FORENSIC BIOLOGY,

SEROLOGY & DNA

Satyam Singh
2K11/BT/025
Delhi Technological University
Delhi

Submitted to:-
Dr Shyam Bihari Upadhayaya
Director,
Forensic Science Laboratory, U.P.
Lucknow

1

ACKNOWLEDGEMENT

I would like to express my gratitude to all those who gave me the
possibility to complete this project report. I want to thank Dr Shyam Bihari
Upadhayaya for giving me permission to commence training and to use
departmental data. I have furthermore to thank Dr Suresh Chandra Deputy
Director (Division of Biology), and Dr Archana Tripathi (Deputy Director,
Division of Serology & DNA) who gave and confirmed this permission and
encouraged me to go ahead with my training. I am deeply indebted to my
supervisor Dr F. A. Khan, Dr Farhat Shaheen, Mr S. N. Yadav, from Division of
Biology and from Division of Serology & DNA whose help, stimulating
suggestions and encouragement helped me in all the time of training.









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Table of contents
Acknowledgement
Table of contents
Introduction 3
Case Opening 4
Exhibits 5
Hair 5
Fibres 8
Bones 9
Blood stains 10
Benzidine test 11
Seminal stains 13
Saliva stains 18
Diatom 20
Blood 21
DNA 24









3

Introduction
Forensic science is the scientific method of gathering and examining information
about the past. This is especially important in law enforcement where forensics is done in
relation to criminal or civil law, but forensics are also carried out in other fields, such as
astronomy, archaeology, biology and geology to investigate ancient times.
The State Forensic Science Laboratory (SFSL), Lucknow is a wing of the Uttar Pradesh
Police, which fulfils the forensic requirements in the state. There are other SFSLs in Uttar
Pradesh as well but being in the state capital SFSL Lucknow has its own importance.
At present this laboratory is being headed by Dr Shyam Bihari Upadhyaya.






















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Case opening
Local police investigating the crime collects sample (as per the case) from the crime
scene and after sealing it sends the bundles to the forensic science laboratory for further
analysis.
The queries are mentioned by the investigating authorities in the report.
The common queries mentioned are:
1) Are the stains found on the exhibits are those of blood?
2) To whom the hair found on the crime scene belong to?
3) If the found weapon on the crime scene used for murder (in case of a murder case)?
4) If the blood found on the crime scene is of human origin?
5) Does the given viscera contain any type of poison or toxic substance?
In case opening we were trained to collect samples of the cases, examine and analyse
samples with techniques and expertise and reach to a conclusion for question asked about
sample.


















5

Blood Stains
The most important biological stains which require identification and
characterization in the investigation of crimes is immense value in the reconstruction
of criminal process involving injury and also in connecting a criminal with the crime.
When presence of blood is suspected the material should be carefully examined
in presence of light. If necessary, a magnifying lens should be used. Occasionally
examination may be required under ultra-violet light. Blood stains appear as dark
patches under ultra-violet light. The suspected areas should be demarcated and if
possible be photographed. The following particulars should be noted about the
stains:
i. Locationthe exact location of stain in the garments, persons or
other places should be noted.
ii. Shapevarious types of shapes may be found depending on the
conditions. streaks are caused by venous haemorrhage; a large stain of
irregular shape indicator massive haemorrhage; circular stains are found
when blood falls somewhat perpendicularly on a horizontal surface from a
small heightcrenation appear in the periphery of circular stains when the
height increases; smears are found when blood is wiped off; splashes
sometimes like note of exclamations are found when there is projection of
arterial blood or when a pool of blood is trampled on or when a bloody
weapon is quickly pulled out; club like or oval stains may be found when
blood falls on a surface obliquelythe more oblique the falls the more
elongated are the stains. Apart from these bloody finger prints, palm prints
or foot prints may be found.
iii. ColourStudy of the colour may provide useful informations
regarding the age of blood stains. The significance of this study is that it helps
in determining the age of blood stains.
iv. AmountEstimation of the amount of blood involved may also be
useful helping in quantitative determination of blood.
Phenolphthalein Test
This test is also known as Kastle-Meyer Test. It was first described in 1903. Chemical
indicator Phenolphthalein is used to detect the possible presence of hemoglobin.it relies on
peroxidase like activity hemoglobin in blood to catalyse the oxidation of phenolphthalein
(the colourless reduced form of phenolphthalein) into phenolphthalein, which is visible as
bright pink colour.
Chemicals Required
Ethanol
Phenolphthalein
3% H
2
O
2
6

Procedure
Standard preparation
Standard includes a known blood stain(positive control) and a known blood-
free sample(negative control). These controls are tested prior to analysis on a daily basis.
Testing of stain
The suspected stain is rubbed with a folded piece of filter paper or a cotton
swab.
The following chemicals are added onto the filter paper or cotton swab in
order
One drop ethanol
One drop phenolphthalein
One drop of 3% H
2
O
2

A positive reaction is indicated by the development of a pink colour inn 5
seconds of adding H
2
O
2.
Reactions occurring after 5 seconds of the addition of hydrogen peroxide are
inconclusive.
Important points to remember
This test is performed on any stain that visually appear to be blood, even if blood
analysis is not requested.
If a sample is weak, cut a small portion of the sample and place this cutting on filter
paper and add the chemicals directly to the sample.









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Benzidine Test
The test depends on the presence of an enzyme peroxidase in the haemoglobin.
Peroxidase acts as the catalytic agent to celebrate the liberation of oxygen from hydrogen
peroxide present in the reagent and thereby causing oxidation of benzidine to give rise to a
pale blue colour. In presence of plant peroxidase this test may be positive which however
can be eliminated by heating or on standing. Heating at a temperature of 100C for 5
minutes would inactivate plant peroxidase. Reactions due to plant peroxidase usually
disappears on standing for about a week.
Chemical oxidants (oxides of iron, potassium permanganate etc.) & chemical
catalysts like salts of copper and nickel especially when those are in strong solution may
give rise to positive benzidine test. Blue colouration due to chemical oxidants can be
eliminated by use of two solution tests where instead of using a mixture of benzidine and
hydrogen peroxide, benzidine is first added and then followed by hydrogen peroxide if no
colouration develops.
The positive benzidine reaction that occurs in connection with the chemical catalysts
is different in its character from that produced by blood. The faint blue colour is usually
produced before the addition of hydrogen peroxide. After addition of hydrogen peroxide
the faint blue colour disappears followed by very slow development of deep blue
colouration. There is no evidence to indicate that other body fluids could give rise to
positive benzidine test independently.
Procedure:
There have been innumerable variations of reagents and techniques that are
followed in performing benzidine test.
The reagent usually consists of mixture of benzidine, glacial acetic acid and hydrogen
peroxide.
Solution I
Benzidine (Reagent quality) 1.5 gm
Glacial acetic acid (Pure) 13 ml
Re-distilled water 57 ml
Solution II
Hydrogen peroxide 20 volume
Preparation of benzidine solution 13 ml of pure glacial acetic acid is taken in a suitable
conical flask and warmed on a water bath at 50C. 1.5 gm of benzidine is then added into
the flask, and after benzidine has dissolved, the flask is removed from the water bath and 57
8

ml of re-distilled water is added. The solution thus prepared may be stored in an amber
coloured reagent bottle.
Procedure of testing the stain to be tested is rubbed lightly with one end of a dry filter
paper. A drop of benzidine solution prepared as above is applied and if no colour develops
within 15 to 30 seconds hydrogen peroxide is dropped in the same area. Positive reaction is
indicated by immediate development of deep blue colour. There may be situations where
instead of using a filter paper it may be convenient to collect the material with a small
cotton swab (as in case of finger nails) or by scraping (as in case of hard irregular objects) or
by cutting a small piece (as in case of delicate fabrics). This test may also be applied to
screen the presence of blood materials where the stain is not apparent e.g. when the blood
is suspected to be present in powdered earth, dust or debris.












