SAINT XAVIERS SENIOR SECONDARY SCHOOL JAIPUR

BIOLOGY PROJECT
DNA FINGERPRINTING

PREPARED BY:

Name: Nandaditya Swami

Class: XII-C

Class Roll Number: 25

Session: 2016-2017

Board Roll Number: ____________

 CERTIFICATE

This is to certify that Nandaditya Swami of class XII-C has completed the
project on DNA FINGERPRINTING successfully under our guidance.
This project is complete in all respects and is upto our expectations.

_______________ ______________

Mrs. Kshama sharma Mr. Ravindra Sharma

(Biology Teacher) (Lab Incharge)

 ACKNOWLEDGEMENT

"There are times when silence speaks so much more loudly than words
of praise to only as good as belittle a person, whose words do not
express, but only put a veneer over true feelings, which are of gratitude
at this point of time."

I express my heartfelt gratitude to my teacher Mrs. Kshama Sharma and

lab incharge Mr. Ravindra Sharma without whose vital support, guidance
and encouragement It would not be possible to finish this project.
 INDEX

Introduction

Tandem Repeats

DNA Profiling

Stages of DNA Profiling

Uses of DNA Fingerprinting

Criminal cases
Medical cases

Details of Analysis

Percentage Accuracy
 DNA FINGERPRINTING

What is DNA Fingerprinting?

It is a method of identifying an individual by unique characteristics of

that persons DNA.

A Historical Perspective

In the course of his research on variability in human DNA, Alec Jeffreys

developed a method of forensic DNA typing. This method was termed as
DNA Fingerprinting, was used for the first time to solve two criminal
cases in the UK in 1987. Jeffreys was knighted in 1994 for Services to
Science, and has been the recipient of numerous other honours.

Before going into a details of DNA Fingerprinting we first define the DNA
fragments on which this whole technique is based i.e.

The TANDEM REPEATS

Two important categories of tandem repeat have been used widely in

forensic genetics: minisatellites, also referred to as variable number
tandem repeats (VNTRs); and microsatellites, also referred to as short
tandem repeats (STRs). The general structure of VNTRs and STRs is
the same. Variation between different alleles is caused by a difference
in the number of repeat units that results in alleles that are of different
lengths and hence tandem repeat polymorphisms are known as length
polymorphisms. Another category is single nucleotide polymorphism
(SNPs)

Variable number tandem repeats (VNTRs)

VNTRs are located predominantly in the subtelomeric regions of

chromosomes and have a core repeat sequence that ranges in size from
six to one hundred base pairs. The core repeats are represented in
some alleles thousands of times; the variation in repeat number creates
alleles that range in size from 500 base pairs to over 30,000 base pairs.
The number of potential alleles can be very large: the MS1 locus, for
example, has a relatively short and simple core repeat unit of nine base
pairs with alleles that range from approximately 1,000 to over 20,000
base pairs which means that there are potentially over 2,000 different
alleles at this locus.

Short tandem repeats (STRs)

STRs are currently the most commonly analysed genetic polymorphism

in forensic genetics. They were introduced according to the number of
repeats that they contain tool for just about every forensic laboratory in
the worldthe vast majority of forensic genetic casework involves the
analysis of STR polymorphisms. There are thousands of STRs that can
potentially be used for forensic analysis. STR loci are spread throughout
the genome including the twenty two autosomal chromosomes and the X
and Y sex chromosomes. They have a core unit of between one and six
base pairs and the repeats typically range from 50 to 300 base pairs.

Single nucleotide polymorphisms (SNPs)

The simplest type of polymorphism is the SNP: single base differences

in the sequence of the DNA. SNPs are formed when errors (mutations)
occur as the cell undergoes DNA replication. Some regions of the
genome are richer in SNPs than others. For example, chromosome one
contains an SNP on average every 1,450 base pairs compared with
chromosome nineteen, where SNPs occur on average every 2,180 base
pairs. SNPs normally have just two alleles: for example one allele with a
guanine and one with an adenine, and therefore are not highly
polymorphic.

Preference of STR over VNTR and SNP

Although VNTRs were the first polymorphisms used in DNA profiling and
they were successfully used in forensic casework for several years. The
use of VNTRs was, however, limited by the type of sample that could be
successfully analysed because a large amount of DNA was required.
Interpreting VNTR profiles could also be problematic. Their use in
forensic genetics has now been replaced by short tandem repeats
(STRs) SNPs are so abundant throughout the genome that it is
theoretically possible to type hundreds of them. This will make the
combined power of discrimination very high. It is estimated that to
achieve the same discriminatory power that is achieved using ten STRs,
fifty to eighty SNPs would have to be analysed. With current technology,
this is much more difficult than analysing ten STR loci.

