0.0367
0.0000
0.0105
0.0000
0.0000
0.0026
0.0026
Totals 382 1.0000
FB-IIIE-3
Page 52 of 74
Version: 3.02072
STRs
D21S11 Southwestern
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Range
<24.2-
24.2-
24.3-
26-
27-
28-
29-
29.2-
30-
30.2-
30.3-
31-
31.2-
32
32.1-
32.2-
33-
33.2-
34-
34.2-
35-
35.2-
36-
>36-
(alleles)
<24.2
24.2
24.3
26
27
28
29
29.2
30
30.2
30.3
31
31.2
32
32.1
32.2
33
33.2
34
34.2
35
35.2
36
>36
Count
0
1
0
0
4
28
83
1
134
13
0
28
35
5
0
55
0
17
0
2
0
0
0
0
Fraction
0.0000
0.0025
0.0000
0.0000
0.0099
0.0690
0.2044
0.0025
0.3301
0.0320
0.0000
0.0690
0.0862
0.0123
0.0000
0.1355
0.0000
0.0419
0.0000
0.0049
0.0000
0.0000
0.0000
0.0000
Totals 406 1.0002
FB-IIIE-3
Page 53 of 74
Version: 3.02072
STRs
D18S51 Black
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Range
<11-
11-
12-
13-
13.2-
14-
14.2-
15-
15.2-
16-
17-
18-
19-
20-
21-
21.2-
22-
>22-
(alleles)
<11
11
12
13
13.2
14
14.2
15
15.2
16
17
18
19
20
21
21.2
22
>22
Count
2
2
21
20
2
23
0
60
0
68
59
47
28
20
4
0
2
2
Fraction
0.0056
0.0056
0.0583
0.0556
0.0056
0.0639
0.0000
0.1667
0.0000
0.1889
0.1639
0.1306
0.0778
0.0556
0.0111
0.0000
0.0056
0.0056
Totals 360 1.0004
FB-IIIE-3
Page 54 of 74
Version: 3.02072
STRs
D18S51 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Range
<11-
11-
12-
13-
13.2-
14-
14.2-
15-
15.2-
16-
17-
18-
19-
20-
21-
21.2-
22-
>22-
(alleles)
<11
11
12
13
13.2
14
14.2
15
15.2
16
17
18
19
20
21
21.2
22
>22
Count
5
5
50
48
0
68
0
50
0
42
61
36
14
10
2
0
1
0
Fraction
0.0128
0.0128
0.1276
0.1225
0.0000
0.1735
0.0000
0.1276
0.0000
0.1071
0.1556
0.0918
0.0357
0.0255
0.0051
0.0000
0.0026
0.0000
Totals 392 1.0002
FB-IIIE-3
Page 55 of 74
Version: 3.02072
STRs
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Range
<11-
11-
12-
13-
13.2-
14-
14.2-
15-
15.2-
16-
17-
18-
19-
20-
21-
21.2-
22-
>22-
(alleles)
<11
11
12
13
13.2
14
14.2
15
15.2
16
17
18
19
20
21
21.2
22
>22
Count
3
6
52
45
0
50
0
73
0
54
41
21
18
11
4
0
3
1
Fraction
0.0079
0.0127
0.1361
0.1178
0.0000
0.1309
0.0000
0.1911
0.0000
0.1414
0.1073
0.0550
0.0471
0.0288
0.0105
0.0000
0.0079
0.0026
Totals 382 1.0001
FB-IIIE-3
Page 56 of 74
Version: 3.02072
STRs
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Range
<11-
11-
12-
13-
13.2-
14-
14.2-
15-
15.2-
16-
17-
18-
19-
20-
21-
21.2-
22-
>22-
(alleles)
<11
11
12
13
13.2
14
14.2
15
15.2
16
17
18
19
20
21
21.2
22
>22
Count
2
5
43
69
0
69
0
56
0
47
56
21
15
7
8
0
3
5
Fraction
0.0049
0.0123
0.1059
0.1700
0.0000
0.1700
0.0000
0.1379
0.0000
0.1158
0.1379
0.0517
0.0370
0.0172
0.0197
0.0000
0.0074
0.0123
Totals 406 1.0000
FB-IIIE-3
Page 57 of 74
Version: 3.02072
STRs
D5S818 Black
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<7-
7-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<7
7
8
9
10
11
12
13
14
15
>15
Count
0
1
18
5
23
94
128
88
2
0
1
Fraction
0.0000
0.0028
0.0500
0.0139
0.0639
0.2611
0.3556
0.2444
0.0056
0.0000
0.0028
Totals 360 1.0001
D5S818 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<7-
7-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<7
7
8
9
10
11
12
13
14
15
>15
Count
0
0
0
12
19
160
138
57
3
1
0
Fraction
0.0000
0.0000
0.0000
0.0308
0.0478
0.4103
0.3539
0.1462
0.0077
0.0026
0.0000
Totals 390 1.0002
FB-IIIE-3
Page 58 of 74
Version: 3.02072
STRs
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<7-
7-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<7
7
8
9
10
11
12
13
14
15
>15
Count
0
11
5
24
22
189
152
74
3
0
0
Fraction
0.0000
0.0229
0.0104
0.0500
0.0458
0.3938
0.3167
0.1542
0.0063
0.0000
0.0000
Totals 480 1.0001
Bin
1
2
3
4
5
6
7
8
9
10
11
Range
<7-
7-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<7
7
8
9
10
11
12
13
14
15
>15
Count
0
25
1
22
27
171
118
39
2
1
0
Fraction
0.0000
0.0616
0.0025
0.0542
0.0665
0.4212
0.2906
0.0961
0.0049
0.0025
0.0000
Totals 406 1.0001
FB-IIIE-3
Page 59 of 74
Version: 3.02072
STRs
D13S317 Black
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
13
10
18
85
173
45
13
1
0
Fraction
0.0000
0.0363
0.0279
0.0503
0.2374
0.4832
0.1257
0.0363
0.0028
0.0000
Totals 358 0.9999
D13S317 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
39
30
20
125
121
43
14
0
0
Fraction
0.0000
0.0995
0.0765
0.0510
0.3189
0.3087
0.1097
0.0357
0.0000
0.0000
Totals 392 1.0000
FB-IIIE-3
Page 60 of 74
Version: 3.02072
STRs
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
55
55
37
147
110
52
24
0
0
Fraction
0.0000
0.1146
0.1146
0.0771
0.3063
0.2292
0.1083
0.0500
0.0000
0.0000
Totals 480 1.0001
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
27
89
41
82
88
56
23
0
0
Fraction
0.0000
0.0665
0.2192
0.1010
0.2020
0.2168
0.1379
0.0567
0.0000
0.0000
Totals 406 1.0001
FB-IIIE-3
Page 61 of 74
Version: 3.02072
STRs
D7S820 Black
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<6-
6-
7-
8-
9-
10-
10.1-
11-
11.3-
12-
13-
14-
>14-
(alleles)
<6
6
7
8
9
10
10.1
11
11.3
12
13
14
>14
Count
0
0
3
73
66
136
0
94
0
38
8
2
0
Fraction
0.0000
0.0000
0.0071
0.1738
0.1571
0.3238
0.0000
0.2238
0.0000
0.0905
0.0191
0.0048
0.0000
Totals 420 1.0000
FB-IIIE-3
Page 62 of 74
Version: 3.02072
STRs
D7S820 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<6-
6-
7-
8-
9-
10-
10.1-
11-
11.3-
12-
13-
14-
>14-
(alleles)
<6
6
7
8
9
10
10.1
11
11.3
12
13
14
>14
Count
0
1
7
66
60
118
0
82
0
57
12
3
0
Fraction
0.0000
0.0025
0.0172
0.1626
0.1478
0.2906
0.0000
0.2020
0.0000
0.1404
0.0296
0.0074
0.0000
Totals 406 1.0001
FB-IIIE-3
Page 63 of 74
Version: 3.02072
STRs
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<6-
6-
7-
8-
9-
10-
10.1-
11-
11.3-
12-
13-
14-
>14-
(alleles)
<6
6
7
8
9
10
10.1
11
11.3
12
13
14
>14
Count
0
1
5
68
60
128
0
109
0