Exhibits
These are the evidences sent to any forensic science laboratory for the identification
and comparison purpose.
Hair
It is a filamentous, biological material that originates from follicles found in dermis. It
is made up of protein (keratin) by the process of keratinisation. Hair is one of the defining
characteristics of mammals, but also found in other animals. It is found all over the body.
The human body apart from the areas of globular skin is covered in follicle which produce
9

thick, terminal and fine vellus hair. It is black, brown and grey in colour.

Human hairs are unique in that its medullary index (the fraction of the hair shaft's
diameter that the medulla occupies) is very small: generally less than 1/3. Other species will
have ratios much larger, usually at least 1/2. The medulla's presence in human hair and the
patterns of which differ both from person to person and from strand to strand on a single
person's head. One hair may have an absent medulla while another from the same person
may have a complete or fragmented medulla. For this reason, medulla patterns are not very
useful as forensic evidence and are typically only used for determining the species of the
subject. The medulla contains large amounts of mitochondrial (as opposed to cytoplasmic)
DNA. Mitochondrial DNA is passed from the mother to her offspring. As such, mDNA is of
forensic value.

Morphological structure
Morphologically a hair has following parts:
a. Root
b. Shaft, and;
c. Apex
Anatomical structure
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Anatomically hair consists of following:
a. Cuticle
The hair cuticle is the outermost part of the hair shaft. It is a hard
shingle-like layer of overlapping cells, some five to twelve deep. It is formed
from dead cells which form scales that gives the hair shaft strength and do
the best job of providing protection for it.
b. Cortex
The cortex of the hair shaft is located between the hair cuticle and
medulla and is the thickest hair layer. It also contains most of the hair's
pigment, giving the hair its colour.
c. Medulla
The medulla is the innermost layer of the hair shaft. This nearly
invisible layer serves as the pith or marrow of the hair, it is primarily an air
space that is more prominent in pigmented (grey or white) hair.

Classification of hair
Hair can be classified in mainly two domains:
a. Human hair
b. Animal hair
Determination of origin of hair
Following characteristics can be used to determine the origin of hair:
S. No. Characteristic
feature
Human hair Animal hair
1. Cuticle Imbricated, similar
along shaft from root
tip
Often showing
variations in structure
along shaft
2. Cortex Occupying most of the
width of shaft greater
than medulla
Usually less than
width of medulla
3. Medulla Less than 1/3
rd
width of
shaft, amorphous and
discontinuous
Greater than 1/3
rd
width of shaft,
continuous
4. Texture Smooth Coarse
5. Pigment
distribution
Even toward cuticle Central towards
medulla
Determination of age
Age can be determined from hair on the basis of following
characteristics:
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Analysis of hair:
1. Physical examination
Spread the exhibit on clean surface under proper illumination.
With hand magnifier carefully locate any loose hair/fibre and
collect by picking up using forceps or may be transferred to
adhesive tape.



2. Microscopic examination
Make a temporary mount of the hair sample on a clean slide with
distilled water/glycerine. Cover with coverslip. Examine under
microscope from one end to other for general appearance
length, colour, presence and absence of root, tip & shaft
characteristics, treatment by dye or bleached etc.
Fibres
Fibres of various type are involved in crimes as physical evidence. If a piece of
a worn garment is found on the crime scene its physical fit and textile resemblance
with the remnant of the garment seized from any accused, is very valuable evidence.
Types:
1. Natural fibres: silk, cotton, wool etc.
2. Synthetic fibres: nylon, polyester, acrylic etc.
Examination of fibres:
1. Microscopic examination(temporary mount)
Microscopy of fibres is carried out using temporary preparation of the fibres in
distilled water or glycerine or chloral hydrate.
i. Place small quantity of fibres on a slide:
ii. Add a drop of distilled water or glycerine or chloral hydrate and place a
coverslip over it
iii. Pass the slide over a micro burner flame until the liquid boils.
iv. Let it cool down to room temperature.
S. No. Characteristic
feature
Child/Young Adult
1. Smoothness Fine Coarse
2. Pigmentation Less More
3. Hair root Dissolves in alc.
KOH
Does not dissolves in alc.
KOH
4. Dye Generally absent Often present
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v. Examine under microscope. A study of cell wall & lumen, crystals, fibre
and dislocations & cross markings, cells from tissue other than
sclerenchyma will be identification of plant fibres.
2. Solubility tests
Solubility of fibres in different regions like 80% sulphuric acid, conc. HCl, acetone,
phenol & cuprammonium hydroxide is checked for identification purposes.












Bones
When bones or skeletons are found, they are taken to a forensic laboratory
for examination. A forensic expert examines the bones, to possibly deduce the
gender, age height, race as well as medical history and manner of death.
The first step during the examination of bones, is to find out whether the
bones are of human or animal. Once this has been determined, the next step is
finding the age of the bones by noting the growth and decay that has occurred in the
bones.
Examination of bones
Growth rate
Teeth that have or not have grown can also reveal the age of skeleton as
young children will not have lost their milk teeth and at the age of 18 , wisdom teeth
first appear. During the teenage years, bones become thicker & larger, and fuse
together in a process known as ossification.
At the age of 17 in males & 20 in females, the lower bone plate & the radius
fuse together, and soon after, the upper bone plate & the radius fuse together. The
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bone in the body that finishes growing last is collarbone, which ceases growth at 28
years. In the bones of elderly, degeneration begins to occur.
Gender
The skull has three points in determining gender. These are:
I. Ridges located above the eyes,
II. Bone situated just below the ear and
III. Occiput, the bone located at lower back of the skull.
The latter two bones are muscle attachment sites, all of which are more
prominent in men, including greater strength. The difference in hips is very obvious,
as a mans hip are narrower and a womans hips are wider, being built for child
bearing.
Height
Determining the height of a skeleton involves reassembling the skeleton and
measuring the length of significant bones. The human height measures roughly 2,
2/3
rd
the length of femur, though it also depends on the race and gender of the
skeleton.
Bone defects
Disease, injury & birth defects are also revealed in the bones. Birth defects
such as bifida, some infectious diseases, poor diet & cancer can all be damaging to
bones. The bones of a deceased person break differently compared to the bones of
an alive person and healing at the edge of a fracture indicates injuries during life.










