12.1: Forensic Genetics

• The foundations of forensic genetics were laid down when Karl Landsteiner described ABO blood group systems in 1901.

• The detection of other red cell antigens, serum proteins and erythrocyte enzymes made the serological analyses of blood and other body fluids possible.

• By 1980, a battery of conventional blood grouping tests were available which considerably improved the forensic utility especially when used in conjunction with the white blood cell antigen system, HLA.

• In 1985, a breakthrough came when Sir Alec Jeffrey’s of United Kingdom described that a set of DNA markers called Variable Number of Tandem Repeats (VNTR) were much more variable among humans and these were immediately applied to forensic cases about human identification.

12.2: DNA

• DNA is the biological blueprint of life.

• The structure of deoxyribonucleic acid (DNA) was described by James Watson and Francis Crick in 1953.

• DNA was determined to be a right-handed double helix.

• DNA is composed of repeating subunits called nucleotides.

• Nucleotides are further composed of a phosphate group, a sugar, and a nitrogenous base.

  • Adenine

  • Guanine

  • Cytosine

  • Thymine

• Nuclear DNA is inherited equally from both mother and father.

• Mitochondrial DNA is inherited only from the mother, and therefore it can be used to match with the maternal lineages.

• The genetic makeup of every individual established at the time of conception is unique.

  • It defines that an individual’s genetic characteristics contain many polymorphisms that can be used for human identification.

Advantages of Using DNA for Identification

• DNA is ubiquitous, it is present in all the nucleated cells of the body.

• The DNA makeup of a person is the same in all the cells of the body and cannot be altered.

• The DNA of every person is unique in its profile.

• DNA can be extracted from all body fluids and all the tissues of the body.

• In post-mortem cases, DNA can be obtained from body tissues.

• In cases where the body has been buried, DNA can still be obtained from body tissues.

• In burnt and charred remains, DNA can be obtained from hard tissues like bones and teeth.

• DNA can be stored in small quantities easily as compared to other evidentiary materials.

• DNA can be stored for very long periods without deterioration if stored appropriately.

• DNA test detects genetic makeup whereas blood or protein tests are genetic products.

• DNA methods avoid any complications of dominance or recessives.

• DNA does not combine and thus can detect the number of persons at the crime scene if they have contributed to it.

12.3: The Basics of Molecular Biology

• There are 3 billion base pairs (bp) in a single copy of the human genome.

  • These are arranged in compact structures, which we all know as chromosomes.

• There are 23 pairs of chromosomes in all the cells of humans and so are called diploid cells.

• Only in the gametes, one copy of each chromosome is present and these are called haploid cells.

• The DNA in the chromosomes is arranged as coding and non-coding regions.

• Introns — functional portions of the genes.

• Exons — non-functional regions of the genes.

• Loci — polymorphic markers have been detected in the areas of the human genomes.

• Alleles — an alternative form of the marker at a particular locus.

• Homozygote — it is if the alleles are the same.

• Heterozygote — it is if the alleles are different.

• Genotype of the Person — allelic configuration at a locus.

• Profile of the Person — genotypes at different loci.

12.4: Tandemly Repetitive DNA

• Tandemly Repetitive DNA: Segments of DNA are arranged as a particular sequence being repeated more than once, a sequence GGGCCCTTAA might be repeated many times.

• Variable number of tandem repeats (VNTRs) — minisatellite polymorphisms in many tandemly repeating units of a particular sequence, typically 16-80 bp long.

• Polymerase Chain Reaction (PCR) — an invitro-molecular photocopying process that generates millions of copies of the target DNA sequence, the boundaries of which are defined by synthetic oligonucleotide primers that are complementary to the 3' ends of the sequence.

• Denaturation of DNA: The two strands of DNA are wrapped around each other, for replication the DNA has to unwind to have a single strand available for the synthesis of the new strand.

• AmpFLPs — A smaller minisatellite loci that had alleles from 9-15 bp, composed of short core repeat units.

• Short Tandem Repeats (STRs) — microsatellites that are an abundant class of DNA polymorphisms.

  • Occurring every 300 to 500 kb in the human genome, these markers have a repeat unit of 1-6 bp in length and are highly polymorphic.

  • STRs form the basis of forensic DNA analysis.

12.5: Short Tandem Repeat DNA Profiling

• Multiplex PCR — this is where each primer pair would amplify a specific sequence or STR.

• The first multiplex PCR kit was developed by the Forensic Science Services (FSS) of the UK and it comprised four STR loci, THO1, vWA, FES/FPS, and F13A1.

• The FSS then launched its second generation multiplex (SGM) comprising of six STR loci THO1, FGA, D8S1179, D18S51 and D21S11.

12.6: Classification of STRs

• STRs can be classified on the basis of length of the repeat unit so there are di, tri, tetra and penta nucleotide STRs, depending on the repeat unit size of two, three, four, or five nucleotides.

