1

CHAPTER - 1

INTRODUCTION

1.1 INTRODUCTION

Crime is as old as the human civilization. But so is man's ingenuity in

investigating crime. About 149 years ago (1860), finger prints (thump prints) were used

for the first time by a British administrator, Sir William Herschel, in order to prevent

large-scale swindling of funds by illiterate military soldiers (Laljisingh, 2009). This

sensational discovery attracted the attention of eminent biologist all over the world

including Francis Galton and Edward Henry. Edward Henry provided a detailed

classification of finger prints, which is used even today all over the world. Although

probability of two people having identical fingerprints is 1 in 1010

(world population is

approximately 6.9X109), criminals started adopting sophisticated strategies and the

investigation agencies couldn't get finger impressions. After the sensational discovery

of ABO system of blood grouping (Landsteiner, 1901), Rh factors and human

lymphocyte antigen (HLA), Forensic scientists adopted them for establishing identity

of individuals in crime investigation but positive identification would be only with

certainty of 99.7%. More than 18 different tests including Rh factors, isozymes,

Serological markers, soluble proteins within the plasma, were used for Forensic

identity (James et al., 2003). One of the soluble blood serum protein marker,

phosphoglucomutase (PGM) was used in Forensic hematogenetics by many

workers. (Hopkinson&Harris, 1968, and Spencer et.al., 1994). However the necessity

to provide probability of at least 99.999% was fulfilled by the discovery of DNA finger

printing by British Scientist Professor Alec Jeffreys of Leicester University in 1985.

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1.2 DNA FINGER PRINTING

Alec Jeffreys provided irrefutable evidence that there is a class of DNA which is

organized differently in each individual in such a way that it gives individual specific

pattern. In the DNA of each person millions of bases are present and its sequence varies

in every person. As the DNA molecule has many millions of base pairs, scientists

developed a shorter method based on baspairs repeating pattern in DNA to differentiate

the individuals. The arrangement of baspairs, for example GATA varies in each

individual. In the DNA of one individual there may be 10 repeats of uninterrupted

GATA repeats on a particular chromosome. In another individual these repeats may be

more or less than 10. This variation in number of base pair repeats between individuals

is used by DNA finger printing. Alec Jeffreys isolated a number of non-coding (junk)

DNA sequences and used them as probe for DNA finger printing.

The centre for Cellular and Molecular Biology (CCMB) India had isolated a

class of repetitive DNA consisting of GATA repeats (545 base pairs) from a highly

poisonous snake, the banded Krait (Bkm) and used it by labeling with radioactive p32

and hybridized it with DNA of different individual. The DNA of different individual

was cut with appropriate restriction enzymes, size fractioned on agarose gel by

electrophoresis, transfered it on nylon membrane and hybridized with p32 labelled

BKM and exposed this on X-ray film. Some of the bands formed on the X-ray film may

be common and some may be different indicating the individuality. (Laljisingh, 2009).

These bands are used in Forensic investigation. For example in a family consisting of

father, mother and a child, every band present in child accounted for, either being

inherited from mother or father. Normally maternity is certain. If maternity is known,

one can establish paternity. If paternity is known maternity can be established. If father

and mother are known one can establish the identity of the child.

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This DNA finger prints between brothers and sisters are different. In case of

identical twins, DNA finger printings are identical. For DNA finger printing, sufficient

amount of biological material is essential but in Forensic examination it is difficult to

get sufficient sample. After the discovery of thermal cycler based on Polymerase chain

reaction (PCR), third generation technology, it was possible to amplify a small sample,

from one molecule to millions of molecules, in a short time.

After the advent of human genome profiling, the fourth generation DNA finger

printing got a new dimension. The human genome consists of approximately 3 billion

base pairs (bp) of DNA in which there is estimated to be 100000 genes. The average

length of a gene is 5-10 thousand bp. Approximately 30% of the non -coding DNA is

in the form of repetitive sequences and much of this is arranged in tandem repeats. The

tendemly repeated sequences have been the focus of Forensic use and fall into two

types depending upon the size of repeat element, Variable Number Tandem Repeat

(VNTR) and Short Tandem Repeat (STR).