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Seminal Stains
Seminal stain is an important biological which requires identification and
characterisation in the investigation of sexual offence like rape, sodomy, bestiality etc. the
stains are usually found on the clothing of the victim and the suspects especially on their
undergarments. They may be found on the bed-sheets, on pillow cases, on carpets, on floor
or on leaves, grass etc. where the offence was committed. They may also be found on the
person of the victim and the suspect especially on their perineum, thighs and pubic hairs.
Vaginal and anal swabs and smears of the suspects are often required to be examined in the
laboratory for the detection and characterisation of the semen.
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Physical Examination
On naked eye examination in ordinary light dry seminal stains appear as greyish white or
yellowish grey stains having irregular outlines. Stains on black clothing appear as whitish
stains. The stained areas on fabrics are generally stiff to feel. On examination under
ultraviolet light the stains may show a bluish white fluorescence.
The use of ultraviolet examination in screening of suspected seminal stains is not very
satisfactory because of the following reasons:
i. all seminal stains may not fluoresce;
ii. stains other than semen may also fluoresce;
iii. certain clothing may have very bright fluorescence which may mask the fluorescence
due to semen;
iv. some of the modern detergents used for the washing of clothing strongly fluoresce
under ultraviolet light; the fluoresce due to the residue of such washing materials
may make the fluorescence of the stain invisible.
Chemical examination
1. Acid phosphatase testthe test is based on the presence of an enzyme acid
phosphatase in semen. The enzyme is present in semen in much higher percentage
in any other body fluids. If the test is found positive it is suggestive of the presence
of semen. As such this test can be used as a very good screening tests for the
examination of seminal stains.
Reagents
Sodium Chloride 230 gm
Glacial acetic acid 5 ml
Anhydrous sodium acetate 12 gm Or Trihydric sodium acetate 20 gm
Brentamine fast blue B salt 0.5 gm
Calcium -naphthyl phosphate 0.5 gm
1% sol. of Teepol (or Aerosol) 10 ml
Distilled water 900 ml

16

A buffer is made by dissolving sodium chloride, glacial acetic acid and sodium
acetate in distilled water. Brentamine fast blue is dissolved in small amount of this
buffer. A fine suspension of calcium -naphthyl phosphate is prepared by grinding
the same in Teepol or Aerosol with pestle and mortar. The resulting suspension is
added to the remaining amount of buffer. The Brentamine solution is then added to
the solution of calcium -naphthyl phosphate and the mixture is filtered and stored
in an amber coloured bottle. The reagent lasts about 6 weeks when stored in
refrigerator.
The reagent may also be prepared by using -naphthyl phosphoric acid
instead of calcium -naphthyl phosphate in the following way:

Preparation of -naphthyl phosphoric acid50 g -naphthol and 10 g calcium
chloride are dried over P
2
O
5
are dried over several days. They are then refluxed
under anhydrous conditions with 225 g (135 ml) POCl
3
(fresh) for 24 hours. Pressure
is reduced to 15 mm and fraction distilling at 180C is collected. The compound is
then hydrolysed in one litre of distilled water at room temperature for 24 hours with
occasional stirring. The free acid is extracted from aqueous solution by one litre of
ether; 200 ml of 10% sodium bicarbonate are shaken with this ether solution, the
bicarbonate layer is run off, acidified and re-extracted with ether. Evaporation of this
ether after drying with anhydrous Na
2
SO
4
leaves the pure oil. This is then made into
a solution with acetone in the concentration of 1g in 5 ml and as such kept
indefinitely in refrigerator.
The acid phosphatase reagent is made by evaporating off the acetone from
10 ml of this solution with a hair dryer (all the acetone must be evaporated), adding,
in dilute aqueous solution in order, 10 ml glacial acetic acid, 40 gm of sodium acetate
trihydrate (or 23.4 gm of Brentamine fast Blue B salt, and making to 2 litres with
distilled water. The final solution should be clear amber colour.

TestA sheet of filter paper is thoroughly moistened with normal saline and is
pressed over the area to be examined for a couple of minutes. The paper is lifted off
and the reagent is sprayed to it. In about to 1 minute the areas corresponding to
seminal stains appear as purple red.

Mechanism of the testthe enzyme phosphatase of semen hydrolyses the calcium
-naphthyl phosphate/-naphthyl phosphoric acid liberating -naphthol. This
couples with the dye forming material (Brentamine fast blue) to give the purple red
dyestuff.
Microscopic examination
Microscopic detection of spermatozoa is a confirmatory evidence for the presence of
semen in a suspected stain.
Morphology of human spermatozoahuman spermatozoon consists of an oval and
flattened head (pear shaped when seen in profile), with a long slender tail connected with
17

the head by means of a very shot neck. The total length varies from 45-55 microns, the head
being about 5 microns in greater diameter and 3.5 microns in lesser diameter and the tail
being about 40 microns in length.
A few small cuttings from the stained area of the fabric is moistened with a few drops of
0.01N HCl and kept for hr. Individual threads are then teased apart with the aid of
stainless steel dissecting needles. A drop or two of the liquid is then squeezed out on a
microscopic slide and a thin smear is prepared thereon. The slide is then allowed to dry and
thereafter it is stained as follows.
i. Haematoxylin-Eosin
a) Haematoxylin stain:
Haematin 0.4 gm
Potash alum 5 gm
Glycerine 30 ml
Distilled water 70 ml
Haematin is first rubbed up in little glycerine. Potash alum is then dissolved in
distilled water and the remaining amount of glycerine is added to it. The stain thus prepared
is very stable.
b) Eosin stain:
Eosin (water soluble yellowish) 1 gm
Distilled water 100 ml
The slide is stained with
(a) Haematoxylene for 10 to 15 minutes and then washed in running water. A few
drops of dilute solution of ammonia (3 to 5 drops in a litre of distilled water) are
poured on the slide and then rinsed. Counterstaining is done with Eosin stain for
one minute. The slide is then washed, dried and examined under oil immersion.
By this stain the posterior part of the head stains deep purple and the anterior
part pink and this differential staining is very characteristic. Nuclei of epithelial
cells as well as those of leucocytes are also stained deep purple with this stain.

ii. Malachite green-Eosin
(a) Malachite green 1gm
Distilled water 100 ml
(b) Eosin stain : as before
The slide is first stained with Malachite green stain for 10 to 15 minutes followed by
through washing with distilled water. The slide is counterstained with Eosin stain for one
minute. Stained in this manner posterior part of the head stains purple and the anterior part
pink; neck and tail of the spermatozoa assume green colour. With this stain epithelial cells
take uniform pink colour and the leucocytes remain almost unstained.
iii. Aniline Blue Eosin B solution.
Aniline blue aqueous 1 gm
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Eosin B 0.5 gm
Phenol 1% aqueous 10 ml
Distilled water 30 ml
Smears are prepared as usual and fixed with a mixture of equal volumes of absolute
alcohol and ether for about 3 minutes and then allowed to dry in air. Fixed smear is stained
with its compound Aniline blueEosin B solution for 5 minutes at 40C to 50C. Stained
slide is washed carefully with distilled water. Slide is ready for careful for microscopic
examination as soon as it is dried up.
The technique produces good differentiation and preserves the cytological structure
with its details undisturbed and easily recognisable. Anterior part of the head is stained
bright pink. The neck and tail are stained dark blue and blue and blue respectively.
iv. Methylene BlueEosin :
(a) Methylene Blue stain
Saturated solution of Methylene blue in absolute alcohol 30 ml
Distilled water 69 ml
1/10,000(N) solution of KOH 1 ml
(b) Eosin Stain :
Eosin (Water soluble-yellowish) 1 gm
Distilled water 100 ml
The smear is fixed by slight heating. It is then stained with Methylene blue stain for
15 minutes. The slide is washed in running water and then counterstained with eosin stain
for 5 minutes. Thereafter it is again washed in running water and dried in air. The slide is
now ready for microscopic examination. With this stain posterior on-third of the head
appears unstained or faintly stained blue; neck and tail also assume pink colour.
Identification of an intact spermatozoon with head and tail is no doubt the most
reliable proof of the presence of semen. The detection of a number of heads taking
characteristic differential stains is also perfectly reliable.
Serological examination
Serological examination may be used for
(i) The detection of semen
(ii) Identification of species, and
(iii) Identification of group specific substances in seminal stains.