Types of STRs

1. Simple consisting of 1 repeating sequence — an STR locus has several alleles but each allele differs only in several repeats but the sequence of the repeats is the same.

2. Simple with non-consensus alleles — consider two alleles of locus HUMTHO1.

   • Allele 3: [AATG] 3

   • Allele 4: [AATG] 4

3. Compound with non-consensus alleles — considers two alleles of the locus vWA.

   • Allele 16: TCTA [TCTG] 4[TCTA] 11TCCATCTA

   • Allele 16: TCTA [TCTG] 3[TCTA] 12TCCATCTA

4. Complex repeats — consider two alleles of locus D21S11

   • Allele 25: [TCTA] 4 [TCTG] 3 [TCTA] 3 TA [TCTA] 3 TCA [TCTA] 2 TCCA TA [TCTA] 10

   • Allele 26: [TCTA] 4 [TCTG] 6 [TCTA] 3 TA [TCTA] 3 TCA [TCTA] 2 TCCA TA [TCTA] 8

5. Hypervariable repeats: SE33. Few loci have not only different repeat regions; they also have them arranged in many different ways. SE33 is an example.

12.7: STR Mutational Mechanism

• The forensic value of STRs is due to their high levels of polymorphisms. These polymorphisms are in part due to the process of mutation occurring in these genomic sequences which generate different alleles at a locus.

• Sometimes, ‘slipped strand mispairing’ leads to a mutation in regions of the genome, having abundant simple repetitive sequences which have been recognized as the major mechanism involved in their generation.

• During this process, the gain or loss of a repeat region occurs depending on the looping out of the newly synthesized strand or the template strand.

• It is important to understand that the actual mutation rates for the STRs are difficult to determine as the mutations are rare events and generally there is no consensus regarding them.

12.8: STR Resolution Techniques

• Electrophoresis has remained the cornerstone of STR allele detection, although the STR alleles can be separated by agarose gel electrophoresis with ethidium bromide staining, polyacrylamide gel electrophoresis (PAGE) gives better separation of the alleles.

• DNA sequence using fluorescent dyes in 1986.

• The automated systems such as ABI GeneScan® allowed electrophoretic information to be stored and tabulated as the alleles migrated through a gel matrix and pass a laser detection window.

• Multiplexing — a technique where several STR loci are amplified in a single tube PCR.

• Allelic ladder — consists of several alleles of an STR system and is used as a reference to designate the alleles.

  • It can be thought of as a ruler which can measure a segment; an allelic ladder measures the size of the allele reference to the allele present in the ladder.

• Genotyper — the program would call the allele based on the established windows, which is called the ‘absolute window method’.

  • The same method could be used to designate the alleles where the crime scene sample is to be compared to control samples.

12.9: Environmental Contamination

• The sensitivity PCR renders the microsatellite systems prone to contamination.

• Possible sources of contamination include, environment, contamination from a previous PCR and between samples during preparation.

• The latter two sources of contamination could be controlled by appropriate laboratory procedures and designated working areas.

• Environmental contamination is limited.

12.10: Acceptance of DNA Evidence at the Legal Forum

• The use of DNA analysis in forensic investigation offers the potential for the identification or exclusion of a suspect with specificity, which was rarely possible in the past.

• The tool is especially useful in those difficult cases in which the only evidence recovered is biological, such as seminal fluid, or blood.

  • Maternity identification — is necessary to identify the mother in cases of child abandonment or infanticide or swapping of neonates.

  • Paternity identification — is important in cases of sexual assault in which pregnancy occurs and pregnancy is either terminated or goes to full term.

• In 1985, the forensic use of DNA began in the UK and it was applied to a civil immigration case and then to a criminal case which established the ground for the application of DNA analysis to forensic cases.

• By 1990, the systems for DNA profiling were standardized and most laboratories on both sides of the Atlantic were using them.

  • The methodology was termed sound in the first report of the National Academy of Science’s National Research Council. UK pioneered DNA technology, and acceptance of the technique by the legal forum was initially smooth.

• As the DNA evidence became acceptable in the UK the FSS quickly moved to PCR-based technology and developed a quadruple system consisting of four STR loci.

  • The Home Office, UK then commissioned the FSS for the development of a National DNA Database, and using a more discriminating Second Generation Multiplex system, the database was started to be developed in 1995.

  • The FSS has started profiling criminal cases using the 11 loci AMPFLSTR® SGM Plus kit.

  • It also provided the necessary powers to the FBI Director to improve the standard of DNA analysis by ensuring quality assurance, appointment of a DNA advisory board, and constructing a national DNA databank.

12.11: Standardization of STR Nomenclature and Technique

• There was a need for a common nomenclature for the widespread use of STR systems.

• The basic recommendations of the International Society of Forensic Genetics are that the alleles would be designated according to the number of the repeats.

• The International Society of Forensic Genetics as well as the European Network of Forensic Science Institutes has been performing a pivotal role in these standardization efforts through regular inter-laboratory exercises and publication of its recommendations.

