Forensic DNA Profiling - 2 Chapter Notes | Forensic Medicine and Toxicology (FMT) - NEET PG PDF Download

Environmental Contamination

Environmental Contamination is a concern when using microsatellite systems due to the sensitivity of PCR. Contamination can arise from various sources, including:

• Environmental factors
• Previous PCR processes
• Cross-contamination between samples during preparation

While the last two sources of contamination can be controlled through proper laboratory protocols and designated work areas, environmental contamination has been found to be minimal.

Acceptance of DNA Evidence in Legal Proceedings

The introduction of DNA analysis in forensic science has revolutionized the ability to identify or eliminate suspects with a level of precision that was not possible in the past. This method is especially crucial in difficult cases where the only available evidence is biological in nature, such as blood or seminal fluid.

Maternity and Paternity Identification

• Maternity identification is essential in situations involving child abandonment, infanticide, or cases where infants are mistakenly swapped.
• Paternity identification plays a vital role in sexual assault cases that result in pregnancy, regardless of whether the pregnancy is terminated or carried to term.

The forensic application of DNA began in the United Kingdom in 1985, initially in a civil immigration case before being applied in a criminal context. This pioneering work set the stage for the use of DNA analysis in forensic investigations. The technique underwent scrutiny for its forensic potential and was found to be appropriate. By 1990, DNA profiling systems became standardized, with widespread adoption in laboratories across the UK and the US. The methodology received validation in the early report by the National Academy of Sciences’ National Research Council (NRC Report I, 1992). While the UK made significant strides in utilizing DNA for forensic purposes, it is crucial to acknowledge that the technology's development was a global endeavor.

Initially, legal forums embraced DNA evidence without major issues. However, difficulties emerged regarding the statistical methodologies employed in various cases, including R. v Andrew Deen (Times Law Report 1994), R. v Denis Adams (Cr. App. R. 1996), and R. v Alan James Doheny and Gary Adams (Cr. App. R. 1997). These landmark cases played a pivotal role in establishing the protocols for presenting and accepting DNA evidence in UK courts.

As DNA evidence gained traction in the UK, the Forensic Science Service (FSS) quickly integrated PCR-based technology, creating a quadruplex system utilizing four STR loci. The UK Home Office subsequently commissioned the FSS to develop a National DNA Database, which commenced in 1995 with the advanced Second Generation Multiplex system. In recent developments, the FSS has been profiling criminal cases using the 11 locus AMPFLSTR® SGM Plus™ kit from Applied Biosystems in the USA.

In the United States, the passage of the DNA Identification Act of 1994 by Congress played a significant role in the acceptance of DNA evidence. This legislation enabled the FBI Director to improve DNA analysis quality through various measures, including quality assurance, the establishment of a DNA advisory board, and the creation of a national DNA databank. Consequently, the CODIS database for STRs was established in the US, comprising 13 loci, including a gender marker.

Standardization of STR Nomenclature and Technique

STR (Short Tandem Repeat) technology has become increasingly important in forensic science, necessitating the standardization of its various aspects. This standardization ensures that laboratories across different countries and regions can share and utilize data for legal and population genetics purposes.

Nomenclature is a crucial aspect of this standardization. The International Society of Forensic Genetics (ISFG) and the European DNA Profiling Group (EDNAP) established guidelines for naming alleles in STR systems. Alleles should be identified by the number of repeats they contain. In cases of incomplete motifs or complex repeats, these should be represented by the number of complete repeats, followed by a decimal point and the number of bases in the incomplete motif.

STR Classes

STRs are classified into various classes based on the arrangement of the repeat region in the alleles of a specific system. The classes include:

• Simple
• Simple with non-consensus alleles
• Compound
• Complex
• Complex hypervariable systems

The resolution of STRs is further enhanced by:

• Fluorescent technology
• Using sequenced allelic ladders to designate alleles
• Employing standard multiplex sets of STRs

These standardization efforts by the forensic community significantly boost the confidence of both the scientific and legal communities in STR technology. The ISFG and the European Network of Forensic Science Institutes play a vital role in these efforts through regular inter-laboratory exercises and by publishing recommendations.

Population Genetics

Forensic identification using DNA markers relies on accurate estimates of how frequently these markers occur in specific populations. Since obtaining exact figures is impractical due to the large number of individuals, a sample of the population is assessed to estimate these frequencies for practical use. These frequencies are then stored in a database, with a recommendation to include at least 100 profiles to represent all regional populations.