i) Serological Detection of semen
Agar gel precipitation test already described in connection with the determination of
origin of blood stains can also be used for the detection of semen in suspected stains.
The anti-human semen serum may be used for this purpose. The test is not absolutely
specific for semen, but in cases of aspermia (complete lack of semen) the use of anti-
human semen serum is recommended in addition to various chemical tests.
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ii) Identification of species
The precipitin tests described for the identification of species of blood may also be
successfully used for the identification of species of semen. The same anti-human sera
which are used for the determination of origin of blood may also be used here.
iii) Identification of group specific substances
Group specific substances like A, B and H can be determined in seminal stains
derived from secretor individuals by using absorption inhibition technique precisely in
the same manner as described for blood stains. The absorption elution as well as the
mixed agglutination techniques recommended for ABO grouping of dried blood stains
may also be used for this purpose. Absence of any reaction against anti-A, anti-B and
anti-H indicates that either the individual concerned is a non-secretor or the stains have
deteriorated to such an extent that the group specific substances have become
undetectable. In forensic practice the seminal stains are often found contaminated with
vaginal secretion. As such, the interpretation of the results of grouping of seminal stains
should always be made with due caution.
Age of seminal stain
An idea about the approximate age of seminal stains can be made on the basis of the
following factors:
1. Surface character of the stain
(i) Stains on unabsorbent fabric-examined under low power
stereomicroscope:
(a) Recent stains appear shining translucent and structure less ;
(b) Stains of few days old appear whitish and slightly granular ;
(c) Stains of about a week old look like fine sugary deposit ;
(d) Stains which are more than a month old look greyish yellow.
(ii) Stains on absorbent fabric examined under low power stereomicroscope:
(a) Recent stains appear as colourless with a slight greyish tinge at the
edge ;
(b) Stains of few days old appear greyish yellow ;
(c) Stains which are more than month old look somewhat brownish.
2. Structural change in spermatozoa;
3. Spreading of chlorides.
Saliva Stains
Stains of saliva sometimes encountered in the investigation of criminal cases and it
becomes necessary to detect such stains and characterise them as far as possible.
For the detection of saliva a number of different tests can be applied. Human saliva
contains a fairly large amount of ptyalin, an enzyme responsible for the conversion of starch
to dextrines and maltose. Ptyalin is also present in plant materials as well as in saliva of
20

certain other animals like pig, rodent etc. Saliva stain can be identified satisfactorily by
detecting its ptyalin content.
Chemical Examinations
Methoda piece of filter paper moistened with distilled water is pressed over the
adjacent unstained area for about 5 minutes. Thereafter the filter paper is sprayed with 1%
solution of soluble starch and is then incubated at 37C for 30 minutes keeping the paper in
a moist chamber. The filter paper is now sprayed with Lugols iodine. Presence of saliva
(Ptyalin) will be indicated by development of light brown colour in the portion of filter paper
corresponding to suspected stained area. This is because the ptyalin transferred from the
stain has hydrolysed the starch. The remaining portion of the filter paper will turn blue due
to reaction between starch and iodine. The test is fairly sensitive and reliable.
Microscopic examination
A smear is prepared with the deposits obtained after centrifuging a normal saline
extract of the suspected stain. The smear is then stained with methylene blue and eosin and
examined under microscope. If the stain is that of saliva the smear may be found to contain
the following:
(i) Large buccal epithelial cells with well-defined nuclei;
(ii) Salivary corpuscles;
(iii) Leucocytes;
(iv) Fission fungi;
(v) Bacteria;
(vi) Fat drops.
Grouping of saliva stains
Saliva from secretor individuals contains large amount of blood group specific
substances (ABH). Traces of blood groups specific substances are present also in the saliva
of non-secretor individuals. The concentration of ABH substances in saliva of the secretor
individuals is about four times to these present in the blood cells. As such, A, B and H
antigens in saliva stains are readily detectable. The absorption inhibition technique or one
of the direct methods such as mixed agglutination or absorption elution may be used
conveniently for the purpose. The direct methods are more sensitive. By these methods it is
also possible to detect the traces of A, B and H antigens present in the saliva of non-
secretors. From forensic point of view this is very useful because 20-25 % stains
encountered in connection with criminal cases are likely to be from non-secretor
individuals. As a rule while grouping a saliva stain central part of the stain is to be included
in the test material as it has been observed that the H substance is not evenly distributed
throughout the stain but tends to be concentrated in the central region.

21

Diatom
Diatoms are a major group of algae, and are among the most common types
of phytoplankton. Most diatoms are unicellular, although they can exist as colonies in the
shape of filaments or ribbons, fans, zigzags, or stars. Diatoms are producers within the food
chain.
A unique feature of diatom cells is that they are enclosed within a cell wall made
of silica (hydrated silicon dioxide) called a frustule. These frustules show a wide diversity in
form, but are usually almost bilaterally symmetrical, hence the group name. The symmetry
is not perfect since one of the valves is slightly larger than the other allowing one valve to fit
inside the edge of the other.
Fossil evidence suggests that they originated during, or before, the early
Jurassic Period. Only male gametes of centric diatoms are capable of movement by means
of flagella. Diatom communities are a popular tool for monitoring environmental conditions,
past and present, and are commonly used in studies of water quality.
The main goal of diatom analysis in forensics is to differentiate a death by
submersion from a post-mortem immersion of a body in water. Laboratory tests may reveal
the presence of diatoms in the body. Since the silica-based skeletons of diatoms do not
readily decay, they can sometimes be detected even in heavily decomposed bodies. As they
do not occur naturally in the body, if laboratory tests show diatoms in the corpse that are of
the same species found in the water where the body was recovered, then it may be good
evidence of drowning as the cause of death.
Further matching of diatoms from bone marrow and drowning site can strengthen
this supportive evidence and a positive conclusion can be drawn whether a person was
living or not when submerged.











22

Blood
Blood is a complex fluid consisting of different blood cells suspended in a yellowish liquid
called plasma. The blood cells comprise a mixture of red cells (erythrocytes erythrocytes), white cells
(leukocytes leukocytes) and platelets (thrombocytes). The plasma contains many different proteins,
chemical substances, clotting (coagulation coagulation) factors and numerous metabolic substances.
Blood serves as a transport medium for carrying all its different components to the different organs
of the body.
Red Blood Cells (Erythrocytes)
Red cells appear under the microscope as biconcave discs (see Figure 1). They are extremely
small, with a diameter of 7.2 microns. There are approximately 5 million of these cells in each cubic
millimetre (mm
3
) of blood (5 x 10
12
/L). After this, they break down and are removed by cells of the
reticuloendothelial system. These cells are highly specialized and are scattered throughout the body.
They are found mainly in the bone marrow, liver, spleen and lymph glands. Red cells are filled with a
substance called haemoglobin and their primary function is to carry oxygen to the body tissues.
Haemoglobin
Haemoglobin is a large complex molecule made up of iron-containing molecules called haem
which are attached to polypeptide chains called globin. Haemoglobin is a red fluid found in the red
blood cells. It has the ability to combine reversibly with oxygen and carbon dioxide. Its main function
is to transport oxygen to the various tissues to provide the body with its energy and heat.
Basic blood group immunology
Antigens
An antigen is any substance that, when introduced into a body and recognized as foreign,
will bring about an immune response. This might result in the production of an antibody that will
react specifically with that antigen in some observable way.
Antibodies
An antibody is a product of an immune response and will react with that antigen in some
observable way. Antibodies are immunoglobulins (Ig) and are found in the gamma globulin part of
plasma proteins. There are five categories of immunoglobulins: Ig G, Ig M, Ig A, Ig D and Ig E. In this
module, we shall concentrate on Ig G and Ig M. Antibodies are proteins that are formed from amino-
acid molecules held together by peptide bonds; these are referred to as amino-acid chains. The Ig G
antibody has only four of these chains: two of them are small and are referred to as light chains,
while the two larger chains are referred to as heavy chains. This antibody is, however, very small
when compared with the Ig M antibody which is made up of 10 light chains and 10 heavy chains.
Look at Figure 2 which shows the difference between the two antibody molecules.
23