12.12: Population Genetics

• It is important to understand that forensic identification using DNA markers can only be done if a real estimate of the frequencies of those markers in the populations concerned is possible.

• Since real estimates are not possible due to large numbers, a sample of the population is examined and population frequencies of the markers are estimated for application to that population.

• These frequencies are kept as a database, and it has been recommended that at least 100 profiles should constitute such databases of all regional populations.

• The frequencies of the markers are important, as these are to be used in calculating the probability of discrimination and/or match by multiplying them together.

• In 1989, some leading scientists, who started a debate on the statistical basis of calculating the population frequencies, argued that the use of a general racial database is incorrect and databases from the relevant ethnic population should be used in all cases.

  • It was also stated that the frequencies of the genotypes could be multiplied to generate a profile frequency if the loci were independent.

12.13: Y-Chromosome Polymorphism

• Most of the Y-chromosome does not undergo recombination during meiosis, the paternally inherited chromosome bears the genetic prints from the father to son along the whole paternal lineage.

• In forensic practice, male-specific Y-chromosome markers could be used as an adjunct to other markers on the autosomes and could prove to be a new and useful forensic tool on their own as well.

• Y-chromosome contains a few polymorphic minisatellites like MSY1 and 2 as well.

• These markers are hypervariable but due to the simplicity of the use and availability of several Y-chromosomes, STR systems have not gained widespread use for forensic purposes.

Development of Y-Chromosome Specific STR Systems

• DYS19: Described as the first STR on the human Y-chromosome, which was found to be polymorphic and suitable for sex and paternity determination in deficiency cases.

• Most of these loci were found to be polymorphic and the haplotypes defined by using all of them could achieve very high levels of individualization in males.

• The haplotypes were designated as Yh1 to Yh5 depending on the markers used to obtain the haplotype.

12.14: Forensic Applications of Y-STRs

• Y STR analysis can be valuable in mixture interpretation in multiple rape cases or the detection of male specific profile in azoospermia/vasectomized male suspects when spermatozoa are not available.

• It could determine the male component in the male/ female mixture specimens in cases where the specimen was small, or the differential lysis failed, or in other body fluid mixtures where the differential lysis could not be attempted.

• The value of Y- Y-chromosome STR polymorphisms has been determined as excellent for paternity testing in deficiency cases.

• The Y-linked loci have been proven to be better exclusionary tools in paternity cases than the autosomal markers, though their value in identification might not have an edge over the autosomal loci.

12.15: Y-STR Multiplexing Strategy

• Multiplex amplification of several loci allows simultaneous amplification of many STR systems, which conserves the usually small samples submitted for forensic analyses.

• The multiplex systems are mostly used as both are robust and reproducible and, together, allow seven Y STRs to be analyzed.  These systems are therefore favored at the moment.

• A new multiplex capable of amplifying seven Y STR loci, the ‘Y-PLEXTM 6’ has been recently developed by a commercial firm Reliagene, Technologies, Inc. USA.

12.16: Phylogenetic Value of Y-Chromosome Specific STRs

• Y-chromosome markers have been utilized as a tool for studying phylogenetic relationships and haplotypes based on Y markers have shown greater differentiation than autosomal or mtDNA markers.

• Population specific Y haplotypes have been described based on such markers, which is of interest for phylogenetic as well as forensic purposes.

12.17: Detection of DNA

Areas to Look for DNA on or off the Crime Scene

• Fingernails or nail clippings.

• Tissues, paper towels, cotton swabs, or ear swabs.

• Toothpicks, cigarette butts, straws, and anything which might have come in contact with the mouth like cellular phones.

• Blankets, pillows, sheets, dirty linen, caps, and headgear of any type.

• Eyeglasses, contact lenses.

• Used stamps, and envelopes.

• Ligatures found on the body or the scene.

• Bullets that have passed through a body.

Collection, Storage, and Transport of DNA Evidence

1. Wear clean latex gloves while collecting each item of evidence.

2. Each item of evidence must be packed in a separate container or envelope.

3. Blood, semen, saliva, urine, and other stains must be air-dried before packaging. Items of evidence having such stains might be dried using a hair dryer or a fan. After drying, the samples should be packed in a paper bag or envelope.

4. In the case of condoms, these should be placed in a sterile tube.

5. If stains are to be removed from an unmovable surface:

   1. Photograph the surface with a ruler using black and white as well as color film.

   2. Using a sterile moist swab, rub the swab on the stain till it is transferred to the swab; more than one swab might be required for this purpose.

12.18: Appendix I

1. Match Probability — the probability that the two randomly selected individuals will have identical genotypes. Where PM is the match probability, pk represents the frequency of each distinct genotype, and m is the number of distinctive genotypes.

2. Power of Discrimination — the probability that two randomly selected individuals will have different genotypes. This is the reciprocal of the probability of a match.