Importance of Marker Frequencies

• The frequencies of the markers are crucial for calculating the probability of a match or discrimination by multiplying these frequencies together.
• Population genetics plays a vital role in forensic genetics.

In 1989, prominent scientists initiated a discussion about the statistical methods for calculating population frequencies. They argued against relying on a general racial database, advocating instead for the use of databases from the relevant ethnic groups in each case. They also noted that if the loci were independent, the frequencies of the genotypes could be multiplied to create a profile frequency. This led to challenges regarding DNA evidence, although the technology and its statistical foundation withstood legal scrutiny.

National Academy of Sciences Report

• The National Academy of Sciences in the USA, in its second report (NRC Report II: The Evaluation of Forensic DNA Evidence, 1996a), further examined population genetic issues.
• It recommended methods for assessing population substructure and the likelihood of coincidental DNA matches.
• It also established standards for the collection, preservation, and presentation of DNA evidence.

Y-Chromosome Polymorphisms

• Recombination during meiosis is a process that usually does not occur in most of the Y-chromosome. This is important because it means that the Y-chromosome inherited from the father carries genetic information that has been passed down from father to son throughout the paternal lineage without change.
• In forensic practice, markers specific to the Y-chromosome can be used to identify individuals along with other markers found on the autosomes. These Y-chromosome markers can also be useful on their own in forensic investigations.
• The Y-chromosome contains a few polymorphic minisatellites, such as MSY1 and MSY2. These markers are highly variable, meaning they can differ a lot between individuals. However, their forensic use is limited because of their simplicity and availability.
• Y-STR systems, which are used in forensic contexts, involve analysing short tandem repeats on the Y-chromosome. However, the application of these systems may be restricted in some cases.

Development of Y-Chromosome Specific STR Systems

• 60 per cent of the Y-chromosome region on the long arm contains interspersed tandem repeats.
• DYS19 was the first STR identified on the human Y-chromosome, recognised for being polymorphic and useful in sex and paternity determination in specific cases.
• Various Y STRs were later identified, with most loci being polymorphic as well.
• The haplotypes (the profile of an individual at two or more loci) created using these STRs can provide a very high level of individualisation for males.
• Understanding the terminology for Y STR haplotypes (a set of STRs) is essential.
• These haplotypes were named based on the specific markers used to create them.
• The power of discrimination for a haplotype made up of seven STRs— DYS19, 389I, 389II, 390, 391, 392, and 393 (Yh1)—is high across different populations.
• A ten-marker haplotype (Yh4), which includes the Yh1 loci and DYS385, YCAII, is also suitable for human identification.
• Most of these markers have been used to create population databases worldwide for forensic applications.
• Recent studies have described various new Y STRs and validated them for forensic use over the past two years.

Forensic Applications of Y STRs

Y STR analysis is beneficial for forensic identification in situations where autosomal STRs are less effective. Its applications include:

• Interpreting mixtures from multiple rape cases.
• Detecting male-specific profiles from azoospermic or vasectomized male suspects when spermatozoa are not available.
• Determining the male component in male/female mixture specimens in cases with small specimens, failed differential lysis, or other body fluid mixtures where this method is not feasible.

Paternity Testing

Y-chromosome STR polymorphisms are highly useful for paternity testing in cases where there is a deficiency:

• Y-linked loci can serve as important exclusionary tools in paternity cases, although their effectiveness may vary compared to autosomal markers.
• In situations where the father is unavailable, other paternal relatives can be tested using Y STRs.

Table 12.1 presents some of the Y-chromosome STR markers validated for forensic purposes.

Y STR Multiplexing Strategies

• Multiplex amplification enables the simultaneous amplification of multiple Y STR systems, which is crucial for conserving the typically small forensic samples used in forensic analyses.
• The multiplex systems developed by Prinz, M. et al. and Kayser, M. et al. are preferred due to their robustness and reproducibility.
• These systems facilitate the analysis of seven Y STRs.
• A new multiplex system, called ‘Y-PLEX™ 6’, has been recently introduced by Reliagene Technologies, Inc., USA, capable of amplifying seven Y STR loci.

Phylogenetic Value of Y-Chromosome Specific STRs

Y-chromosome markers are valuable for studying phylogenetic relationships because of their unique inheritance pattern. These markers show greater variation in haplotype data compared to autosomal or mitochondrial DNA markers. Population-specific Y haplotypes, derived from Y-chromosome markers, are crucial for both phylogenetic and forensic research.