Ig G antibodies
Ig G antibodies make up approximately 73% of our total immunoglobulins. They have a
molecular weight of only 150 000. Ig G antibodies do not cause agglutination of red cells containing
their antigen when suspended in saline; they have the ability only to coat or sensitize them. The life
span of the Ig G immunoglobulin is approximately 6070 days.
Ig M antibodies
Ig M antibodies comprises approximately 8% of our total immunoglobulins. They are much
larger antibodies than Ig G antibodies, with a molecular weight of 900 000. They readily agglutinate
red cells suspended in saline and have a life span of only 10 days. The Ig M antibody often activates
complement during an antigenantibody reaction and, as a result, will cause haemolysis of the red
cells rather than agglutination.

Agglutination Test
Principle:
When red cells are mixed with various reagent antisera (soluble antibody), agglutination will
occur on the slides containing cells positive for (possessing the antigen) the corresponding antigen.
No agglutination will occur when the red cells do not contain the corresponding antigen. One
primary application of this principle is blood typing.
Limitations of the Procedure:
a. Carefully dispense the red cell suspension and antibody in separate areas of the slide.
Adding red blood cells directly to the drop of antisera may cause splash back, resulting in
contamination of the red blood cells.
b. When mixing the antibody with the red blood cells make sure the mixture covers the
entire bottom of the testing area. Inadequate spreading of the mixture will make
interpretation difficult and may result in misinterpretation of results.
c. It is critical to read the results at the end of the designated time. Allowing the reaction to
go beyond the allotted time may result in the drying of the reactants, resulting in a false
positive.
Material:
a. Antibody A & B
b. Red blood cells (#1 and #2)
c. Slides
d. Applicator sticks
24

e. Pipets
Procedure:
a. On the section of slide labelled anti-A place one drop of antibody A.
b. On the section of slide labelled anti-B place one drop of antibody B.
c. Place one drop of cells in each antibody containing circle.
d. Carefully mix each solution with a separate applicator stick.
e. Tilt slowly for one minute.
f. Record results.
Interpretation:
a. Agglutination (clumping) of the red blood cells is positive.
b. No agglutination is negative.
c. It is critical to read the results immediately as false positives can occur when the
mixture begins to dry on the side.


25

DNA
Deoxyribonucleic acid, or DNA, is the molecule of life. It is the chemical code
specifying our function, appearance and pedigree and is unique for all individuals except
identical twins. An individuals DNA is formed by combination of DNA from his or her
parents with half coming from the mother and half from the father. For this reason, DNA
testing can be used as evidence of paternity of a child.
DNA is found in most cells of the body, including white blood cells, semen, hair roots
and body tissue. Traces of DNA can be detected in body fluids such as saliva and
perspiration. Mitochondrial DNA, which follows the maternal line of an individual, can be
extracted from hair and bone samples. This can be used to examine relatedness and
common ancestry between individuals, and to verify the identity of buried remains.
Forensic Science utilises the properties of DNA in several ways. The adage every
contact leaves a trace indicates the importance of a technique able to type trace amounts
of genetic material left during the commission of a crime. Hairs or saliva left on a balaclava
worn during a robbery, semen located at a rape scene, blood collected from an assault,
perspiration on clothing, traces of assailants skin under a victims fingernails, can often be
DNA profiled. This genetic information can then be used to include or exclude suspects as
being the source of the genetic material.
It is not yet possible to test the whole of an individuals DNA. Forensic analysis
involves the testing of regions of an individuals DNA. Databases have been compiled which
list the abundance of a particular fragment of DNA in the population. From this information,
an estimate of the abundance of combinations of DNA at several regions can be made and
compared to the DNA of victims or suspects. In this way, an individual can be included or
excluded as a possible source of DNA found in relation to a criminal investigation. Statistical
interpretation of the information can be made to estimate the likelihood of material coming
from a particular individual relative to coming from a random member of the population. In
New Zealand, forensic DNA testing is carried out at the Institute of Environmental Science
and Research Ltd. (ESR), where a Bayesian approach to statistical interpretation is used.

Structure of DNA
The DNA molecule is a vast ladder in which the vertical pieces consist of alternating
sugar molecules and phosphate groups, and the rungs are complementary bases. The 4
major bases available for the deoxyribonucleotide are adenine (A), guanine (G), thymine (T)
and cytosine (C) (Figure 1). The backbone of the molecule consists of phosphodiester bonds
connecting the 3'- hydroxyl of the deoxyribonucleotide to the 5'-hydroxyl of the next sugar.
Two backbone chains, running in opposite directions, are paired complementarily by the
interaction of a purine (A or G) with a pyrimidine (T or C). The pairing of a purine and
pyrimidine results in a regular structure. A can only bond with T, and G can only bond with C
due to steric hindrance, so if the structure of one side of the chain is known, the other can
be determined.
26


In vivo, DNA is a template for reproduction of genetic material and cellular
information. The molecule splits along its central axis, providing access to one side of the
complementary chain. Enzymes then add the appropriate bases to each chain, giving a new
exact copy of the complementary sequence. This ability of DNA to repair and replicate itself
is exploited in the DNA profiling techniques used in forensic science.
Parts of the DNA known as genes contain codes to make a specific product, usually a
protein. These compose only a small part of the total DNA. Most of the remainder has no
known function, and can be referred to as spacer or packer DNA between the genes. Much
of this is highly repetitive sequences of DNA repeated end-on-end a varying number of
times. These regions are referred to as minisatellites. Smaller repetitive sequences of, for
example, 4 base pairs are referred to as microsatellites. The number of times this sequence
is repeated is determined in the type of profiling called Short Tandem Repeats (STRs).
DNA PROFILING TECHNIQUES
Two of the common types of DNA profiling used for Forensic analysis are described.
Restriction fragment length polymorphism (RFLP)
This technique is outlined in Figure 2. Double stranded DNA is extracted from blood
or semen. The DNA is cut into small pieces by a sequence-specific enzyme, i.e. an enzyme
that cuts the DNA wherever a particular sequence of bases occurs. The fragments are then
separated out by a process called electrophoresis: the sample is put at one end of a bath of
a jelly-like substance called agarose gel and a voltage is applied. The fragments are charged,
and the voltage is applied in such a way as to encourage the fragments to migrate to the
other end of the gel. Small fragments move much faster than large ones, so separation on
the basis of molecular weight occurs.
After electrophoresis the gel and fragments are exposed to 0.25M HCl to depurinate
the DNA and nick the sugar phosphate backbone, as this assists in fragment transfer. The
depurinated sites are then cleaved by washing in NaOH/NaCl. The denatured DNA is then
transferred to a nylon membrane and the variable minisatellites region of the DNA
examined by
32
P- radiolabelled short pieces of single stranded DNA called probes. A probe
binds to its complementary sequence on the membrane. The radiolabelled membrane is
exposed to film to produce an autoradiograph.
27

Successive regions of the DNA are examined. The distribution of each of the probed
regions of DNA within the population is estimated from a population database to give an
indication of the probability that the sample comes from a given suspect.