90
17
2
0
Fraction
0.0000
0.0021
0.0104
0.1417
0.1250
0.2667
0.0000
0.2271
0.0000
0.1875
0.0354
0.0042
0.0000
Totals 480 1.0001
FB-IIIE-3
Page 64 of 74
Version: 3.02072
STRs
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
Range
<6-
6-
7-
8-
9-
10-
10.1-
11-
11.3-
12-
13-
14-
>14-
(alleles)
<6
6
7
8
9
10
10.1
11
11.3
12
13
14
>14
Count
0
1
9
41
20
128
0
121
0
80
16
2
0
Fraction
0.0000
0.0024
0.0215
0.0981
0.0479
0.3062
0.0000
0.2895
0.0000
0.1914
0.0383
0.0048
0.0000
Totals 418 1.0001
FB-IIIE-3
Page 65 of 74
Version: 3.02072
STRs
D16S539 Black
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
15
83
46
123
78
69
4
0
0
Fraction
0.0000
0.0359
0.1986
0.1101
0.2943
0.1866
0.1651
0.0096
0.0000
0.0000
Totals 418 1.0002
D16S539 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
8
42
27
110
137
66
13
1
0
Fraction
0.0000
0.0198
0.1040
0.0668
0.2723
0.3391
0.1634
0.0322
0.0025
0.0000
Totals 404 1.0001
FB-IIIE-3
Page 66 of 74
Version: 3.02072
STRs
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
11
70
46
135
122
78
17
1
0
Fraction
0.0000
0.0229
0.1458
0.0958
0.2813
0.2542
0.1625
0.0354
0.0021
0.0000
Totals 480 1.0000
Bin
1
2
3
4
5
6
7
8
9
10
Range
<8-
8-
9-
10-
11-
12-
13-
14-
15-
>15-
(alleles)
<8
8
9
10
11
12
13
14
15
>15
Count
0
7
33
72
131
119
43
10
1
0
Fraction
0.0000
0.0168
0.0793
0.1731
0.3149
0.2861
0.1034
0.0240
0.0024
0.0000
Totals 416 1.0000
FB-IIIE-3
Page 67 of 74
Version: 3.02072
STRs
TH01 Black
Bin
1
2
3
4
5
6
7
8
9
10
Range
<5-
5-
6-
7-
8-
8.3-
9-
9.3-
10-
>10-
(alleles)
<5
5
6
7
8
8.3
9
9.3
10
>10
Count
0
0
46
185
78
0
61
44
6
0
Fraction
0.0000
0.0000
0.1095
0.4405
0.1857
0.0000
0.1452
0.1048
0.0143
0.0000
Totals 420 1.0000
TH01 Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
Range
<5-
5-
6-
7-
8-
8.3-
9-
9.3-
10-
>10-
(alleles)
<5
5
6
7
8
8.3
9
9.3
10
>10
Count
0
0
92
70
51
1
67
124
1
0
Fraction
0.0000
0.0000
0.2266
0.1724
0.1256
0.0025
0.1650
0.3054
0.0025
0.0000
Totals 406 1.0000
FB-IIIE-3
Page 68 of 74
Version: 3.02072
STRs
TH01 Southeastern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
Range
<5-
5-
6-
7-
8-
8.3-
9-
9.3-
10-
>10-
(alleles)
<5
5
6
7
8
8.3
9
9.3
10
>10
Count
0
0
102
121
50
0
89
113
5
0
Fraction
0.0000
0.0000
0.2125
0.2521
0.1042
0.0000
0.1854
0.2354
0.0104
0.0000
Totals 480 1.0000
Bin
1
2
3
4
5
6
7
8
9
10
Range
<5-
5-
6-
7-
8-
8.3-
9-
9.3-
10-
>10-
(alleles)
<5
5
6
7
8
8.3
9
9.3
10
>10
Count
0
1
97
141
34
0
43
101
1
0
Fraction
0.0000
0.0024
0.2321
0.3373
0.0813
0.0000
0.1029
0.2416
0.0024
0.0000
Totals 418 1.0001
FB-IIIE-3
Page 69 of 74
Version: 3.02072
STRs
TPOX Black
Bin
1
2
3
4
5
6
7
8
9
10
Range
<6-
6-
7-
8-
9-
10-
11-
12-
13-
>13-
(alleles)
<6
6
7
8
9
10
11
12
13
>13
Count
0
36
9
154
76
39
94
10
0
0
Fraction
0.0000
0.0861
0.0215
0.3684
0.1818
0.0933
0.2249
0.0239
0.0000
0.0000
Totals 418 0.9999
TPOX Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
Range
<6-
6-
7-
8-
9-
10-
11-
12-
13-
>13-
(alleles)
<6
6
7
8
9
10
11
12
13
>13
Count
0
0
1
221
50
150
103
16
0
0
Fraction
0.0000
0.0000
0.0025
0.5443
0.1232
0.0370
0.2537
0.0394
0.0000
0.0000
Totals 406 1.0001
FB-IIIE-3
Page 70 of 74
Version: 3.02072
STRs
TPOX Southeastern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
Range
<6-
6-
7-
8-
9-
10-
11-
12-
13-
>13-
(alleles)
<6
6
7
8
9
10
11
12
13
>13
Count
0
2
1
243
40
30
133
31
0
0
Fraction
0.0000
0.0042
0.0021
0.5063
0.0833
0.0625
0.2771
0.0646
0.0000
0.0000
Totals 480 1.0001
TPOX Southwestern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
Range
<6-
6-
7-
8-
9-
10-
11-
12-
13-
>13-
(alleles)
<6
6
7
8
9
10
11
12
13
>13
Count
0
2
2
232
14
14
114
39
1
0
Fraction
0.0000
0.0048
0.0048
0.5550
0.0335
0.0335
0.2727
0.0933
0.0024
0.0000
Totals 418 1.0000
FB-IIIE-3
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Version: 3.02072
STRs
CSF1PO Black
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Range
<6-
6-
7-
8-
9-
10-
10.3-
11-
12-
12.1-
13-
14-
15-
>15-
(alleles)
<6
6
7
8
9
10
10.3
11
12
12.1
13
14
15
>15
Count
0
0
18
36
14
114
0
86
126
0
23
3
0
0
Fraction
0.0000
0.0000
0.0429
0.0857
0.0333
0.2714
0.0000
0.2048
0.3000
0.0000
0.0548
0.0071
0.0000
0.0000
Totals 420 1.0000
FB-IIIE-3
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Version: 3.02072
STRs
CSF1PO Caucasian
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Range
<6-
6-
7-
8-
9-
10-
10.3-
11-
12-
12.1-
13-
14-
15-
>15-
(alleles)
<6
6
7
8
9
10
10.3
11
12
12.1
13
14
15
>15
Count
0
0
1
2
8
103
1
122
132
0
29
6
2
0
Fraction
0.0000
0.0000
0.0025
0.0049
0.0197
0.2537
0.0025
0.3005
0.3251
0.0000
0.0714
0.0148
0.0049
0.0000
Totals 406 1.0000
FB-IIIE-3
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Version: 3.02072
STRs
CSF1PO Southeastern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Range
<6-
6-
7-
8-
9-
10-
10.3-
11-
12-
12.1-
13-
14-
15-
>15-
(alleles)
<6
6
7
8
9
10
10.3
11
12
12.1
13
14
15
>15
Count
0
0
1
2
6
122
0
142
171
0
33
2
1
0
Fraction
0.0000
0.0000
0.0021
0.0042
0.0125
0.2542
0.0000
0.2958
0.3563
0.0000
0.0688
0.0042
0.0021
0.0000
Totals 480 1.0002
FB-IIIE-3
Page 74 of 74
Version: 3.02072
STRs
CSF1PO Southwestern Hispanic
Bin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Range
<6-
6-
7-
8-
9-
10-
10.3-
11-
12-
12.1-
13-
14-
15-
>15-
(alleles)
<6
6
7
8
9
10
10.3
11
12
12.1
13
14
15
>15
Count
0
0
1
0
3
106
0
111
164
0
27
4
2
0
Fraction
0.0000
0.0000
0.0024
0.0000
0.0072
0.2536
0.0000
0.2656
0.3923
0.0000
0.0646
0.0096
0.0048
0.0000
Totals 418 1.0001
Accepted Date: October 2, 2000
FB-IIIB-2
Page 1 of 7
Version
3.00277
Procedure: Slot Blot
PROCEDURES MANUAL
METHOD: DNA Quantitation
PROCEDURE: SLOT BLOT
Prepared by:

