27 DNA Profiling and Individualization

Forensic DNA (Deoxyribonucleic acid) analysis

DNA is a chemical code which determines all the genetic attributes of a person. It is known that DNA is unique for all individuals except identical twins. Forensic DNA analysis revolves around this property of DNA. Moreover DNA can be obtained from biological samples such as blood, saliva, hair etc found at crime scene. This can be used to examine relationship between two individuals and to verify identity of deceased person.

DNA is a double helix structure which consists of deoxyribose sugar and phosphate backbone held together by cross links based on complementary base paring between bases (Adenine pairs with thymine and guanine pairs with cytosine).

Forensic DNA analysis involves use of DNA marker obtained from biological sample found at crime scene. DNA database have been compiled by legal authorities of various countries which list abundance of a particular fragment of DNA in the population. From this information, comparison of DNA marker obtained from crime scene (either suspects or victims) with DNA marker of known suspect can be made to reveal identity of suspect. Statistical interpretations are required to estimate the thus likelihood that material obtained from crime scene belongs to particular individual aiding in individual identification.

Therefore, forensic DNA analysis aids in identification of perpetrators especially involved in crimes such as gang rape cases, disaster victims. It also plays a crucial role in paternity testing.

DNA profiling

DNA profiling basically refers to sequencing of an individual’s DNA. Also known as DNA typing, it is a revolutionary technique utilized by forensic scientist for individual identification based on their DNA characteristics. It is different from full genome sequencing. DNA profiles are generated from biological material found at crime scene. DNA profile can be generated by techniques which basically utilize repetitive sequences which are highly variable such as Short Tandem Repeats (STRs).

When the DNA profile of a reference sample found at the scene of crime matches with DNA profile of a known suspect included in national forensic DNA databases, then the suspected DNA profile is termed as an “inclusion” and if DNA profile of suspect does not match profile obtained from biological sample found at crime scene then it is termed as “exclusion”.

This use of DNA for identification is referred to as “DNA profiling”.

Individualization

When the two samples i.e. an evidence item and reference item belong to common source of origin, then evidence item is said to have been individualized & the process is called as individualization.

It is important to differentiate between class characteristics and individualizing characteristics. Class characteristics are developed as a result of controlled process while individualizing traits are not created by controlled process but are result of random actions.

Fingerprint of two individuals are not identical, this is true even for uniovular twins. Thus matching of latent print found at crime scene with “reference print” stored in computer database provides a means of identification of perpetrator with reduced chances of error. Similarly if DNA profiles developed from biological material, match with DNA profile of known suspects accurately, this is termed as inclusion. Mutations give rise to genetic variability in DNA and hence no two individuals have identical DNA configurations.

Therefore individualization is based on characterization of those individuals which are so rare that cannot be duplicated by chance alone.

History of DNA Profiling (or DNA Typing)

Alec Jeffery’s (1984) developed the technique of DNA fingerprinting based on RFLP (Restriction Fragment Length Polymorphism) analysis aiding in personal identification. The development of Polymerase Chain Reaction (PCR) technique further diversified the role of molecular biology in forensic science. PCR – based DNA profiling procedure technique have become integral part of many forensic science laboratories all over the world, this method was more easier than RFLP technology & less error prone than other blood – typing methods; moreover limited amount of DNA could be analyzed accurately to identify the suspect.

DNA profiling or DNA typing procedures were first utilized to solve paternity disputes & immigration cases. DNA was first used to aid in criminal investigation through DNA fingerprinting technique in 1986. The technique was used to link semen stain sample collected from two different crime sites in UK. The result obtained linked the two semen samples and caused conviction of Colin Pitchfork.

Mitochondrial DNA (mtDNA) and Y- chromosome DNA help to develop profile which not only have immense importance in forensic DNA analysis but also are important tools to study ancient migratory pattern of our ancestors. There DNA typing systems play a crucial role in solving so called volume crimes and aid in identification of deceased victims in mass disasters & serial homicides. These markers also help in resolving paternity disputes and help in establishing kinship.