The RFLP profiling technique is outlined in Figure 2.
Short tandem repeat profiling (STRs)
This is the new generation of DNA profiling. In this technique microsatellite
repeating regions are examined. The technique is based on the polymerase chain reaction -
the cycle of reactions in which DNA is split, replicated then split again for replication. This
amplification gives an exponential increase in the number of copies of the original template
(Figure 3 on next page). The reaction is under kinetic control, reaching a plateau which is
dependent on competition, the ultimate inactivation of the catalytic enzyme and the
original number of template molecules, primer and dNTPs (deoxynucleotide triphosphates -
the building blocks of DNA).
The DNA is denatured (split) at approximately 94
o
C. Short strands of DNA, called
primers, attach to the target DNA at a specific site. Bases are added enzymatically to the end
of the primers to form a new complementary strand. Approximately thirty such cycles are
carried out to produce many copies of the original material. Since the amount of original
28

material is increased, this technique is particularly suited to the analysis of trace amounts of
DNA.
The amplified DNA is separated by electrophoresis through an ultra-thin denaturing
polyacrylamide gel. This technique can be performed manually, with repeats visualised
using silver staining, or automatically, with multiple loci visualised simultaneously using
fluorescent dyes. The number of repeats for a particular individual is determined at several
loci. The manual method currently in use examines three loci, with an extra male/female sex
test. The automated method currently examines four loci. Statistical analysis on the
abundance of the observed patterns in the population is carried out.
29


30

PhenolChloroform Method
In general, this method involves disruption and lysis of the stain material, digestion
of cell components and removal of contaminants by organic solvents. The DNA is finally
recovered by alcohol and salt precipitation and subsequent rehydration. An alternative
protocol involves the purification of the extracted DNA by means of column based methods
instead of the alcohol precipitation. Cell lysis is performed using an enzyme- and detergent-
based buffer (Proteinase K with sodium dodecyl sulfate). Organic extraction is performed by
adding an equal volume of water-saturated, buffered phenol to the aqueous DNA sample,
vigorously vortexing the mixture, and centrifugation to allow phase separation. The upper,
aqueous layer is carefully removed to a new tube, avoiding the phenol interface. This step is
followed by the addition of chloroform to extract residual phenol from the aqueous phase.
The DNA is concentrated by ethanol precipitation in the presence of salt. After washing with
70% ethanol, the pellet DNA is dried in a speed vac and dissolved in low salt buffer. This
method is suitable for the extraction of DNA from a wide range of cell types and stain
materials.
Materials
1. Sterile distilled water.
2. Hydrogen peroxide 30%.
3. 1 M Tris-HCl solution, pH 8.0.
4. 1 M Tris-HCl solution, pH 9.0.
5. 0.5 M EDTA disodium salt.
6. 5 M Sodium chloride.
7. Ethanol 100% & 70%.
8. Proteinase K stock solution: 20 mg/mL (dissolve 100 mg of proteinase K in
5 mL of sterile distilled water; store frozen in 1-mL aliquots).
9. 1M dithiothreitol (DTT) solution.
10. Phenol solution (+ bottle of equilibration buffer for pH 8.0).
11. Chloroform: isoamylalcohol solution 24:1.
12. 3M sodium acetate buffer solution, pH 5.2.
13. 10% sodium dodecyl sulfate solution.
14. Linear polyacrylamide (LPA; 5 mg/mL, Ambion).
15. 1M Calcium chloride solution.
16. Extraction buffer (EB):
i. 10 mM Tris-HCl, pH 8.0,
ii. 10 mM EDTA disodium salt, pH 8.0;
iii. 100 mM sodium chloride; and
iv. 2% sodium dodecyl sulfate.
17. Extraction buffer for hairs (EBH):
i. 10 mM Tris-HCl, pH 8.0;
ii. 10 mM CaCl2
iii. 100 mM sodium chloride,
iv. 2% sodium dodecyl sulfate.
18. Sterile Petri dishes.
31

Procedure
Extraction of DNA from All Types of Cells and Stain Materials except Hairs and Sperm-
Cell/Nonsperm-Cell Mixtures
1. To the sample add 500 L of extraction buffer plus 20 L of proteinase K (20 mg/mL).
2. Vortex and incubate at 56C overnight with agitation in a Thermomixer.
3. Add an equal volume of buffered Phenol (pH 8.0; see Notes 1, 2), vortex, and spin for
10 min at maximum speed in a micro centrifuge.
4. Transfer the aqueous (upper) phase to a new micro centrifuge tube with 0.5 mL of
chloroform (see Note 3).
5. Vortex and spin for 10 mins at maximum speed in a micro centrifuge.
6. Transfer the aqueous (upper) phase to a new micro centrifuge tube with 50 L of 3M
NaAc, pH 5.2.
7. Add 0.8 mL of 100% EtOH, vortex, and precipitate at 20C for at least 1.5 h (see
Notes 4, 5).
8. Recover DNA by centrifugation for 30 min at maximum speed in a micro centrifuge
and decant the supernatant.
9. To the pellet add 1 mL of 70% EtOH and spin for 20 min at maximum speed in a
micro centrifuge.
10. Dry pellet in a vacuum centrifuge for 30 min (see Note 6).
11. Dissolve pellet in 50 L of Tris buffer (10 mM, pH 9.0).

Extraction of DNA from Sperm-Cell/Nonsperm-Cell Mixtures
1. To the stain add 500 L of extraction buffer plus 20 L of proteinase K (20 mg/mL).
2. Vortex and incubate at 37C for 30 min with agitation in a Thermomixer.
3. Remove stain material with tweezers sterilized in 10% H2O2 and spin for 5 min at
maximum speed in a micro centrifuge (see Note 7).
4. Transfer supernatant (= nonsperm-cell fraction) to a new tube and proceed with
Subheading 3.1.1., step 2.
5. Add 1 mL of sterile water to the pellet, vortex and spin for 5 min at maximum speed
in a micro centrifuge, and remove the supernatant.
6. Repeat step 5 twice.
7. Add 500 L of extraction buffer plus 20 L of proteinase K (20 mg/mL) plus 20 L DTT
(1 M) and proceed with Subheading 3.1.1., step 2.
8. Vortex and incubate at 56C overnight with agitation in a Thermomixer.
9. Add an equal volume of buffered Phenol (pH 8.0; see Notes 1, 2), vortex, and spin for
10 min at maximum speed in a micro centrifuge.
10. Transfer the aqueous (upper) phase to a new micro centrifuge tube with 0.5 mL of
chloroform (see Note 3).
11. Vortex and spin for 10 mins at maximum speed in a micro centrifuge.
12. Transfer the aqueous (upper) phase to a new micro centrifuge tube with 50 L of 3M
NaAc, pH 5.2.
13. Add 0.8 mL of 100% EtOH, vortex, and precipitate at 20C for at least 1.5 h (see
Notes 4, 5).
32

14. Recover DNA by centrifugation for 30 min at maximum speed in a micro centrifuge
and decant the supernatant.
15. To the pellet add 1 mL of 70% EtOH and spin for 20 min at maximum speed in a
micro centrifuge.
16. Dry pellet in a vacuum centrifuge for 30 min (see Note 6).
17. Dissolve pellet in 50 L of Tris buffer (10 mM, pH 9.0).