1.3 VARIABLE NUMBER TANDEM REPEAT (VNTR)

Minisatellites or variable number tandem repeats were the first regions, or loci,

of DNA to be used in Forensic science and paternity studies. These DNA loci range in

size from 500 to more than 2000bp. A core unit typically between 9 and 60 bp, is

repeated tandemly along the chromosome. It is the number of repeats that varies

between chromosomes and therefore between individuals. Each repeat length at a

particular VNTR locus is termed a allele. Each repeat length for a particular VNTR

locus can be large, with none of the alleles present at a particularly high frequency.

Jeffreys (1985) used Restricted Fragment Length Polymorphism (RFLP) technique to

detect VNTR loci to identify the criminal in a case. This multilocus probing was

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replaced by single locus probing (SLP) that utilized a probe specific to a single VNTR

locus, there by producing results that were simple to interpret compared to Multi Locus

Probes (MLPs).

VNTR was however, superseded by micro satellite DNA analysis after the

utilization of thermal cyclers (James et al., 2003).

1.4 SHORT TANDEM REPEATS (STRs)

Short tandem repeats (STRs) consist of simple tandemly repeated sequences,

commonly between 2 and 6 bp in length, which are widely dispersed throughout the

genome. STRs are highly abundant: there are as many as half a million STR loci in the

human genome, occurring on average every 6-10 kb (Beamann & Weber 1992). The

STR loci so far analyzed fall into three categories: simple repeats where the repeat

element is repeated in sequence identically; compound repeats comprising two or more

simple repeats; and complex repeats comprising several blocks of variable unit length

and variable sequence (Oldroyd et al., 1995).

The use of STRs has many advantages over VNTR DNA typing. The amount of

starting DNA required can be considerably less, and even a high level of degradation

can be tolerated. STR typing can be performed on samples with less than 1 ng of

degraded DNA, whereas multi- and single-locus probing techniques require 50 ng of

high molecular weight DNA. Also the speed of analysis can be rapid as there is no

requirement for a hybridization process; instead the products of the PCR can be

accurately sized by conventional polyacrylamide electrophoresis.

Although there is a large element of size variation, STRs span segments of

DNA small enough to be amenable to PCR amplification and which can still be

detected in highly degraded DNA (Hagelberg et al., 1991, Jeffreys et al., 1992).

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Although it is estimated that there are as many as half a million STR loci in the

human genome, not all of them are suitable for use in Forensic science. STR loci are

chosen when they meet a range of criteria. The primers used in the amplification

reaction must anneal (i.e. bind) to the DNA of all the members of the population, which

means that the primers used in an amplification will produce PCR products from

members of every ethnic group. The loci can be highly polymorphic with an even

frequency distribution and a high level of heterozygosity. The allelic windows are small

due to the short repeat units, preventing allelic dropout when the DNA is highly

degraded and larger fragments of DNA are no longer present.

An example of a commonly used STR is called vWA (Kimpton et al., 1992),

which is found near to the gene sequence for von Willebrand factor. It has the repeat

sequence TCTA (TCTG) 3-4 (TCTA)n. The repeat sequence alters from TCTA to three

or four repeats of the sequence TCTG, after which there are a variable number of

TCTG repeats. The sequences TCTA and TCTG comprise the repeat unit. The smallest

commonly found allele has a total of 11 repeats and the largest allele has a total of 20

repeats. In between there are eight alleles (Jeffreys et al., 1992, Kimpton et al., 1992,

Sharma & Litt 1992,Puers et al., 1993,Wiegand et al., 1993, DNA recommendations

1994, Bar et al., 1997,Evett et al., 1997).