Assistant R&D Laboratory Director
Approved by:

Susan H. Johns Date
FB-IIIB-2
Page 2 of 7
Version
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INTRODUCTION
DNA analysis is dependent on the quantity and the quality of the DNA present in an evidentiary
sample. The purpose of quantification is to determine the amount of high molecular weight DNA present,
the extent of degradation and the specificity of the DNA sample (i.e., human or bacterial). Once the above
determinations are made, the DNA analysis method that utilizes the most information is chosen. DNA
quantification can be accomplished by the yield gel method or the slot blot method.
Slot Blot: This method is used under the following circumstances: 1) when there are small amounts of
DNA present, 2) to determine if the DNA recovered is human in origin, and 3) for all samples to be
subjected to PCR testing.
Warning: Treat all reagents/samples as potential biohazards
Warning: The following are considered hazardous reagents:
Sodium Dodecyl Sulfate (SDS) potentially causes burns when in contact with the skin or eyes. It
causes irritation if inhaled and may cause severe digestive tract irritation if ingested. Accidental
exposure will be treated according to routine laboratory safety procedures (give milk or water if
ingested). When preparing solutions using this chemical, proper personal protective equipment,
including a dust mask or respirator, should be employed. Use of the fume hood is required.
Sodium Hydroxide is a corrosive chemical which may be fatal if absorbed through the skin. It
causes severe eye and skin burns, gastroinstestinal burns if ingested and severe irritation if inhaled.
Accidental skin or eye contact will be treated with routine laboratory safety practices. Seek immediate
medical aid. Wear appropriate personal protective equipment and use the fume hood when using this
chemical.
PREPARATIONS
DNA Quantitation Reagents:
AmpliType/Slot Blot Hybridization Solution
PCR-20% SDS 25 ml
PCR-SSPE 20x 250 ml
ddi H
2
O 725 ml
FB-IIIB-2
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Citrate Buffer
Sodium Citrate Na
3
C
6
H
5
O
7
.
2H
2
O 92 g
adjust pH to 5.0 with Citric acid monohydrate C
6
H
8
O
7
.
H
2
O. 30 g
ddi H
2
O to 5 l
500 mM EDTA pH 8.0
EDTA-2H
2
O@Na
2
930.5 g
ddi H
2
O 4.0 l
NaOH pellets 5-100 g
When fully dissolved, add more NaOH to bring the pH to 8.0 (EDTA does not go into solution until the
pH nears 8.0.) Adjust the volume to 5.0 liter. Autoclave.
4M NaOH
NaOH 800 g
ddi H
2
O 4200 ml
Adjust volume to 5.0 liter.
PCR-SSPE 20X
500 mM EDTA pH 8.0 200 ml
NaCl 1050 g
NaH
2
PO
4
.
H
2
O (monoydrate) 138 g
pH to 7.4
ddi H
2
O to 5 l
Prewetting Solution (store at room temperature)
ddi H
2
O 85 ml
4M NaOH 10 ml or 400 mM NaOH
20% SDS
PCR-SDS 1000 g
ddi H
2
O to 5 l
Heat gently to about 65C and stir to dissolve. Use a nuisance mask. Prepare the reagent in the
fume hood.
Slot Blot Standards
from Quantiblot kit
ECL Chemi-luminescent reagents
Amersham RPN 2106
Prepares as instructed
Slot Blot Wash Solution
PCR-20% SDS 25 ml
PCR-SSPE 20x 75 ml
ddi H
2
O 900 ml
FB-IIIB-2
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Version
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Spotting Solution (store at room temperature)
ddi H
2
O 85 ml
4M NaOH 10 ml
500 mM EDTA ph 8.0 5 ml
Bromthymol blue from kit 200 :l
INSTRUMENTATION
Included in the Perkin-Elmer Quantiblot Kit is a human DNA standard and two DNA calibrators.
This human DNA standard is used to prepare seven DNA standards. The seven DNA standards represent
the following quantities: 10, 5.0, 2.5, 1.25, 0.625, 0.3125, and 0.15625 ng. Results are interpreted by
comparing the signal intensity of the DNA test sample to the signal intensity obtained from the DNA
standards. The DNA calibrators are used to provide DNA of a known concentration to verify that the
DNA standards were correctly diluted and are providing correct results for the test samples.
Critical Reagents:
These are slot blot reagents that need to be quality controlled. See Quality Assurance Manual
(Appendix VI) for instructions.
1. DNA standards. The seven DNA standards ranging in concentration from 10ng to 150pg serve
as a guide in the quantification of DNA.
2. Charged membrane. This membrane is the surface to which DNA binds. Proper binding of the
DNA is required for accurate DNA quantitation.
FB-IIIB-2
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Slot Blot:
Before proceeding with the slot blot, a review of the Perkin - Elmer kit procedure is recommended.