DNA Fingerprinting and DNA Profiling - same or different?

DNA fingerprinting, as developed by Sir Alec Jeffreys, targeted

particular repeating sequences of DNA (9-80 base pair long) found at a
number of loci (locus). Jeffries described the pattern produced in a
fingerprint as unique to an individual. Technology at the time (1985)
required good DNA samples and took 1 - 2 weeks for a result.
Advances in technology have led to DNA profiling , using smaller short
tandem repeats ( STR s) also from a number of loci. The smaller STRs
are more likely to survive DNA degradation, use less DNA (because of
PCR technology), and can be processed within 24 hours.

Stages of DNA Profiling

Stage 1: Cells are broken down to release DNA If only a small amount of
DNA is available it can be amplified using the polymerase chain reaction
(PCR) technique.

Stage 2: The DNA is cut into fragments using restriction enzymes. Each
restriction enzyme cuts DNA at a specific base sequence. The sections
of DNA that are cut out are called restriction fragments. This yields
thousands of restriction fragments of all different sizes because the base
sequences being cut may be far apart (long fragment) or close together
(short fragment).

Stage 3: Fragments are separated on the basis of size using a process

called gel electrophoresis. DNA fragments are injected into wells and an
electric current is applied along the gel. DNA being negatively charged is
attracted to the positive end of the gel. The shorter DNA fragments move
faster than the longer fragments hence DNA fragments are separated on
the basis of size. A radioactive material is added which stains the DNA
fragments to produce a fluorescent image when irradiated by ultra-violet
radiation(DNA is not visible in normal light even after staining).
A photographic copy of the DNA bands is obtained.

Stage 4: The pattern of fragment distribution is then analysed.

Uses of DNA Profiling

DNA profiling is used to solve criminal, medical and medicolegal

problems.

Crime Forensic science is the use of scientific knowledge in legal

situations. The DNA profile of each individual is highly specific. The
chances of two people having exactly the same DNA profile are 30,000
million to 1 (except for identical twins).

Biological materials used for DNA profiling are Blood, Hair, Saliva,
Semen, Body tissue and/or cells. DNA samples have been obtained
from vaginal cells transferred to the outside of a condom during sexual
intercourse.

DNA Profiling can solve crimes. The pattern of the DNA profile is
compared with those of the victim and the suspect. If the profile matches
the suspect it provides strong evidence that the suspect was present at
the crime scene (but it doesnt proves that they committed the crime). If
the profile doesnt match the suspect then that suspect may be
eliminated from the enquiry.

Example: A violent murder occurred. The forensics team retrieved a

blood sample from the crime scene. They prepared DNA profiles of the
blood sample, the victim and three suspects as follows:

The repeating sequence obtained from site are exactly the same as that
of suspect i.e. the suspect with even little variation can be neglected.
As the DNA fingerprint obtained from site matches that of suspect 2.
Hence it can be confirmed that suspect 2 was there at the crime scene.

Solving Medical Problems DNA profiles can be used to determine

whether a particular person is the parent of a child. A childs paternity
(father) and maternity (mother) can be determined. This information can
be used in Paternity suits Inheritance cases Immigration cases.

As in a child repeating sequences are a combination from both parents

so fingerprints are not identical but it matches at maximum places. From
above figure it is clear that dad3 is his father.

An Example Using EcoR1 for a Question of Paternity EcoR1 cuts a

similar section of DNA i.e. cuts only at GAATTC. After the DNA is cut
with EcoR I fragments are placed in different lanes on an agarose gel
the fragments are then subjected to an electric field The smaller
fragments move faster, the larger ones move slower This process of
separating the fragments by length is called gel electrophoresis.

Resulting Picture after Gel Electrophoresis the bigger fragments are

near the top

Paternity Test In general the childs DNA must be a combination of

Mothers DNA and Fathers DNA.

What is analysed in the DNA?

DNA profiling depends on regions of non-coding DNA that show great

variability between individuals (are polymorphic which means many
forms) Modern profiling uses Short Tandem Repeats , STRs These
 are short sequences of DNA, usually 2-5 base pairs (bp) long, that
repeat, or stutter many times.