The International Society for Forensic Haemogenetics (ISFH) has provided

guidelines for the nomenclature of the loci and alleles (DNA recommendations 1994,

Bar et al., 1997). Some of the first STR loci reported were named after the gene

sequence to which the locus was nearest. This led to names such as vWA (von

Willebrand factor; Kimpton et al., 1992), THO1 (tyrosine hydrolase; Puers et al., 1991)

and F13A1 (coagulation factor XIII A; Polymeropoulos et al., 1993). STR loci without

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any connection to a protein coding sequence are named after their chromosomal

location such as D3S1358 (Li et al., 1993) and D19S253 (Weber et al., 1993), on

chromosomes 3 and 19, respectively

Each allele is given a number based upon the number of repeats, therefore

THO1 5 has five tetranucleotide repeats. When, as in the case of THO1 9.3, there is an

incomplete number of a repeat, the number of individual bases (three in the case of

THOI 9.3) is used. Another example of this is the FGA locus, where there is a 22 and a

22.2 allele. The 22.2 allele contains 22 complete tetranucleotide repeat sequences and

an incomplete repeat sequence containing only two bases. As each person has two

alleles, one inherited from the mother and one inherited from the father, a DNA profile

is given with both alleles. Should a person have a THO1 6 and THO1 9.3, his or her

correct genotype is THOI 6, 9.3. A person who inherits by chance THO1 6 from both

parent’s THO1 6, 6.

Using one locus in an STR can yield a low level of discrimination. When a

second locus is used the levels of discrimination can be multiplied provided that the

two loci are not genetically linked. In Forensic cases, between four and 13 STR loci

are commonly amplified.

1.5 Y-CHROMOSOME STRs

The STR loci most commonly used in Forensic science are found on the idio -

or autosomal chromosomes. However, in the course of the last few years, STR loci

found on the male specific Y chromosome are becoming increasingly important. For

Forensic purposes Y-chromosome STR loci are particularly useful in sexual assaults

where male fractions from spermatozoa are mixed with female epithelial cells. Using

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autosomal STR loci, a mixed profile is commonly encountered, but Y-chromosome

STR loci will only' detect the male fraction.

In paternity studies Y chromosomes have recently found a limited but useful

role. In cases where the paternity of a son is under dispute, and the alleged father is

either deceased or cannot be traced, an uncle or grandfather can be used as a source of

DNA. In these instances the STR loci on the Y chromosome should be the same on all

the descendants through the paternal line, assuming that no mutational events have

occurred. The most famous sample involved the allegation that Thomas Jefferson, the

third President of the United States fathered a child by one of his slaves, Sally Hemings

(Foster 1998).Y-chromosome analysis was undertaken on the male line of Thomas

Jefferson's paternal uncle and the paternal lines of Sally Hemings' sons. One of Sally

Hemings sons male lines matched the DNA profile with the male line of Jefferson's

uncle, thereby supporting the hypothesis that Thomas Jefferson did father a child by

one of his slaves. This case also illustrated one of the drawbacks of the Y chromosome

as a Forensic tool: even though 19 markers were examined, likelihood the match could

only be given as > 100.This is because all the markers are linked and therefore the

values for each loci cannot be multiplied together as with autosomal STRs.

1.6 PATERNITY TESTING

A DNA laboratory can be asked to determine whether a man is the biological

father of a child. The genetic relationship of the mother is rarely questioned, but

disputes over who may be the true biological father occur with some frequency.

Previously, Serological techniques were used in which a battery of blood group systems

were needed to attain the level of discrimination to determine paternity beyond

reasonable doubt. Serological systems were superseded by DNA profiling methods

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such as multi-locus probes (MLPs) and then single-locus probes (SLPs). SLPs are still a

common method of analysis in paternity testing as six SLP probes produce a very high

probability of paternity. More recently STRs have been used, although a large number

of STR loci are required. The reason for the change from conventional typing of blood

group markers to DNA methods is the higher degree of probability obtained and the

automation of the test. Using STRs multiplexes, a large number of polymorphic loci

can be analyzed in one reaction, with one set of equipment, and the result is obtainable

in within 24 hours.