1. Prepare the slot blot standards.
2. Label tubes: A, B, C, D, E, F, G, BLANK, CAL1, CAL2, YOUR SAMPLES.
3. Place at least 150 :l spotting solution in all tubes.
4. Place 5 :l of the corresponding slot blot standards or calibrators into the tubes marked A through
G and Cal 1 & 2.
5. Place known volumes of your samples into the appropriate tubes. The goal is to load
approximately 1 ng of DNA . A yield gel will aid in this effort.
6. Wet the membrane (charged nylon, Bio B cut to the appropriate size) in prewetting solution and
assemble the slot blot apparatus.
7. Apply the vacuum to the seal.
8. Load the samples into the center of the wells being careful not to puncture the membrane or
introduce bubbles. Load spotting solution into empty wells to aid in even vacuum pressure.
9. Turn on the vacuum to the wells.
10. When all of the wells go dry turn off the vacuum. Record any samples that do not have a uniform
blue bar. These samples will be unreliable.
11. Place the membrane in AmpliType/Slot blot hybridization solution. At this point, the membrane
may be stored in hybridization solution in the refrigerator.
12. Process up to 4 membranes together in one container. Prehybridize the membranes in a box
containing 100 ml prewarmed hybridization solution and 5 ml 30% H
2
O
2
at 50C for 15 minutes
with rotation at 50-60 RPM. Pour off the hybridization solution.
FB-IIIB-2
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13. Add 30 ml AmpliType/Slot blot hybridization solution to the box. Tilt the box to pool the solution
to one end and add 20 :l D17Z1 probe. Incubate at 50C for 20 minutes with rotation at 50-60
RPM. Pour off the solution.
14. Briefly rinse membrane in slot blot wash solution.
15. Add 30ml prewarmed slot blot wash solution. Tilt box to pool the wash solution at one end and
add 90 ul HRP-SA enzyme conjugate. Incubate at 50 degrees C for 10 minutes with rotation
at 50-60 RPM. Pour off the solution.
16. Rinse the membrane for 1 minute with 100 ml prewarmed (50 degrees C) slot blot wash solution
at room temperature with rotation. Repeat.
17. Rinse the membrane for 10 minutes with 100 ml prewarmed (50 degrees C) slot blot wash
solution at room temperature with rotation.
18. Rinse briefly in 100 ml citrate buffer. Drain off as much solution as possible.
19. Process membranes individually. Mix 5 ml of ECL reagent 1 with 5 ml ECL reagent 2 and pour
mixture over membrane. Rotate for 1 minute so that entire membrane is saturated.
20. Place membrane in plastic, wipe off excess solution, and heat seal.
21. In a darkroom, tape heat sealed plastic containing membrane in a cassette and place a piece of
X-RAY film over it (close cassette) for 30 minutes.
22. Develop film in processor.
23. Quantitative estimates of DNA must only be made by interpolation between standards of known
quantity. Values above the highest visible standard must be designated as greater than (>) the
highest visible standard. Such samples must be diluted and rerun. Values below the lowest
visible standard must be designated as less than (<) the lowest visible standard.
REPORT WORDING
See Appendix I.
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REFERENCES
1. Walsh, P.S.; et al. A Rapid Chemiluminescent Method for Quantitation of Human DNA.
Nucleic Acids Research. 1992, Vol 20 (19).
2. Ways, J.S.D.; Presley, L.A.; Budowle, B.; Shutler, G.G.; Fourney, R.M.; A Simple and
Sensitive Method for Quantifying Human Genomic DNA in Forensic Specimen Extracts.
BioTechniques. 1989, Vol 7, No 8.
3. Ways, J.S.D.; Michaud, D.; Bowen, J.H.; Fourney, R.M. Sensitive and Specific Quantification
of Human Genomic Deoxyribonucleic Acid (DNA) in Forensic Science Specimens: Casework
Examples. Journal of Forensic Sciences. July 1991, Vol 36,
No 4, 1198-1203.
Also see Instructions for Perkin-Elmer Slot Blot Kit.
Accepted Date: December 3, 2001
FB-IIIB-1
Page 1 of 5
Version 2.01337
Procedure: Yield Gel
PROCEDURES MANUAL
METHOD: DNA Quantitation
PROCEDURE: YIELD GEL
Prepared by:

Approved by:

FB-IIIB-1
Page 2 of 5
Version 2.01337
INTRODUCTION
DNA analysis is dependent on the quantity and the quality of the DNA present in an evidentiary
sample. The purpose of quantification is to determine the amount of high molecular weight DNA
present, the extent of degradation and the specificity of the DNA sample (i.e., human or bacterial).
Once the above determinations are made, the DNA analysis method that utilizes the most information is
chosen. DNA quantification can be accomplished by the yield gel method or the slot blot method.
Yield Gel: The purpose of this method is to determine the quantity and the quality of the DNA present.
The use of the yield gel before a slot blot is optional and is up to the analysts discretion. However, the
yield gel may assist in determining the amount of each sample to add to the slot blot.
Warning: Hazardous Reagents:
Ethidium Bromide is a hazardous chemical that may be harmful if ingested and is irritating if
exposed to mucous membranes and upper respiratory tract, eyes and skin. It is a mutagen and
moderately toxic. Proper personal protective equipment including gloves should be used when
making and working with solutions that contain this dye. In addition, yield gels must be run in a
dedicated area of the laboratory.
PREPARATIONS
DNA Quantitation Reagents:
1% Agarose with Ethidium Bromide
Agarose 1.5 g
1X TAE 150 ml
Dissolve agarose by boiling in microwave oven
add ethidium bromide solution 15 :l
Ethidium Bromide Solution
Ethidium bromide 5 mg
ddi H
2
O 1.0 ml
Protect from light. Mutagenic substance. Wear gloves.
FB-IIIB-1
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Version 2.01337
Loading Buffer
Glycerol 5 ml
Bromophenol blue 0.02 g
20X TAE 0.5 ml
ddi H
2
O 4.5 ml
20X TAE
Tris base 96.6 g
Glacial acetic acid 22.8 ml
500 mM EDTA pH 8.0 40.0 ml
Adjust volume to 1.0 liter.
1X TAE
20X TAE 50 ml
ddi H
2
O 950 ml
TE
1.0 M Tris pH 7.5 10 ml
ddi H
2
Yield Gel Standards
Lambda phage DNA at 250 g/ml = stock
Carry out serial doubling dilutions of the stock with TE to obtain the solutions shown below.
0.125ml stock+ 0.875 ml TE = 31.3 g/ml
0.5 ml 31.3 g/ml + 0.5 ml TE = 15.6 g/ml
0.5 ml 15.6 g/ml + 0.5 ml TE = 7.8 g/ml
0.5 ml 7.8 g/ml + 0.5 ml TE = 3.9 g/ml
0.5 ml 3.9 g/ml + 0.5 ml TE = 1.9g/ml
0.5 ml 1.9 g/ml + 0.5 ml TE = 0.9 g/ml
Combine 0.5 ml aliquots of the diluted standards with loading buffer as shown.
0.5 ml at 31.3 g/ml + 0.25 ml loading buffer = 125 ng/6 :l
FB-IIIB-1
Page 4 of 5
Version 2.01337
INSTRUMENTATION
Yield gel standards are made from dilutions of lambda phage DNA. There are 6 standards of
known quantity: 125, 63, 31, 15, 8, and 4 ng. The purpose of these standards is to estimate the
amount of DNA present in the samples. Results are interpreted by comparing the signal intensity of the
DNA samples to the signal intensity obtained for the DNA standards.
Yield Gel:
1. At this stage the DNA has been solubilized in TE
-4
. Briefly spin the tubes before opening.
2. Prepare a minigel with 1% agarose with ethidium bromide. Insert one or two combs,
depending on how many lanes are needed. When the gel is set, add 1X TAE without
ethidium bromide and remove the combs.
3. Combine 4 :l of each sample with 2 :l loading buffer.
4. In the first six wells load 6 :l of each yield gel standard (4-125ng). Next, place the samples
mixed with loading buffer (6 :l total volume) into the remaining wells.
5. Electrophorese at 200 volts for 8 minutes or equivalent. The bromophenol blue tracking dye
should move 1-2 cm from the origin.
FB-IIIB-1
Page 5 of 5
Version 2.01337
6. After electrophoresis is complete, remove the gel from the tank. Examine and photograph the
gel using an ultraviolet light transilluminator. DO NOT EXPOSE YOURSELF TO THE UV
LIGHT FOR AN EXCESSIVE AMOUNT OF TIME. ALWAYS WEAR UV
PROTECTION WHEN WORKING WITH THE TRANSILLUMINATOR.
7. From the photograph, assess the quantity of DNA in test specimens by comparison with the
DNA standards. The standards and high molecular weight DNA will migrate as a band only
a short distance. A smear from the origin toward the dye front indicates that the DNA has
been fragmented.
8. Quantitation extimates of DNA must only be made by interpolation between two standards of
known quantity. Values above the highest visible standard must be designated as greater than
(>) the highest visible standard. Such samples must be diluted and rerun. Values below the
lowest visible standard must be designated as less than (<) the lowest visible standard.
REPORT WORDING
See Appendix I.
REFERENCES
See ISP Validation Studies (listed by number):
1. Validation Study I.
2. Quantitation of DNA by spectrophotometry versus yield gels.
Accepted Date: October 2, 2000 FB-IIIA-9 Procedure: Isolation of
Next Review Date: July 31, 2006 Page 1 of 4 DNA From Chewing Gum
Forensic Biology/DNA Procedures Manual Version 2.00277
PROCEDURES MANUAL
PROCEDURE: ISOLATION OF DNA FROM
CHEWING GUM
Prepared by:

Approved by:

Susan H. Johns Date
INTRODUCTION
Refer to Introduction under the DNA Isolation Methods section.
Refer to safety considerations under the DNA Isolation Methods section.
PREPARATION
Refer to Preparation under the DNA Isolation Methods sections.
INSTRUMENTATION
Refer to the Minimum Standards and Controls under the DNA Isolation Methods section.
Chewing Gum:
1. Cut the gum into smaller pieces. Prior refrigeration of the sample may aid in this process.
2. Add: 400 Fl Stain Extraction Buffer
10 Fl Proteinase K (20 mg/ml)
5 Fl 390 mM DTT
Mix and spin briefly to force gum into the liquid.
3. Incubate at 56C overnight.
4. Place the cuttings in the basket of the Spin-Ease tube. Remove the stain extraction buffer out
of the material by spinning for 5 minutes at 10,000 x g (Spin-Ease). Retain the cuttings.
Transfer the liquid into an eppendorf tube.
5. Phenol Extraction: Add 500 l phenol/chloroform/isoamyl alcohol. This step must be done
in the fume hood. Vortex for approximately one minute to achieve a milky emulsion in the tube.
Spin the tube for two minutes at maximum speed. Note: Do not use microfuge tubes
provided for use with Microcon filters for organic extractions.
6. At the analysts discretion, the aqueous layer and interface may be re-extracted. Transfer the
aqueous phase (top layer) to a new tube. Discard the old tube containing the phenol into the
appropriate waste container in the fume hood.
7. Repeat steps 3 and 4, using 500 l chloroform/isoamyl alcohol. Do not re-extract the
interface.
8. Microcon 100 Filtration
A. Place the aqueous phase from the phenol/chloroform/isoamyl or chloroform/isoamyl mixture
in a Microcon 100 tube. Follow the manufacturer's recommendations for centrifugation
speed and time or spin at 500 x g for 8 to 10 minutes. Centrifuge for additional time if
liquid remains on the filter. It may be necessary to increase speed to 1500 x g for some
samples. Add 50-100 l of TE and centrifuge as before to wash residual extraction
components from the DNA. Examine the filter unit to verify that no tearing or cracking has
occurred.
Do not use microfuge tubes provided for use with Microcon filters for organic
extractions or high-speed centrifugation. They are not designed to withstand these
conditions.
B. Add the appropriate volume of TE (30l - 100Fl) (depending on anticipated DNA
-4
recovery), invert filter, vortex 15 seconds, and spin out liquid at 3500 x g for 3 min.
C. Incubate the sample to resolubilize the DNA for at least one hour at 56EC.
Note: Retain filtrate until you have confirmed expected recovery of DNA.
REPORT WORDING
See Appendix I.
REFERENCES
Refer to the References under the DNA Isolation Methods section.
Reviewed Date: October 2, 2000 FB-IIIA-8 Procedure: Isolation of
Next Review Date: July 31, 2006 Page 1 of 3 DNA From Bone
Forensic Biology/DNA Procedures Manual Version 2
PROCEDURES MANUAL
BONE
Prepared by:

Approved by:

Susan H. Johns Date
INTRODUCTION
PREPARATION
INSTRUMENTATION
Bone:
Bone which has been submerged or in the environment for a long period of time may not yield
DNA which is amenable to nuclear DNA testing. Therefore, ask for entire bone samples (i.e., ribs
or femurs). Request also that the medical examiner does NOT saw on the bone. Note: If soft
tissue is present try extracting 2-5mm before beginning bone extraction procedure.
2
1. Begin by cleaning the bone and removing all of the flesh. Soak the bone in 10% bleach for 1-2
minutes. Next, rinse and dry the bone thoroughly. Then, using sandpaper, remove the outer
layer of tissue. Avoid the marrow and the sawed ends of the bone because these may be
contaminated.
2. Drape a piece of clean plastic over a vise and between the jaws of the vise. Close the vise on
the piece of bone. Using a hack saw with a NEW blade, generate between 5 and 15 g of bone
dust.
3. Place up to 1 g of bone dust in a 15 ml conical bottom tube or 3 g in a 50 ml conical bottom
tube.
4. To decalcify the bone: Fill the tube nearly to the top with 500 mM EDTA pH 8.0. Incubate
the tube with rocking for 24 hr at 4EC. After the overnight incubation spin the tube for 15
minutes at 2000 xg. Discard the supernatant. Refill the tube with 500 mM EDTA pH 8.0 to
resuspend the pellet. Repeat 5 times.
5. Resuspend the pellet with ddi H O and spin 15 minutes at 2000xg. Repeat this process two
2
times.
6. Resuspend the pellet in twice the pellet volume with Stain Extraction Buffer (including proteinase
K (20Fg/Fl) and 390 mM DTT). Incubate overnight at 56EC. Rock periodically until the
pellet dissolves.
7. Extract the suspension three times with phenol/chloroform/isoamyl alcohol solution. Repeat until
the aqueous phase becomes relatively clear (more than 3 extractions may be required). A
chloroform/isoamyl extraction may be performed at the analyst's discretion. Note: If the
solution isnt clear the filter may become plugged.
8. Finally, remove the DNA from the aqueous phase in Microcon 100s or Centricon 100s. Rinse
the filters twice with TE before collecting DNA.
Note: Since decayed or buried bones may contain large amounts of non-human DNA, slot blot
quantitation is required.
REPORT WORDING
See Appendix I.
REFERENCES
Accepted Date: October 2, 2000 FB-IIIA-7 Procedure: Isolation of DNA
Next Review Date: July 31, 2006 Page 1 of 4 From Cigarette Butts
PROCEDURES MANUAL
CIGARETTE BUTTS
Prepared by:

Approved by:

Susan H. Johns Date
INTRODUCTION
PREPARATION
INSTRUMENTATION
Cigarette Butts:
1. Remove 1 - 2 cm of the paper from the mouth end of the cigarette butt. Cut this into smaller
pieces and place into a Spin-Ease tube.
2. To the tube add:
400 l Stain Extraction Buffer
10 l Proteinase K (20 mg/ml)
5 l 390 mM DTT
Mix and spin briefly to force the cutting into the liquid.
3. Incubate at 56EC overnight.
4. Place the cuttings in the basket of the Spin-Ease tube. Remove the stain extraction buffer out of
the material by spinning for 5 minutes at 10,000 x g (Spin-Ease). Retain the cuttings. Transfer
the liquid into a microfuge tube.
6. At the analysts discretion, the aqueous layer and interphase may be re-extracted in a new tube.
Discard the old tube containing the phenol into the appropriate waste container in the fume
hood.
7. At the analysts discretion, step 5 may be repeated using 500 l chloroform/isoamyl alcohol.
A. Place the aqueous phase from the phenol/chloroform/isoamyl or isoamyl/chloroform mixture
occurred.
conditions.
B. Add the appropriate volume of TE (30l - 100Fl) depending on anticipated DNA
-4
recovery, invert filter, vortex 15 seconds, and spin out liquid at 3500 x g for 3 min.
REPORT WORDING
See Appendix I.
REFERENCES
Next Review Date: July 31, 2006 Page 1 of 5 DNA From Envelope
Forensic Biology/DNA Procedures Manual Version 2.00277 Flaps and Stamps
PROCEDURES MANUAL
ENVELOPE FLAPS AND
STAMPS
Prepared by:

Approved by:

Susan H. Johns Date
INTRODUCTION
Refer to Safety Considerations under the DNA Isolation Methods Section
PREPARATION
Refer to Preparation under the DNA Isolation Methods Sections.
INSTRUMENTATION
Envelope Flaps and Stamps:
For analyzing envelope flaps and stamps forensic biologists and latent print examiners must work
together. As a result of the evidence being exposed to different analyses, each section must take the
following precautions to prevent the evidence from becoming compromised: forensic biologists should
handle the envelope carefully to avoid destroying latent fingerprints, and latent print examiners should
not expose the evidence to ninhydrin or physical developer prior to the removal of the DNA from the
envelope or stamp. It is best if the stamp or the glued surface of the envelop flap can be cut from the
envelope.
When the glued surface can be cut from the envelope:
1. Cut out the stamp or stamp-sized portions of the glued envelope flap. Because individuals vary
in the amount of DNA in their saliva, the total amount of glued flap required will vary as well.
Cut these pieces up into smaller pieces and incubate in 1 ml sterile distilled HO in a Spin-Ease
2
tube for 30 minutes at room temperature. This will loosen the glue. Use more than one tube if
necessary.
2. Vortex 30 seconds to separate the glued papers.
3. Place the papers in the Spin-Ease basket and spin for 5 minutes to pellet the cells.
4. Remove supernatant and discard. Do not disturb the pellet.
5. Add 500 l stain extraction buffer, 10 l proteinase K (20 Fg/Fl) and 5 l 390 mM DTT,
incubate at 56EC overnight. Transfer the liquid to a microfuge tube.
7. At the analysts discretion, the aqueous layer and interphase may be re-extracted in a new tube.
hood.
A. Place the aqueous phase from the phenol/chloroform/isoamyl or chloroform/isoamyl
mixture in a Microcon 100 tube. Follow the manufacturer's recommendations for
centrifugation speed and time or spin at 500 x g for 8 to 10 minutes. Centrifuge for
additional time if liquid remains on the filter. It may be necessary to increase speed to
1500 x g for some samples. Add 50-100 l of TE and centrifuge as before to wash
residual extraction components from the DNA. Examine the filter unit to verify that no
tearing or cracking has occurred.
extractions or high-speed centrifugation. They are not designed to withstand
these conditions.
-4
When the envelope cannot be cut up:
1. Open the envelope or remove the stamp by steaming.
2. Collect any epithelial cells which may be present by swabbing the glued surfaces with a sterile
swab moistened with water.
3. Place the swab into a Spin-Ease tube and add 500 l Stain Extraction Buffer, 10 l proteinase
K (20 Fg/Fl) and 5 l 390 mM DTT, incubate at 56EC overnight.
4. Place the cuttings in the basket of the Spin-Ease tube. Remove the stain extraction buffer
out of the material by spinning for 5 minutes at 10,000 x g (Spin-Ease). Transfer the liquid
into a microfuge tube.
in the fume hood. Vortex for approximately one minute to achieve a milky emulsion in the
tube. Spin the tube for two minutes at maximum speed. Note: Do not use microfuge
tubes provided for use with Microcon filters for organic extractions.
6. At the analysts discretion, the aqueous layer and interface may be re-extracted in a new tube.
hood.
mixture in a Microcon 100 tube. Follow the manufacturer's recommendations for
centrifugation speed and time or spin at 500 x g for 8 to 10
minutes. Centrifuge for additional time if liquid remains on the filter. It may be necessary
to increase speed to 1500 x g for some samples. Add 50-100 l of TE and centrifuge as
before to wash residual extraction components from the DNA. Examine the filter unit to
verify that no tearing or cracking has occurred.
extractions or high-speed centrifugation. They are not designed to withstand
these conditions.
-4
REPORT WORDING
See Appendix I.
REFERENCES
Next Review Date: July 31, 2006 Page 1 of 4 DNA From Hair With
Forensic Biology/DNA Procedures Manual Version 2.00277 Roots
PROCEDURES MANUAL
PROCEDURE: ISOLATION OF DNA FROM HAIR
WITH ROOTS
Prepared by:

Approved by:

Susan H. Johns Date
INTRODUCTION
Refer to Safety Considerations under the DNA Isolation Methods Section.
PREPARATION
INSTRUMENTATION
Hair with Roots:
Unmounted hairs
1. Place the hair in a microfuge tube with sterile distilled HO and shake for 1 hour at room
2
temperature. This will remove any debris adhering to the hair.
2. Rinse the hair briefly in fresh sterile distilled HO.
2
3. Cut a 2 cm segment from the hair. Extract in 500 l stain extraction buffer with 50 l 390 mM
DTT and 15 l proteinase K at 56EC overnight.
4. Vortex the hair/stain extraction buffer mixtures for 30 seconds and add an additional 50 l of
390 mM DTT and 15 l of proteinase K (20 Fg/Fl), incubate at 56EC overnight, vortex 30
seconds.
5. Spin the microfuge tube at maximum speed to sediment pigments. Transfer the
supernatant to fresh tube.
7. At the analysts discretion, the aqueous and interface layer may be re-extracted. Transfer the
aqueous phase (top layer) to a new tube. Discard the old tube containing the phenol into the
appropriate waste container in the fume hood.
occurred.
conditions.
-4
C. Incubate the sample to resolubilize the DNA for at least one hour.
Mounted hairs: There are two methods of removal of mounted hairs from a slide. Both of these
methods will be conducted in a fume hood.
Freezing Method
1. Place slide in -80C freezer for a few minutes. Pry off the cover slip.
2. Add a drop of xylene to the hair to dissolve the mounting medium.
3. Remove the hair and soak in about 10-20 ml xylene for a few minutes to remove residual
mounting medium.
4. Rinse the hair briefly in 100% ethanol to remove xylene.
5. Follow Step 1 under Unmounted Hairs.
Xylene Method
1. Soak the slide in Xylene or Xylene Substitute for several hours until the cover slip can be pried
from the slide.
2. Remove the hair and soak in about 10-20 ml Xylene for a few minutes to remove residual
mounting medium.
3. Rinse the hair briefly in 100% ethanol to remove Xylene.
4. Follow step 1 under unmounted hairs.