DNA typing techniques are also used to generate DNA profiles in case of endangered animals such as cheetah to track their migration & breeding pattern.

DNA profiling techniques

Various techniques and systems have been utilized for forensic DNA typing of reference sample. Initially RFLP technology was utilized, but with advent of PCR technique new system have developed which has revolutionized forensic DNA analysis. Nowadays Short Tandem Repeats (STRs) (included both autosomal and Y-chromosome) multiplex systems are frequently used for forensic DNA analysis. The techniques used for DNA profiling can be grouped broadly as:

(1) RFLP analysis

(2) PCR based analysis

1. RFLP analysis

RFLP (Restriction Fragment Length Polymorphism) analysis was first method employed for forensic DNA analysis of reference sample which was recovered from biological material (found at crime scene); followed by restriction digestion of the DNA sample. The resultant DNA fragments produced were of different sizes (because of variation between DNA sequences of different individuals). These DNA fragments were separated using gel electrophoresis. The separated fragments were transferred to filter membrane (nylon or nitrocellulose). This process is referred to as Southern blotting. Radio- labelled probes are added which bind to DNA fragments containing repeat sequences. On exposure to X– ray film, probe – bound DNA fragments appear as dark bands on the film. Therefore, this technique determines variation in length of particular DNA fragment. RFLP Loci exhibit many variations at a particular locus; thus RFLP analysis exhibit highest degree of discrimination per locus. But the Southern blot technique is difficult to operate and takes much more time in comparison to recent techniques i.e. it is also a laborious technique. It also requires good amount of non-degraded DNA sample thus has limited utility in forensic science because DNA samples obtained from biological material found at crime scene are often limited in both quality and quantity.

2. PCR  based Analysis

Polymerase chain reaction technique was developed by Kary Mullis in 1983; for which the inventor received Nobel Prize in 1993. It is an enzymatic process that allows a targeted sequence of the DNA molecule to be amplified across several orders of magnitude; without affecting the surrounding regions. It is basically a molecular photocopier. PCR based DNA analysis exhibits lesser variations per locus in comparison to RFLP analyses. But this technique has immense utility in forensic cases as DNA samples obtained in such cases are of limited quality and quantity. Taq polymerase replicates a DNA template strand with the help of DNA primers and Deoxynucleotide triphosphates (DNTPs).

This enzyme only replicates DNA sequences and does not digest or cut DNA at specific sites. There are three main steps involved in PCR process:

• Denaturation Double stranded (ds) DNA is exposed to high temperature (94 – 9   C) leading to separation of two DNA strands and single stranded (ss) DNA is produced.
• Annealing: The temperature of this step is largely determined by melting temperature of DNA primers. Primers are basically oligonucleotide sequences complementary to three prime (3’) end of both sense and anti- sense DNA strands. Primers bind to DNA template strand at specific – site and form stable DNA – DNA hydrogen bonds as a result of complementary base pairing. Two different primes define the end point of particular targeted sequence that is to be amplified.
• Extension: Taq polymerase replicates DNA sequence with the help of DNTPs to create new DNA strands. The temperature of this step is usually– C for optimal activity of taq polymerase. The nucleotides-adenine, granite, cytosine and thymine- are used as a building block to develop a complimentary strand to template strand. At the end of each PCR cycle,  targeted segment is duplicated. This process/ steps are repeated several times to generate  multiple copies of DNA segment of interest. PCR technique cannot be used for analysis of long strands of DNA containing thousands of bases. DNA profiling procedures improved  significantly as several loci could be analysed  simultaneously  using  PCR  technique.  In   recent times, research in human  DNA quantification has focused on “real-time” quantitative PCR techniques. Thus DNA analysis could be to determine kinship including paternity testing.