Notes
1. For the addition of phenol and chloroform/isoamylalcohol we use a dispenser.
Before use the dispenser pipe is cleaned with 10% hydrogen peroxide solution and
the first two volumes are discarded from the dispenser.
2. Phenol is a hazardous waste material that needs to be disposed properly. Phenol is
highly corrosive and can cause severe burns to skin and damage clothing. Gloves,
safety glasses, and a lab coat are to be worn whenever working with phenol, and all
manipulations should be carried out in a fume hood.
3. The two phases (organic phase and aqueous layer) need to be carefully separated in
that the nucleic acids and proteins tend to be at the interface. Leaving too much of
the aqueous layer behind will cause loss of material and aspirating too close to the
interface can include protein.
4. Precipitation of the DNA at 20C should be performed at least for 1.5 h. It is
possible to place the samples at 20C overnight at this stage.
5. For extractions from stains with small amounts of DNA, use LPA as a co-precipitant.
LPA offers the advantage that it is chemically synthesized and is not derived from
biological sources. Other carriers may contain small amounts of contaminating
nucleic acids, which may cause problems, especially when typing mDNA as seen in
our laboratory when working with glycogen as carrier.
6. While drying the DNA pellet in the vacuum centrifuge, the screw caps of the tubes
are kept in sterile Petri dishes.
7. Concerning DNA extraction from sperm-cell/nonsperm-cell mixtures, when removing
the stain material with tweezers, it is important to squeeze the stain firmly to
prevent loss of liquid and DNA.

Prevention of DNA Contamination
The usage of DNA evidence in the courtroom has exonerated innocent prisoners,
while sending the guilty to a lifetime of incarceration. There is no doubt that DNA evidence
is one of the most powerful tools in forensic science. Many cases have been solved because
of this useful tool. Child support payments have been made, fatherhood has been
confirmed, criminal offenders have been brought to justice, and countless other successes
have occurred due to DNA fingerprinting.
However, using such a powerful tool requires great care and consideration. These errors
led to multiple organizations dictating new guidelines and suggestions for forensic evidence
handling, collection, and transportation. Contamination of DNA can have horrible
33

consequences on DNA analysis; it can lead to evidence being discarded in a court case, or
incorrect results. Contamination occurs when outside material is introduced to the sample,
for example another persons DNA or a chemical that was spilt. Some guidelines to prevent
these errors are highlighted below:
Avoid excessive exposure to heat or humidity refrigerate/freeze if possible
Never handle evidence with bare hands
Never allow two items of evidence to come into contact with each other
Air-dry evidence completely before packaging
Package evidence in paper sacks or envelopes (avoid plastic bags)
Package each item separately
Ship evidence with dry ice or leak-proof ice packet (sample must remain dry)
Following guidelines, systematic procedures, and using care during evidence
collection and transportation greatly lowers the likelihood of contamination. The U.S.
National Institute of Justice suggests that these following steps be taken to avoid
contamination:
Wear gloves. Change them often.
Use disposable instruments or clean them thoroughly before and after handling each
sample.
Avoid touching the area where you believe DNA may exist.
Avoid talking, sneezing, and coughing over evidence.
Avoid touching your face, nose, and mouth when collecting and packaging evidence.
Air-dry evidence thoroughly before packaging.
Put evidence into new paper bags or envelopes, not into plastic bags.
Do not use staples.
Sources of DNA
A large assortment of evidence can be used to collect DNA. Remnants including sweat, skin,
hair, blood, mucus, semen, saliva, urine, and other tissues are commonly left behind at
crime scenes. The National Institute of Justice (1999) lists these following examples as
common pieces of evidence that include genetic material:
34




35

The first method for creating a DNA profile was RFLP, or restriction fragment length
polymorphism. RFLP is not used as often today because it requires a large sample of DNA --
as much as 25 hairs or a nickel-sized spot of bodily fluid -- and can take as long as a month to
complete. It also requires examining multiple sections of the DNA strand to find variations,
which is time-consuming and leaves more room for human error. Some of the steps for RFLP
analysis are also used in other types of DNA profiling. For RFLP, the steps are:
1. Separate white and red blood cells with a centrifuge.
2. Extract DNA nuclei from the white blood cells. This is done by bathing the cells in hot
water, then adding salt, and putting the mixture back into the centrifuge.
3. Cut DNA strand into fragments using a restriction enzyme.
4. Place fragments into one end of a bed of agarose gel with electrodes in it. Agarose
gel is made from agar-agar, a type of seaweed that turns into gelatin when dissolved
in boiling water.
5. Use an electric current to sort the DNA segments by length. This process is
called agarose gel electrophoresis. Electrophoresis refers to the process of moving
the negatively-charged molecules through the gel with electricity. Shorter segments
move farther away from their original location, while longer ones stay closer. The
segments align in parallel rows.
6. Use a sheet of nitrocellulose or nylon to blot the DNA. The sheet is stained so the
different lengths of DNA bands are visible to the naked eye. By treating the sheet
with radiation, an autoradiograph is created. This is an image on x-ray film left by the
decay pattern of the radiation. The autoradiograph, with its distinctive dark-coloured
parallel bands, is the DNA profile.
PCR (polymerase chain reaction) analysis is usually the first step in the creation of a DNA
profile today. PCR can replicate a small amount of DNA to create a larger sample for
analysis. It does this using a repeating process that takes about five minutes. First, a heat-
stable DNA polymerase -- a special enzyme that binds to the DNA and allows it to replicate --
is added. Next, the DNA sample is heated it to 200 degrees F (93 degrees C) to separate the
threads. Then the sample is cooled and reheated. Reheating doubles the number of copies.
After this process is repeated about 30 times, there is enough DNA for further analysis.
PCR is the first step in analysing STRs (Short Tandem Repeats), which are very small,
specific alleles in a variable number tandem repeat (VNTR). Alleles are pairs of genes that
occur alternately at a specific point, or loci, on a chromosome. Analysing STRs is more
accurate than the RFLP technique because their small size makes them easier to separate
and to tell apart.
A variation on STR analysis is Y-STR. Only STRs found on the Y-chromosome (which only
males have) is analysed. STR analysis is useful if the sample has mixed DNA (from both men
and women) or in sexual assault cases with a male assailant. Y-STR is otherwise processed
just like a regular STR.
36

AmpFLP, amplified fragment length polymorphism, is another technique that uses PCR
to replicate DNA. Like RFLP, it first uses a restriction enzyme. Then, the fragments are
amplified using PCR and sorted using gel electrophoresis. AmpFLP's advantage over other
techniques is that it can be automated and doesn't cost very much. However, the DNA
sample must be high quality or errors may result, which is the case with most DNA analysis
techniques. Analysts can have a time telling the longer strands apart because they bunch up
tightly.