REPORT WORDING
See Appendix I.
REFERENCES
Next Review Date: July 31, 2006 Page 1 of 4 DNA From Non-Semen
Forensic Biology/DNA Procedures Manual Version 2.00277 Body Fluid Stains
PROCEDURES MANUAL
PROCEDURE: ISOLATION OF DNA FROM NON-
SEMEN BODY FLUID STAINS
Prepared by:

Approved by:

Susan H. Johns Date
INTRODUCTION
Observe Standard Laboratory Practices.
PREPARATION
INSTRUMENTATION
Non-Semen Body Fluid Stains:
1. Cut the sample into small pieces and place into a Spin-Ease tube. Also, process a
manipulation blank at this time.
2. To the tube add:
10 l Proteinase K (20 mg/ml)
5 l 390 mM DTT
Mix and spin briefly to force the cutting into the liquid.
3. Incubate at 56EC overnight.
4. Place the cuttings in the basket of the Spin-Ease. Remove the stain extraction buffer out of the
material by spinning for 5 minutes at 10,000 x g (Spin-Ease). Transfer the liquid into a
microfuge tube.
6. At the analysts discretion, the aqueous and interface may be re-extracted in a new tube.
hood.
Note: If the DNA pellet from a blood stain is red-brown, the heme has not been completely
eliminated from the sample. The Microcon 100 method will remove heme.
occurred.
conditions.
B. Add the appropriate volume of TE (30 Fl - 100 Fl) (depending on anticipated DNA
-4
recovery), invert filter, vortex 15 seconds, and spin out liquid.
REPORT WORDING
See Appendix I.
REFERENCES
Next Review Date: July 31, 2006 Page 1 of 4 via Differential Extraction of
Forensic Biology/DNA Procedures Manual Version 2.00277 Seminal Stains
PROCEDURES MANUAL
PROCEDURE: ISOLATION OF DNA VIA
DIFFERENTIAL EXTRACTION OF
SEMINAL STAINS
Prepared by:

Approved by:

Susan H. Johns Date
INTRODUCTION
PREPARATION
INSTRUMENTATION
Differential Extraction of Semen Stains:
1. Cut the stain into small pieces or remove the swab from the applicator stick. Place the material
into a Spin-Ease tube. Also prepare a manipulation blank.
2. To the tube add:
5 l Proteinase K (20 g/l)
Add sufficient reaction mixture to have excess liquid visible if substrate is too absorbent.
3. Mix gently and incubate two hours at 37EC.
4. Place the cuttings in the basket of the spin ease tube. Centrifuge for five minutes to spin the
extraction buffer out of the cotton or cloth and pellet the sperm cells. (Spin-Ease tubes
maximum centrifugation speed = 10,000 x g.)
5. Place the swab or fabric material into a new Spin-Ease tube. This tube must be labeled
"FRACTION 3" or "F3".
6. Carefully remove the liquid in the original tube from the cell pellet and place into a new
microfuge tube labeled "FRACTION 1" or "F1". This fraction should contain primarily the
epithelial cell lysate.
7. The remaining cell pellet in the original tube should contain primarily the sperm cells from the
swab. This tube is labeled "FRACTION 2" or "F2".
8. Rinse the pellet (F2) with 1 ml TNE, vortex and centrifuge at maximum speed for 10 minutes.
Remove the TNE buffer and repeat this process four times. Be careful not to disturb the sperm
cell pellet. (Optional: After last wash 1 l of the sperm pellet may be removed for KPIC).
9. To the F2 and F3 tubes add a reaction mixture containing:
40 l 390 mM DTT
10 l Proteinase K (20 Fg/Fl)
Add sufficient reaction mixture to have excess liquid visible if the substrate is too
absorbent. Mix and incubate at 37EC for two hours.
10. Place the cuttings in the basket of the Spin-Ease tube. Remove the stain extraction buffer out of
the material by spinning for 5 minutes at 10,000 x g (Spin-Ease). Transfer the liquid into a
microfuge tube.
12. At the analysts discretion, the aqueous and interface layer may be re-extracted in a new tube.
hood.
13. Optional: At analyst discretion steps 11 and 12 may be repeated using 500 l of
chloroform/isoamyl alcohol.
alcohol into a Microcon 100 tube. Follow the manufacturer's recommendations for
centrifugation speed and time or spin at 500 x g for 8 to 10 minutes. Centrifuge for
additional time if liquid remains on the filter. It may be necessary to increase speed to
1500 x g for some samples. Add 50-100 l of TE and centrifuge as before to wash
residual extraction components from the DNA. Examine the filter unit to verify that no
tearing or cracking has occurred.
conditions.
B. Add the appropriate volume of TE (30Fl - 100Fl) (depending on anticipated DNA
-4
recovery), invert filter, vortex 15 seconds, and spin out liquid.
REPORT WORDING
See Appendix I.
REFERENCES
Accepted Date: March 16, 2001 FB-IIIA-2 Method: DNA Isolation
Next Review Date: July 31, 1006 Page 1 of 5
PROCEDURES MANUAL
LIQUID BLOOD
Prepared by:

Approved by:

Susan H. Johns Date
Accepted Date: March 16, 2001 FB-IIIA-2 Procedure: Isolation of
Next Review Date: July 31, 2006 Page 2 of 5 DNA from Liquid Blood
INTRODUCTION
Refer to Safety Considerations under the DNA Isolation Methods section.
PREPARATION
Refer to Preparation under the DNA Isolation Methods section.
The following Puregene solutions can be stored at room temperature and contain the following
reagents.
RBC Lysis Solution
Ammonium chloride, ethylenediaminetetraacetic acid, sodium bicarbonate
Cell Lysis Solution
Tris[hydroxymethyl]aminomethane ethylenediaminetetraacetic acid, and sodium dodecyl sulfate
Protein Precipitate Solution
Ammonium acetate
Hydration Solution
Tris[hydroxymethyl]aminomethane and ethylenediaminetetraacetic acid
INSTRUMENTATION
Liquid Blood:
1. Collect liquid blood in an EDTA vacutainer tube. The tube contents must be mixed well before
aliquoting. Properly store blood as soon as possible after receipt. For short term storage,
freeze a 300 l aliquot of offender blood in a 1.5 ml microcentrifuge tube at -80C. For long
term storage, spot and freeze a stain sample of offender blood on Schleicher & Schuell filter
paper and a 750 l aliquot of blood.
For Puregene method, go to Puregene Isolation Method for Whole Blood,
Step 9.
2. Add 700 l 1X SSC to the thawed blood and mix well by hand or with a touch to the
vortexer. Spin one minute in microfuge at full speed.
3. Remove and discard the supernatant into a suitable disinfectant (e.g., 10% bleach solution).
4. Add 1.0 ml 1X SSC, vortex, centrifuge one minute at full speed and remove all supernatant
fluid. Discard into 10% bleach solution. Do not remove the pellet.
5. To the pellet add: 375 l 0.2 M Sodium Acetate;
12.5 l 20% SDS
5 l Proteinase K (20mg/ml H O)
2
Vortex and incubate at 56 C for one hour to overnight.
o
6. Add 120 l phenol/chloroform/isoamyl alcohol (be sure to go down to the bottom layer to get
the 120 l); vortex until a milky white emulsion forms. This step must be carried out in the
fume hood! Use extreme caution! Phenol burns! Note: Do not use microfuge tubes
7. Spin two minutes at maximum speed for the tube type. At this point, there should be an
aqueous reddish-brown layer on the top.
8. In the hood, carefully remove the top aqueous layer and place in a new 1.5 ml tube. Do not
remove the layer of denatured protein that collects at the interface. (The interface is
quite stringy. A plastic transfer pipet may aid in the removal of the aqueous layer.) The new
tube now contains the DNA; the old tube containing the phenol and denatured protein must
be discarded into the phenol waste container.
Note: At the analysts discretion, the interface may be re-extracted. Transfer the aqueous
phase (top layer) to a new tube and repeat step 8. Discard the old tube containing
phenol and into a waste container in the hood.
For Microcon method, go to Extraction of Non-Semen Body Fluid Stains,
Step 17; for ethanol method, go to Step 14.
Puregene Isolation Method for Whole Blood
9. Add 900 Fl RBC Lysis Solution to 300 Fl of whole blood. Invert to mix and let stand
five minutes at room temperature. Repeat mixing and incubation steps.
10. Centrifuge for 30 seconds at 13,000-16,000x g. Remove most of the supernatant with a
micropipet leaving behind the visible white pellet and 10-20 Fl of residual liquid.
11. Vortex the tube vigorously to resuspend the white blood cells in the residual supernatant. This
step greatly facilitates cell lysis.
12. Add 300 Fl Cell Lysis Solution and 2 Fl Proteinase K (20 mg/ml) to the tube. Pipet up and
down to lyse the cells. Incubate at 56EC for 30 minutes to one hour.
13. Cool samples to room temperature or below.
14. Add 100 Fl Protein Precipitation Solution to the cell lysate and vortex vigorously at high
speed for 20 seconds.
15. Centrifuge at 13,000-16,000 x g for three minutes. The precipitated proteins will form a tight
dark brown pellet.
16. Pour the supernatant containing the DNA into a clean 1.5 ml tube. Discard the precipitated
protein pellet.
17. Add 300 Fl 100% isopropanol and mix the sample by inverting gently 50 times or until the
white threads of DNA form a visible clump.
18. Centrifuge at 13,000-16,000 x g for one minute; DNA will be visible as a small white pellet.
19. Pour off supernatant and drain tubes on clean absorbent paper.
20. Add 300 Fl 70% ethanol. Invert the tube several times to wash the DNA pellet.
21. Centrifuge at 13,000-16,000 x g for one minute. Carefully pour off supernatant. Centrifuge
quickly and carefully pipet off remaining ethanol.
22. Drain tube on clean absorbent paper and allow sample to air dry 15 minutes.
23. Add 250 Fl DNA Hydration Solution or TE and allow DNA to rehydrate/resolubilize
overnight at 56EC.
REPORT WORDING
See Appendix I.
REFERENCES
Accepted Date: May 14, 2002
FB-App VI (DNA)
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PROCEDURES MANUAL