Analysis of Global Samples

• A global sample was analyzed using biallelic markers and a Y-specific STR (DYS19).
• This analysis aided in tracing early migrations from Africa and at least one migration from Asia.

Initial Findings

• An initial study found that just five Y STRs could differentiate 15 populations.
• This finding allowed for the construction of phylogenetic trees and networks consistent with other data.

Supporting Evidence

• The initial findings have been supported in other populations, demonstrating the utility of Y STRs in population differentiation.

Limitations

• Due to the high mutation rates of Y STRs, they do not maintain a linear relationship over long periods.
• This makes them more suitable for historical population differentiation rather than long-term evolutionary analysis.

Note. Various computer software are used to calculate statistical parameters. Appendix I illustrates how a database of frequencies for specific loci in a population is applied to forensic statistics.

Y-Chromosome STR markers validated for forensic purposes

DNA Detection

Luminol and UV light are effective for detecting DNA because it emits a glow under these conditions.

Potential Sources of DNA at Crime Scenes

DNA can be found in various unexpected places. It is recommended to collect the following items from a crime scene to retrieve DNA:

• Fingernails or nail clippings.
• Tissues, paper towels, cotton swabs, or ear swabs.
• Toothpicks, cigarette butts, straws, and personal items like drinking glasses or utensils.
• Blankets, pillows, sheets, dirty linen, caps, and headgear.
• Eyeglasses and contact lenses.
• Used stamps and envelopes.
• Ligatures found on bodies or at the scene.
• Bullets that have passed through a body.

Collection, Storage, and Transport of DNA Evidence

• Latex gloves should be worn while collecting each item of evidence.
• Each item of evidence must be packed in a separate container or envelope.
• Blood, semen, saliva, urine, and other stains must be air dried before packaging. Items with such stains may be dried using a hair dryer or a fan. After drying, pack the samples in a paper bag or envelope.
• For condoms, place them in a sterile tube. If a sterile tube is unavailable, air dry the condom and then pack it in several layers of paper before placing it in a paper bag.
• To remove stains from an unmovable surface:
  • Photograph the surface with a ruler, using both black and white and colour film.
  • Using a sterile moist swab, rub it on the stain until it transfers to the swab; more than one swab may be needed. Collect two additional swabs from the area adjacent to the stain as substrate controls.
• For proper chain of custody, each package should be marked with the case number, name of the deceased (if required and known), date of collection, item contained, post-mortem number, and any other necessary identification. Seal the package, and the collector must sign across the seal.

Appendix I

This appendix illustrates the calculation of two significant statistical notations using mathematical formulas for those interested.

Match Probability

• Definition: Match probability refers to the likelihood that two randomly selected individuals will possess the same genotypes.

Formula:

Here, pM denotes the match probability, pk indicates the frequency of each unique genotype, and m represents the number of distinct genotypes. The overall match probability across multiple loci is calculated as the product of the individual probabilities for each locus

Example

• The frequencies of blood groups A₁, A₂, B, A₁B, A₂B, and O in the British population were found to be 0.34, 0.08, 0.09, 0.024, 0.006, and 0.46 respectively.
• By squaring these individual frequencies and adding them together, we can find the probability of a match.
• Using this value in the formula, the probability of a match is 0.34 or 34 percent.
• The probability of a match across multiple independent systems is calculated by multiplying the match probabilities of each system.

Power of Discrimination

• Definition: The Power of Discrimination measures the likelihood that two randomly selected individuals will have different genotypes. It is the inverse of the probability of a match.
• Formula: P_d = 1 – pM For several loci the formula is:
  
• Example Calculation: If P_M is 0.34, then the Power of Discrimination is 0.66 (i.e., 1 - 0.34). This indicates the system's effectiveness in distinguishing between different genotypes based on the given probabilities.
• Influence of Genotype Frequencies: The Power of Discrimination (Pd) increases with the number of genotypes and is maximized when the genotypes are present at similar frequencies.
• P_d Values in Punjabi Population: The P_d values for loci D3, vWA, and FGA in the Punjabi population are 0.913, 0.95, and 0.963 respectively.
• Calculating PM Values: By subtracting each P_d value from 1, the P_M (Probability of Match) for each locus is determined to be 0.087, 0.05, and 0.037.
• Combined PM Calculation: Multiplying these P_M values together yields 0.00016. This value is used to calculate the combined Power of Discrimination (Pdcomb), resulting in 0.9998.