HLA DQ-alpha reverse dot blot system

HLA DQ-alpha is name of locus that is analyzed for sequence polymorphism. The variation is detected with help of probes which target particular sub region within this locus. HLA – DQ alpha reverse dot blot strips were easily to use and results were obtained in relatively short span of time (rapid analysis). The pattern of dots between typing strips was compared in order to indicate the same origin of two samples.

Amplitype PM+ DQA1

Under this system several markers at different loci were analyzed at same time; a process porn as multiplexing. The loci involved in this system are associated to sequence polymorphisms that are detected by hybridization to sequence-specific oligonucleotide (SSO) probes. The power of discrimination is more limited than RFLP analysis because limited number of alleles is present in the population. It involves simultaneous PCR amplification of six loci involved using primers that have been labelled with biotin, which is followed by hybridization of PCR products using reverse dot-blot method. At the end, there is colorimetric detection of the hybridized PCR product – biotin / HRP- streptavidin complex. This system is also referred as polymarker.

D1S80

This system was based on amplified fragment length polymorphism (AFLPs) analysis. D1S80 is the DNA locus that is typed. Variations were present in the length of defined DNA fragment but this fragment can be readily amplified by PCR technique; owing to its relatively small size. D1S80 system basically combined the ability to analyse limited quantity and quality DNA through amplification with variations evidenced in length based system. Thus, any allelic differences between individuals will be evidenced by different lengths of the amplification product among individual tested. It involved PCR amplification of a region of DNA containing polymorphic sequences (at D1S80 locus) and separation of amplified products by electrophoresis and analysis of resulting pattern by silver staining or UV transillumination of ethidium bromide to determine inclusion or exclusion.

Short Tandem Repeats (STRs)

STRs are also referred to as microsatellites. They are basically short lengths of non- coding region of human genome having repetitive sequences where each repeat unit is of 2- 5 base pairs (bp), repeated multiple times. As different individuals have different number of repeat unit, STRs can be used to discriminate between unrelated individuals. STRs sequences can be simple, compound or complex based on complexity of repeat unit. STRs are found or both autosomal and sex chromosomes. STRs found on Y – chromosome exhibit less diversity when compared to STRs on other chromosomes become they lack recombination. A particular STR locus is moderately polymorphic and many such loci are used and examined simultaneously.

Different STR – based DNA profiling system are used worldwide. In UK, forensic science services utilize the SGM plus TM system that utilizes l0 autosomal STR markers while FBI utilizes 13 markers. The DNA profiles are then stored on computer database – National DNA database (UK) and Combined DNA Index System (America). The marker sequence can be amplified by PCR and amplification of several loci is performed at same time. Each STR locus is associated with small number of alleles & can be compared to an allelic ladder run on the same gel for comparison & analysis. STRs are most commonly analyzed using florescent detection and automated analysis. STR analysis is less prone to problem of DNA degradation than RFLP analysis; as former technique is based on identification of much shorter sequences than the latter technique.

Commercial testing kits are available for these loci and the kits also include a marker at amelogenin locus to aid in sex determination.

Y – Chromosome Short tandem Repeat (Y- STRs) Markers

Since Y- chromosome is specific to males, Y – STR markers are very useful in sex assault cases which involve mixed stain analysis that contain DNA from both the male perpetrator and the female victim. Y – STR analysis enables DNA profile to be made in case of sexual assault in which man did not sperm because of medical condition. It can also be helpful in determining number of male donors in gang rape case. Commercial kits are currently available for six different Y-STR markers.

Y- STR markers show lesser variability than autosomal STR markers hence their discriminatory power is less. As Y–chromosome lack recombination it helps to generate markers for tracing paternal inheritance. Thus Y – chromosome and mitochondrial DNA markers are good lineage markers used for genealogical and anthropological studies.