QUALITY ASSURANCE

Prepared by:

Approved by:

FB-App VI (DNA)
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APPENDIX VI
Table of Contents
V. Evidence Control
VI. Validations
X. Audits
XI. Reports
XII. Reviews
XIII. Safety
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Overall goals:
Objectives:
product;
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Competency Testing
Proficiency Testing
Corrective Action
FB-App VI (DNA)
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college course work in the following areas prior to beginning DNA supervised casework.
2. Genetics
3. Biochemistry
evaluated.
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C. Experience
D. Certification
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cases.
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preparation.
interpretation.
their laboratory.
laboratory.
additional needs.
standards.
FB-App VI (DNA)
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Procedures Manual.
proficiency tested.
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PCR set up.
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to operation.
from the PCR room.
laboratory.
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Evidence Control
Blood Evidence
Evidence Packaging
Case Tracking
Clean Technique
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and location.
and location.
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V. Stain Storage
A. Blood Standards
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Validations
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1997.
1999;44:133-166.
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1997.
5,552,028.
5,552,028.
39:1254-1269.
TM
1996.
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I. Procedures.
II. Reagents.
the Reagent Log.
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D. Expiration Dates
2. ABAcard kits
3. Nylon membrane
7. Formamide
manual.
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appropriate.
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A. Blood Standards
possible.
B. Dry Blood
C. Semen
secretion.
E. Saliva
F. Urine
FB-App VI (DNA)
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run.

logbook.
2. Semen ID
3. Lugols Stain
test is used.
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test is used.
6. Urea Nitrogen
7. Creatinine Test
properly.
Manual.
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calculation.
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report.
laboratory report.
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2. Procedure:
the manufacturer.
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DATE LOG# NEGATIVE
10NG
POSITIVE
4NG
POSITIVE ANALYST
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Procedures Manual.
2. Procedure:
surrounding wells.
incubation period.
incubation period.
FB-App VI (DNA)
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manufacturer.
Anti-Serum Brand __________________________
Expiration Date __________________________
Date QCd __________________________
Analyst __________________________
ANTI____________________
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2. Procedure:

F. Photograph gel

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Cathodal Origin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Middle Origin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Include Photograph Along With This Sheet
Analyst_________ Date_________
Lot# ____________
Date Received__________________
Date of Dilution if Same
Lot#_________________________
Notes:
QCd?________________
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1. Purpose:
2. Procedure:
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1 2 3 4 5 6
A
B
C
D
E
F
G
H
manufacturer?__________________________________
SAMPLE ID
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2. Procedure:
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CHARGED MEMBRANE
(For Slot Blot)
Date QCd________________________ QCd by______________________
Standards
(ng)
Calibrators
NOTES:
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2. Procedures:
of 20 :L ddi H
2
O.
manufacturer.
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Analyst__________________________ QC Date ___________________
Allelic Ladder ___________ 9947A#_________________

D3S1358
D16S539
TH01
TPOX
CSF1PO
Amelogenin
D7S820
NOTES:
FB-App VI (DNA)
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Analyst__________________________ QC Date ___________________
Allelic Ladder ___________ 9947A#_________________

D3S1358
vWA
FGA
Amelogenin
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
NOTES:
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2. Procedure:
manufacturer.
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Analyst__________________________ QC Date ___________________
Expiration Date______________________
well defined
75
100
139
150
160
200
300
340
350
400
450
490
500
NOTES:
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Annual System Verification
a secondary standard that is traceable to the NIST SRM. The documentation on the traceability of the
secondary standard to the NIST SRM is located at the ISP Indexing Laboratory.
Procedure:
B. Amplify using ISP protocol for appropriate loci.
Assess Results: Compare the results to the known results for the standard. Any discrepancies will be
reanalyzed and resolved.
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A. Matrix
D. Sensitivity
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1. Purpose:
2. Procedure:
locus.
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ABI 310 _____________________________________ Date_________________________
Analyst ______________________________________
1
2
3
4
5
6
7
8
9
10
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A. Matrix:
B. Sensitivity:
Supervisor
Matrix Installation
Sensitivity
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Quality Assurance
I. Calibrations:
1. Procedure
2. Frequency
3. Results
4. Course of action
II. Maintenance
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IV. Frequency:
V. Results:
repairs.
VII. Maintenance
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standard.
III. Maintenance
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Oven Calibrations
I. Calibration
A. Procedure
B. Frequency
C. Results
D. Course of Action
II. Maintenance
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III. Maintenance
for repairs.
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pH Meter
desired pH).
III. Maintenance
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III. Maintenance
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Microscopes
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1. Calibrations:
serviced.
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III. Maintenance
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NIST Thermometer and NIST Calibrated Thermometer Calibration
A. Calibrate the NIST thermometer and NIST calibrated thermometer according to the protocol
provided by the manufacturer.
B. Calibrate annually.
A. If the thermometer reading is +/- 2 degrees from the manufacturers recommended reading, the
NIST thermometer will not be used for any further calibrations.
IV. Maintenance
None
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A. Prepare an ice bath and place the NIST thermometer or NIST calibrated thermometer at an
appropriate depth.
B. Place the thermometer being tested at the same depth as the NIST thermometer or NIST
calibrated thermometer and read the temperature.
Celsius.)
If the thermometer is greater than +/- 2 degrees from the NIST thermometer or NIST calibrated
thermometer, it will not be used for any further calibrations.
IV. Maintenance
None
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I. Procedure
C).
use the water bath.
III. Maintenance
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Proficiency Testing
following:
Competency Tests
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Audits
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Reports/Case Files
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NUMBERED PAGES:
NON-NUMBERED PAGES:
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Reviews
documentation of the review but is not part of the case file.
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generated.
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Safety

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FOREWORD

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