Every child inherits one allele from the mother and one from the father. For each

STR tested in a child, one allele must have come the mother and therefore the other

STR allele present in the child must be present in the father's sample. If a child has the

genotype THO1 6,7 and the mother is THO1 7,9 then the child have inherited allele 7

from the mother. The biological father must posses the THO1 allele 6. A similar

situation must occur for all the STR types tested. In order to carry out a paternity test

blood sample or buccal scrape is obtained from the mother and child (or children) and

alleged father(s). Scrape samples are subjected to amplification of a set of STRs. The

greater the number of STRs, the higher the certainty of paternity. It is routine practice

for at least nine STR loci to be analyzed, although this depends upon the frequency of

the alleles analyzed. More DNA loci can be analyzed if the probability of paternity is

not sufficiently high.

The number of STR loci to be analyzed depends upon the power of discrimination

for each locus, and allied to this is a typical paternity index. The power of identity (Pi)

for a test as described for blood groups is the same for DNA profiles. The formula

described by Jones (1972) holds true for STR loci is given in the following Table.

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Table – 1.1

DNA testing results after typing a mother, child and alleged father using nine STR

loci. Alleles marked with an asterisk in the child's DNA profile could have been

inherited from the alleged father.

STR loci Mother (M) Child (C) Alleged father

(AF1)

D3 14,16 16,17* 14,17

VWA 15,17 15*,17 15,16

FGA 20,22 22,24* 24,24

D8 12,12 12,13* 13,14

D21 28,30 28,30* 30,31.2

D18 14,16 16,17* 14,17

D5 13,13 13,13* 13,13

D13 10,11 9* ,10 9,11

D7 8,10 8,11* 8,11

It is possible for an exclusion to occur in real biological fathers at one or more

of the loci tested. This is caused by the rates of mutation at the loci providing the

exclusion. STRs are known to have a relatively high rate of mutational events

(Brinkman et .al, 1995), with loci such as VWA being 0.2% (Webber &Wang 1993).

When this occurs the frequency of the mutation must be factored into the paternity

index. This inevitably reduces the power of the test, which may require additional loci

to be analyzed. Mutational events normally change a STR allele by one repeat unit; the

change normally makes the repeat unit larger. It is very unlikely that more than one

locus would mutate in spermatozoa from the same male.

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1.7 PATERNITY INDEX (PI)

If an individual cannot be excluded as being the father of a child, then there are

two possibilities: either the alleged father is the biological father of the child, or another

male is the real biological father. If another unrelated man is the biological father, then

there is a coincidental match between the two profiles. The paternity index calculates

how likely it is that the alleged father is the real biological father, compared to another

man being the real biological father. It is common practice to assign a numerical value,

the "paternity index" and probability of paternity, based upon how likely it is that the

alleged father tested is the biological father compared to another unrelated male.

1.8 SINGLE NUCLEOTIDE POLYMORPHISM (SNP)

After sequencing of human genome, scientists have created a database of 2.3

million single nucleotide polymorphisms (SNPs)in the world population. Attempts are

being made to put one million of these SNPs on glass slide, and produce a DNA Chip

that can then be hybridized with the biological samples recovered from the scene of

crime by labeling it with one color of chromophore (red) and the suspect's DNA with

another color of chromophore (green). If all the SNPs detected in the DNA from the

scene of crime are exactly the same as the SNPs detected in the suspect's DNA, they

will produce a third colour (yellow). We can then conclude beyond doubt that the

suspect was involved in this particular case. This will be possible by comparing only a

part of the genome. In a few years, it would be possible to use this technique and

complete the total analysis within a few hours.

The first generation technology of using multilocus probe required larger

quantity of biological material yielding high molecular weight DNA from the scene of

crime. It was technically demanding and used to take nearly 2-3 months to complete

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this analysis. The second generation of the single locus probe also had similar

requirement. The third generation technology based on PCR to amplify DNA from the

samples still required fairly high molecular weight DNA but from small quantity of

biological samples. This also required from months to weeks to complete the test. The

fourth generation of technology which is called STR typing carried out by using

automated DNA sequencer has minimized human error and enables completion of the

analysis within 24 hours. Even if the DNA is highly degraded, it is possible to carry out

DNA fingerprinting test by lengths of STRs do not exceed more than a few 100 bases.