CODIS uses algorithms to compare 13 different STR locations, plus one that determines
the gender of the person in question. It has rules and safeguards to protect the privacy of
people whose profiles are in the database. The matching algorithms - which must be
confirmed by an analyst-can produce leads for law enforcement or even identify a potential
assailant. The downside of using CODIS is that it's only as strong as the number of profiles
included, and there is a backlog of more one million profiles to be entered.
Although most labs use either RFLP or STR techniques for their DNA analysis, there are
situations that require a different approach. One such situation is when there are multiple
male contributors of genetic material, which sometimes happens in sexual assault cases.
The best way to resolve the complex mixture and sort out exactly which men were involved
is Y-marker analysis. As its name suggests, this technique examines several genetic markers
found on the Y chromosome. Because the Y chromosome is transmitted from a father to all
his sons, DNA on the Y chromosome can be used to identify DNA from different males. Y-
marker analysis can also be used to trace family relationships among males.
37


Another situation involves identifying old remains or biological evidence lacking
nucleated cells, such as hair shafts, bones and teeth. RFLP and STR testing can't be used on
these materials because they require DNA found in the nucleus of a cell. In these cases,
investigators often use mitochondrial DNA (mDNA) analysis, which uses DNA from a cell's
mitochondria. Investigators have found mDNA testing to be very useful in solving cold cases,
which are murders, missing-person cases or suspicious deaths that are not being actively
investigated. Cold cases often have biological evidence in the form of blood, semen and hair
that has been stored for a long time or improperly stored. Submitting those degraded
samples for mDNA testing can sometimes break the case open and help detectives find the
perpetrator.
A relatively new technique -- SNP analysis -- is also useful in certain cases where
forensic labs are presented with highly degraded DNA samples. This technique requires that
scientists analyse variations in DNA where one nucleotide replaces another. Such a genetic
change is called a single nucleotide polymorphism, or SNP (pronounced "snip"). SNPs make
excellent markers and are most often used to determine a person's susceptibility to a
certain disease. But forensics labs turn to SNP analysis on occasion.
The main objective of DNA analysis is to get a visual representation of DNA left at the
scene of a crime. A DNA "picture" features columns of dark-coloured parallel bands and is
equivalent to a fingerprint lifted from a smooth surface. To identify the owner of a DNA
sample, the DNA "fingerprint," or profile, must be matched, either to DNA from a suspect or
to a DNA profile stored in a database.
Let's consider the former situation -- when a suspect is present. In this case,
investigators take a DNA sample from the suspect, send it to a lab and receive a DNA profile.
Then they compare that profile to a profile of DNA taken from the crime scene. There are
three possible results:
Inclusions -- If the suspect's DNA profile matches the profile of DNA taken from the crime
scene, then the results are considered an inclusion or non-exclusion. In other words, the
suspect is included (cannot be excluded) as a possible source of the DNA found in the
sample.
38

Exclusions -- If the suspect's DNA profile doesn't match the profile of DNA taken from the
crime scene, then the results are considered an exclusion or non-inclusion. Exclusions
almost always eliminate the suspect as a source of the DNA found in the sample.
Inconclusive results -- Results may be inconclusive for several reasons. For example,
contaminated samples often yield inconclusive results. So do very small or degraded
samples, which may not have enough DNA to produce a full profile.
Sometimes, investigators have DNA evidence but no suspects. In that case, law
enforcement officials can compare crime scene DNA to profiles stored in a database.
Databases can be maintained at the local level (the crime lab of a sheriff's office, for
example) or at the state level. A state-level database is known as a State DNA index system
(SDIS). It contains forensic profiles from local laboratories in that state, plus forensic profiles
analysed by the state laboratory itself. The state database also contains DNA profiles of
convicted offenders. Finally, DNA profiles from the states feed into the National DNA Index
System (NDIS).
To find matches quickly and easily in the various databases, the FBI developed a
technology platform known as the Combined DNA Index System, or CODIS. The CODIS
software permits laboratories throughout the country to share and compare DNA data. It
also automatically searches for matches. The system conducts a weekly search of the NDIS
database, and, if it finds a match, notifies the laboratory that originally submitted the DNA
profile. These random matches of DNA from a crime scene and the national database are
known as "cold hits," and they are becoming increasingly important. Some states have
logged thousands of cold hits in the last 20 years, making it possible to link otherwise
unknown suspects to crimes.




39


Allelic ladder and internal lane standard for PowerPlex 16 run on the DNAscan Rapid DNA Analysis System.
Allelic ladder shows the collection of all alleles commonly found in the population.
40

Bitmap (.bmp file) of an STR profile and ILS automatically generated from the 9947A immortalized cell line
processed on the DNAscan Rapid DNA Analysis System. Results show good balance within each color. All
alleles are designated according to international standards, e.g., 9.3 allele in TH01. Since no primer
sequences have been changed, allele calls are identical to PowerPlex 16 results obtained in the forensic
DNA laboratory.
41



Enlarged view of TH01 locus. Data shows single base resolution achieved on the DNAscan Rapid DNA
Analysis System and separation of alleles 9.3 and 10.
42

DNA evidence plays a pivotal role in the modern criminal justice system, but the same
techniques that prove guilt or exonerate an innocent person are just as useful outside the
courtroom. Here are a few examples:
Paternity testing and other cases where authorities need to prove whether individuals are
related or not -- One of the more infamous paternity cases of late occurred after Anna
Nicole Smith's death in 2007. Five different men claimed to be the father of Smith's baby
daughter, Dannielynn. After a DNA test, Larry Birkhead was proven to be the child's father, a
similar thing happened with Mr ND Tiwari (ex-CM Uttarakhand)
Identification dead bodies -- Police investigators often face the unpleasant task of trying to
identify a body or skeletal remains. DNA is a fairly resilient molecule, and samples can be
easily extracted from hair or bone tissue. Once a DNA profile has been created, it can be
compared to samples from families of missing persons to see if a match can be made. The
military even uses DNA profiles in place of the old-school dog tag. Each new recruit must
provide blood and saliva samples, and the stored samples can subsequently be used as a
positive ID for soldiers killed in the line of duty. Even without a DNA match to identify a
body conclusively, a profile is useful because it can provide important clues about the
victim, such as his or her sex and race.
Studying the evolution of human populations -- Scientists are trying to use samples extracted
from skeletons and from living people around the world to show how early human
populations might have migrated across the globe and diversified into so many different
races. In the 1980s, scientists at the University of California, Berkeley, used mitochondrial
DNA analysis to speculate that all living humans are related to a single woman -- "Eve" --
who lived roughly 150,000 years ago in Africa. Other scientists, using increasingly more
sensitive DNA analysis, have since confirmed this to be true.
Studying inherited disorders -- Scientist also study the DNA fingerprints of families with
members who have inherited diseases like Alzheimer's disease to try to ferret out
chromosomal differences between those without the disease and those who have it, in the
hope that these changes might be linked to getting the disease. DNA testing can also reveal
a person's susceptibility to certain diseases. Several companies, such as 23andMe,
deCODEme and Navigenics, offer at-home genetics tests that can evaluate your risk for
hundreds of diseases and traits, including breast cancer, rheumatoid arthritis and Type 2
Diabetes.
Catching poachers -- Wildlife biologists are now turning to DNA tests to catch people who
hunt illegally. For example, the hunting season for doe on public lands lasts only two days in
many states. If a wildlife official suspects a hunter has shot a female deer after the official
close of the season, he can analyze DNA from the meat and determine the species and
gender of the animal.
Clarifying history -- Historians are turning to DNA evidence to learn more about the past. For
example, Y-chromosome testing was used in 1998 to determine whether Thomas Jefferson,
the third president of theUnited States, fathered children with one of his slaves or not. And
in May 2009, a group of historians asked a Philadelphia museum if they could have access to
a strip of a pillowcase stained with the blood of Abraham Lincoln. Their goal was to analyze
Lincoln's DNA to see if he suffered from a rare genetic cancer syndrome called multiple
endocrine neoplasia type 2B, but the museum's board would not allow the test at the time.
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