Mitochondrial DNA (mtDNA) markers

Mitochondria are intracellular organelles that also contain genetic material besides nucleus and generate energy that is needed by cells to survive. Human mitochondrion DNA (mtDNA) is circular DNA molecule that contains 16569 bp coding for 34 genes which in turn code for synthesis of two ribosomal RNA (rRNA), 13 polypeptides and 22 transfer RNA (tRNA). MtDNA exhibits maternal inheritance i.e. offspring receive mtDNA from their mother. MtDNA sequences are highly variable between unrelated individuals and exhibit high mutation rate. Displacement (D – loop) loop region of mitochondrial DNA exhibits high mutation rate in comparison to nuclear DNA.

Hypervariable region 1 (HV 1) and Hypervariable region 2(HV 2) of D- loop are examined in mt-DNA analysis by PCR amplification using specific primers.

The mt- DNA sequence of all mitochondria in an individual is usually identical; a condition termed as homoplasmy. However in certain individuals, differences in base sequence are found at one or more locations; resulting in two more different types of mitochondria. This condition is termed as heteroplasmy. Heteroplasmic condition can significantly affect forensic investigations.

MtDNA analysis is frequently preferred in cases where samples do not contain much DNA or in case where STR DNA analysis fails. As there are multiple mitochondria in a single cell and each cell contains multiple copies of mtDNA therefore it is possible to extract far more mtDNA than nuclear DNA.  MtDNA is utilized to type the dead cells in hair shafts; bones and teeth.

SNP (Single Nucleotide Polymorphism)

SNPs results from differences in single base unit. They occur throughout the genome including both autosomes & sex chromosomes. Every individual have unique pattern of SNPs. Using mini-sequencing technique the base at a given SNP can be determined and once the bases at several sites at different loci are known; profiles similar to STR profile can be generated. Microarray hybridization technique helps to examine multiple SNP loci at same time. SNP analysis requires small quantity of sample and can yield information even from degraded DNA sample.

Forensic Application of DNA Profiling

DNA profiling with Short Tandem Repeat markers have been utilized in various aspects of human identification. DNA profiling is used to identify both victims and perpetrators involved in serious crimes such as homicide and sexual assaults. Highly similar STR profiles exhibit involvement of closely related individuals, requiring a strong statistical interpretation.

DNA profiling is also used in Disaster Victim Identification (DVI). STR Profiles developed from human remains is matched to ante mortem sample belonging to the victim or profile similarities with genotyped relatives also aid in identification of deceased person.

DNA profiling is also for paternity testing to establish family relationships among people claiming right on property. In numerous cases, individuals can be ruled out from involvement in crimes or family issues based on STR profiles.

DNA databases for particular Y-STRs have been developed in various countries all over the world which is required for statistical interpretation of Y-STR profile matches. Y-STR profiles have ability to specifically identify male individuals involved sexual assault crimes & gang rapes.

DNA profiling can be used in insurance company fraud investigations & to examine wildlife poaching cases.

SUMMARY

DNA profiling helps to identify individual on the basis of individual DNA properties. It basically examines repetitive sequences that are variable such as (VNTRs) variable number of tandem repeats. Earlier RFLP technology was used in DNA profiling but with development of PCR technique forensic DNA analysis was based on PCR – based DNA examination techniques and system.

DNA profiling with sets of autosomal short tandem repeat (STR) marker is commonly applied in today’s world for proper human identification. Moreover DNA profiling with Y – STR markers have been useful for identification of male perpetrators involved in rapes & sexual assaults. Y – STRs and mtDNA markers also help to investigate mass migrations and ancestral origins. DNA profiling or DNA typing system are extremely useful in mass disaster where multiple fatalities are distributed over large areas & this technique helps in identification of deceased victims. Thus a successful identification of skeletal remain can be established by DNA profiling.

A DNA sample obtained from biological material can more accurately establish individualization of biological material of human origin because it is more resistant to degradation than other evidences left at crime scene such as blood, semen, saliva or fingerprints used to establish identification of an individual. But the collection and preservation of evidence plays an important role in establishing the success of forensic DNA analysis.