(Laijisingh. 2009)

These are techniques that are very reliable and are used all over the world. In

addition, it is possible to test the samples in one country and compare its analysis

carried out in another country. In India judicial system has approved the utilization of

DNA base sequence (STR) for Forensic disputes in the year 2008. Forensic Sciences

Department of Government of TamilNadu has been referred to find out the paternity

disputes. And this department has been employing STR typing to solve paternity

disputes to give justice to hundreds of victimized women.

Hence, in the present study an attempt has been made to analyse the STR

studies made over the past five years to find out the validity of STR typing in Indian

scenario and Whether the STR data can be used to study population geneties of

diversified caste system in Tamil Nadu. An indepth analysis of the present data will

shed light on the caste specific STR markers. Also the studies well adjudicate whether

the existing mode of analysis is valid or any improvement is needed for strengthening

the existing techniques.

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1.9 SHORT TANDEM REPEATS - AN OVERVIEW

Short Tandem Repeat (STR) markers are abundant throughout most genomes

and sufficiently polymorphic to serve as effective genetic markers. A number of fields

utilize STRs including gene mapping, disease diagnostics, evolutionary biology and

human identification. The ability to study STR-markers in parallel with multicolour

fluorescence detection technologies has revolutionized the amount of information that

can be collected in a timely fashion (Butler, 2001).

The relatively small size for the tandem 2-6 base pair (bp) repeat regions make

them accessible to amplification using the Polymerase Chain Reaction (PCR) (Butler-

2001).Multiplex PCR, where multiple regions are simultaneously amplified in a single

reaction, has greatly benefited Forensic DNA typing because less DNA material is

required to obtain results from multiple loci (Butler, 2001). In addition, the amount of

labour required to obtain results at all of the markers is reduced since loci are being

typed in parallel rather than sequencially.

1.10 STR ANALYSIS FOR HUMAN IDENTITY TESTING

The exquisite sensitivity of Polymerase Chain Reaction (PCR) now permits

DNA analysis from samples containing as little as single cell (Findlay et al.,1997). This

capability has extended the application of DNA profiling methods to new areas in

Forensic science including the ability to obtain accurate results from finger prints left

on touched objects (Van Oorschot et al., 1997) or badly damaged samples obtained

from a mass disaster (Whitaker et al.,1995).

Since the early 1990s, there are tens of thousands of these STRs that have been

discovered to occur within and between genes along human chromosomes. Due to the

fact that they do not appear to disrupt normal cellular functions, STRs can easily mutate

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giving raise to a high degree of variability between individuals. A dozen or more alleles

are possible for many STR loci.

A small subset of available STRs have been selected and standardized for use in

human identity testing and criminal DNA databasing. In late 1997, the Federal Bureau

of Investigation (FBI) had choosen 13 STR loci for inclusion in the U.S. Combined

DNA Index system (CODIS). These loci CSF1PO, FGA, TH01, TPOX, vWA,

D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51 and D21S11

form the basis of most criminal databases around the world (Budowle et al., 2000).

Commercially available kits enable accurate and relatively rapid characterization of

STR genotypes.

1.11 FORENSIC APPLICATION

STR-DNA typing has moved from research laboratory to wide spread practical

application in Forensic science which has generated considerable excitement both in

criminal justice world and the popular media. Wyman and white (1980) established the

foundation for the concept with the hallmark observation of a polymorphic DNA locus

characterized by a number of variable length restriction fragments called Restriction

Fragment Length Polymorphism (RFLPs). Thereafter Jeffreys et al., (1985) reported

that a repetitive sequence of 33 basepairs found on human myoglobin gene.

Earlier DNA analysis of multilocus system (Krawczak et al.,1993), single locus

system (Hansen and Morling 1993, Morling and Hansen 1993) and the PCR-based

system such as MCT 118, HLA-DQα (Bijerre et al.,1997) had been applied to paternity

testing or parental paternity testing using amniotic fluid (Nata et al.,1993, Arroyo et

al.,1994).