Short Tandem Repeats (STRs) the repeat region is variable between

samples while the flanking regions where PCR primers bind are constant
7 repeats 8 repeats Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another
AATG.

New Technology STR analysis has largely replaced the original RFLP
analysis (DNA Fingerprinting) developed in 1985 by Dr Alec Jeffreys
RFLP analysis requires good amounts of non-degraded DNA but STR
analysis can be done on less than one billionth of a gram (a nanogram )
of DNA (as in a single flake of dandruff).

Some uses of DNA Profiling Forensic work on crime scenes, Parentage

testing (explored in more detail), Victim identification in mass disasters,
Animal identification- e.g. racehorses, Conservation biology and
evolutionary studies.

Parentage testing as conducted at DNA Diagnostics, Auckland.

Why Test? Parentage - e.g. disputes over who is the father of a child and
is thus responsible for child support Determining whether twins are
identical or fraternal Estate cases (these may involve obtaining
pathology samples of deceased individuals) Immigration - establishing
that individuals are the true children/parents/siblings in cases of family
reunification.

Why Test? ctd Bone marrow transplant monitoring - to check that the
transplanted marrow is still present Determination of maternal cell
contamination in chorionic villus sampling (used to investigate the
possibility that a foetus has a severe inherited disease)- is the tissue
sample really fetal? Etc.

Identification is established, by photo ID or by identification by a legal

representative. A consent form is signed and witnessed. A case number
is assigned.
DNA samples are collected- in the case of parentage testing, from the
mother, child and possible father(s) They are usually blood, but a buccal
(cheek cell) swab is acceptable

If the samples need transport they must be sent in leak proof containers
for the couriers safety.

The samples are processed, and DNA is extracted from each Primers for
each locus are added. Each primer is labeled with a fluorescent marker

ctd DNA Diagnostics currently uses an AmpFlSTR Identifiler TM PCR

Amplification Kit which targets 15 STR regions plus a sex specific region.
Kits allow standardization and accuracy, as DNA samples are added to a
pre-made mix.

The DNA and fluorescent primers are run through the polymerase chain
reaction (PCR) to amplify the targeted STR regions on the DNA The
samples are audited continually to ensure accuracy

The amplified DNA in a sample is separated by gel electrophoresis in a

genetic analyser. The analyser has a gel-filled capillary tube through
which the DNA travels (this replaces the gel slab of earlier days). DNA
fragments move through the gel tube by size, smallest first. A laser reads
the fluorescent marked DNA loci.

An ABI Prism 310 Genetic Analyser Capillary tube Sample tray Note-
other models of this Analyser have more capillary tubes and can
process more samples at a time, but this model is sufficient for the
demand for testing to date through DNA Diagnostics

Analysing the Read-out Digital output from the Analyzer is read and
interpreted by genotyping software. Each STR region read has two
peaks, for the regions ( loci ) on an individuals maternal and paternal
chromosomes with that locus. note - if both regions are the same length,
there is one peak. Data is shown numerically Locus D19S433 = 14,15
Locus vWA = 15,16 Locus TPOX= 8,8 Locus D18S51= 13,16 A sample
showing 4 loci

A sample print -out for one person, showing all loci tested. Different
colours help with interpretation.
Whose STR? A child will inherit one of the STRs at each locus from its
mother, and since usually in parentage tests these are determined, then
by elimination the other STRs at each locus come from its father. The
father can donate either of his two STRs at each locus. If a child has
STRs different from those of the putative father, then that man can be
eliminated as a possible father, If a child has a particular STR that is the
same as the putative father, it is necessary to examine possible matches
with other STR loci and examine probability in Parentage Analysis.

Parentage Analysis For each STR tested, the data obtained is used to
calculate a paternity index (the probability of the evidence given that a
particular man is the father versus he is not the father) This is based on
the frequency in the population of the alleles at that locus In New
Zealand there are databases for European, Maori/Cook Islander, Asian
and Tongan/Samoan. International databases are used for other
ethnicities

Analysis II Each STR site index is an independent event, so using

probability law that says the probability that two independent events
may happen together is the product of their individual probabilities , an
overall paternity index is calculated by multiplying together the indices
for each locus

Parentage Analysis II, ctd Paternity index The index in this mans
analysis shows that the DNA evidence is 25 million times more likely that
he is the biological father versus he is not (odds 25 million:1)

Finally further auditing and cross-checking is done to ensure accuracy

Parentage testing results in a report that is sent to all parties tested.