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DNA profiling such as Multi Locus Probe (MLP) and Single Locus Probe

(SLP) had outdated the Serological examination in paternity testing (Forensic

Medicine, 2003)

In the past two decades, numerous advances in DNA-testing technologies had

occurred, most notably among them the development of polymerase chain reaction

(PCR) based typing methods.

Today the Forensic DNA-Typing community had standardized the use of

Short Tandem repeat (STR) markers and the Federal Bureau of Investigation (FBI)

selected 13 STR markers to serve as the core of its Combined DNA Index System

(CODIS) (Butler et al., 2004).

PCR-based STRs had several advantage over conventional southern blotting

techniques of the larger Variable Number Tandem Repeats (VNTRs). Discrete alleles

from STR systems may be obtained due to their smaller size which puts them with size

range where DNA fragments differing by a single basepair in size may be

differentiated. Further smaller quantities of DNA including degraded DNA, may be

typed using STR (http://www.promega.com/pnotes/58/5189c.html).

1.12 POLYMORPHISM IN STR-MARKERS

STR-markers were first described as effective tool for human identity testing in

early 1990s. Polymorphic STR markers are useful for paternity testing, human

identification and genetic mapping.

DNA nucleotide repeats are known by several different names including

microsatellite repeats, Single Sequence Repeats (SSR), Short Tandem Repeats (STR)

and Variable Number Tandem Repeats (VNTR). Thousands of STR-loci have been

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identified in the human genome and have been predicated to occur as frequently as

once every 15kb.

The 13 CODIS loci used in the U.S. are CSF1PO, FGA, THOX, VWA,

D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D8S51 and D21S11

whereas in U.K. and much of Europe utilize 10 core loci that includes additional

markers D2S1338 and D19S433 along with eight overlapping loci FGA, TH01, VWA,

D3S1358, D8S1179, D16S539, D18S51 and D21S11.These loci had become the

common currency of data exchange for human identity testing both in Forensic case

work and paternity testing largely because of their ease of use in the form of

commercial STR kits.(Butler 2006)

Human genome project studies had increased the presence of many more STR

loci than 10 years ago. Infact more than 20,000 tetranucleotide STR loci had been

characterized in human genome and there may be more than a million STR loci present.

However the current core loci had played and will continue to play a vital role

in human identity testing until commercial STR kits exist, which had further increased

the use of these STR loci.

1.13 STR-POPULATION STUDIES

In 2005, 365 population references had been made available in the internet at

http://cstl.nist.gov/biotech/strbase/population/popsurvey.html.

1.14 STR-BASE

The most comprehensive and widely used Internet resources on core STR loci

involved in human identity testing is the National Institute of Standards and

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Technology. Short Tandem Repeat database which was commonly referred as STR-

base (John M.Butler,- 1997, http://www.cstl.nist.gov/biotech/strbase).

The Scientific Working Group on DNA Analysis Methods (SWGDAM

formerly TWGDAM) provided guidelines for validation of PCR based DNA typing

markers, available on STR base.

In addition, Braian Burritt had developed a Microsoft Excel based program

called Omnipop that permits calculation of a user-inputed profile frequency using allele

frequencies from 166 published population survey. Omnipop can be downloaded at

http://cstl.nist.gov /biotech/strbase/population/omnipop150.4.2.xls.

1.15 LEGAL APPLICATION OF STR-DNA

The law enforcement community has greatly benefited from recent

development in the area of DNA testing as the Forensic DNA typing community has

standardized on the use of Short Tandem Repeat (STR) markers in human identity

testing (Butler 2004).

Other cases of widespread media attention were of DNA typing from remains

of victim of the world trade centre Twin Towers collapse following the terrorist attack

of 11 September, 2001, the O.J.Simpson murder trial, the President Clinton-Monica

Lewinsky Scandal and the identification of the remains in the Tomb of the unknown

soldiers (Butler 2005).

DNA-testing had been found to be extremely useful both in civil and criminal

justice system (Symbosis Law School, DNA test, 2007).

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The Great Britain had already started the use of DNA-test in legal cases first in

1987. Later, on most of the developed countries began to use this modern technology

based method in both civil and criminal justice system. (Zahidul Islam -2008)

Today, the vast majority of states in U.S. recognize the power of DNA-

evidence to determine actual innocence, years after other Forensic evidence had

proven flawed. The National Academy of Science and the National Institute of Justice

in U.S., have both established study groups that examine the quality of Forensic

evidence in the wake of wrongful conviction-convictions based on evidence less than

compelling and less accurate than DNA. (Chris Asplen, the DNA connection:

post conviction DNA-Testing,-2008).

In U.S., DNA test was introduced as prosecutorial tool in criminal justice

system in 1986. There are more than 130 labs both at state and local level that can

conduct the Forensic analysis. Other legislation like DNA identification Act 1994,

Transplantation of human organs Act 1994 and the Advancement of justice through

DNA-technology Act 2003. In addition, U.S. National Commission for the future of

DNA evidence was established in 1998.

The U.K. had also enacted Data Protection Act 1998 whereas New Zealand

and Canada had enacted criminal investigation act and DNA identification act 1998

respectively.

India started to avail the benefit of DNA-test in its criminal justice system

long ago. Now New Delhi High Court introduced the use of DNA-test in civil system.

By an order on 14 May 2008, Delhi High Court set legal precedent for the use of DNA-

test for determining paternity in case of child maintenance suit.

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However in India, there is no special enactment dealing with DNA profiling

as is there in other countries. But DNA-technology had made a drastic improvement in

the methodology of disputes of civil and criminal cases. In India, there are 21 well

established Forensic laboratories, four of them being administrated by the Central

Government. (Criminology and Forensic science, ID # 1012, DNA-Test)

1.16 PATERNITY (PARENTAGE) TESTING

Paternity cases are performed where the identity of the father of a child is in

dispute. These cases typically involve the mother, child and one or more alleged

fathers. A number of different laboratories perform parentage testing.

The determination of parentage is made based on whether or not alleles are

shared between the child and the alleged father when a number of genetic markers are

examined. Thus the outcome of parentage testing is simply exclusion or inclusion.

Paternity testing laboratories often utilize the same Short Tandem Repeat (STR)

multiplexes and commercial kits are as employed by Forensic testing laboratories.

However rather than looking for a complete one-to-one match in a DNA-profile, the

some of non-maternal or “obligate paternal allele” at each genetic locus is under

investigation.

1.17 AUTOSOMAL STR-ANALYSIS IN PATERNITY TESTING

Blood samples collected by finger prick on FTA (Flinter’s Technology of

Australia) paper (Burgone et al., 1994) from the paternity disputed individuals were

processed with the recommended protocols for DNA-extraction, quantification,

amplification and then separation and laser induced fluorescent detection of dye-

labelled polymerase products – the STR- amplicons using the capillary electrophoresis

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instrument such as 3100-Genetic analyzer (ABI-Prism 3100) with the help of STR-kits

available commercially, namely Applied Biosystem/promega kits.

The data collected on ABI-3100 Genetic analyzer was converted into

appropriately coloured peaks and STR-genotype information using gene scan and gene-

typer programme. (Butler 2001)

Table-1.2

Characteristics of common autosomal 15 STR-markers (Butler-2007)

STR Loci

Chromosomal

Location

Repeat Motif

Allele Range

PCR Product Sizes

with dye label

CSF1PO

FGA

TH01

TPOX

VWA

D3S1358

D5S818

D7S820

D8S1179

D13S317

D16S539

D18S51

D21S11

D2S1338

D19S433

Amelogenin

(sex-typing)

5q33.1

4q31.3

11p15.5

2p25.3

12p13.31

3p21.31

5q23.2

7q21.11

8q24.13

13q31.1

16q24.1

18q21.33

21q21.1

2q35

19q12

Xp22.22

Yp11.2

TAGA

CTTT

TCAT

GAAT

[TCTG][TCTA]

[TCTG][TCTA]

AGAT

GATA

[TCTA][TCTG]

TATC

GATA

AGAA

[TCTA][TCTG]

[TGCC][TTCC]

AAGG

Not applicable

6-15

17-51.2

4-13.3

6-13

11-24

12-19

7-16

6-15

8-19

8-15

5-15

7-27

24-38

15-28

9-17.2

Not applicable

305-342 bp (6-FAM)

215-355 bp (PET)

163-202 bp (VIC)

222-250 bp (NED)

155-207 bp (NED)

112-140 bp (VIC)

134-172 bp (PET)

255-291 bp (6-FAM)

123-170 bp (6-FAM)

217-245 bp (VIC)

252-292 bp (VIC)

262-345 bp (NED)

185-239 bp (6-FAM)

307-359 bp (VIC)

102-135 bp (NED)

X=107 bp (PET)

Y=113 bp (PET)

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1.18 PATERNITY DISPUTE IN TAMILNADU

Both criminal and civil cases of paternity disputes among population from all

over TamilNadu, received through both criminal and civil judiciary departments are

being undertaken for human identity testing at Forensic Sciences Department, Chennai,

India, a State Government organization that was started doing DNA-testing from 09-

09-1999.

An average of more than 100 paternity cases per annum, are being analyzed

and reported, as human identity testing in the Forensic laboratory of TamilNadu.

Earlier RFLP-based DNA-testing was employed. After 2000, multiplex PCR-

based 13 core STR-loci typing (13 STR-markers) and then 16 core STR loci typing (16

STR-markers) is being followed for paternity testing.

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FIGURE 1.1

SUMMARY OF PROCESS USED FOR FORENSIC DNA-TYPING

WITH STR-MARKERS

Collect sample and store

Extract DNA

Quantify Human DNA

PCR-Amplify

STR-Regions

Separate and detect STR

Amplicons

STR-Genotyping

Interpret results/perform

database search/generate

case work report

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1.19 BACKGROUND AND CENTRAL EFFORT OF THE PRESENT STUDY

The scholar, being employed as Forensic Scientist in Biological Sciences

division of Forensic Sciences Department, TamilNadu, India, having analyzed

biospecimens encountered in criminal cases for the past twenty four years, developed

the interest on the application of modern biotechnological tool-PCR based autosomal

STR-typing, employed in paternity testing for more than a decade, successfully with

good human identity testing results. As the STR-typing has become the work-horse of

modern Forensic DNA-Analysis and vital role as maternal evidence in criminal justice

system with high power of discrimination, it is planned to take up this study on the

comparative analysis of STR-DNA markers application in paternity testing in

TamilNadu with other Forensic testing.

The central effort of the present study was to probe the socio-economic status

of individuals of TamilNadu involved in paternity cases and studied the social

reasoning with caste-wise and age and literacy-wise distribution of paternity disputes.

Secondly, generated STR-DNA markers population data with allele

frequencies, alleles range, variant alleles, rare alleles and genotype frequencies of 15

autosomal STR-markers from the profiles of paternity cases received and reported

during the year 2004-2008 in TamilNadu Forensic Science Department, India.

Similarly, STR-markers based Forensic criminal DNA-typing and its

advantages over the other Biological Forensic testing, were also studied and presented.

Generally, the application of Forensic testing employed in human identity

testing relating to criminal justice system is carried out with the purpose of, not to

convict the guilty but also to exonerate the innocent being punished wrongly. Hence in

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the present study, had been evaluated the amazing potential of Forensic STR-typing

with the other Forensic biological testing.

Thirdly, like the population STR-DNA databases, generated from population

studies, that are already in use both in United States of America and United Kingdom,

STR-DNA population data generated from Tamil populations by the present study

could be used for the future Indian population. The STR-database in caste-wise pattern

could be used for comparison purposes to understand how frequent or how rare a crime

scene DNA profile may be in a particular population, like finger print records.