DNA in Forensic Science: Structure, Genetic Markers, Extraction and Profiling

The double-helix model was published by James Watson and Francis Crick in Nature, April 1953, building on the X-ray diffraction work of Rosalind Franklin and Maurice Wilkins (Photograph 51). The structure is a right-handed double helix: two antiparallel polynucleotide strands held together by hydrogen bonds between complementary bases. Adenine pairs with thymine (two H-bonds), guanine with cytosine (three H-bonds). The sugar-phosphate backbone runs outside, the bases stack inside. Helix diameter is roughly 2 nm; one full turn covers about 10.5 base pairs and 3.4 nm.

For forensic work, the relevant breakdown is coding vs non-coding DNA. Only about 1 to 2 percent of the human genome codes for protein. The rest, often called non-coding or junk DNA, is full of repetitive sequences. Two kinds of repeats matter:

• Tandem repeats: short motifs repeated head-to-tail. VNTRs (10 to 100 bp motifs, repeated 5 to several hundred times) and STRs (2 to 6 bp motifs, repeated 5 to 50 times).
• Single-base variations: SNPs scattered roughly every 1,000 bases.

Forensic profiling targets the non-coding repeats deliberately. They are highly polymorphic (which gives identification power), they do not affect the donor's phenotype (which sidesteps privacy concerns), and the short STR repeats survive partial degradation better than the long VNTRs of the early Jeffreys era. Under Section 39 of the Bharatiya Sakshya Adhiniyam 2023, the resulting profile is admissible as expert opinion in Indian courts, which is why analysts document the marker set and the validation status of the kit in every report.

Four marker classes are tested in NTA papers. Each has a defined chromosomal location, a defined inheritance pattern, and a defined casework niche. Memorise this table; it answers half the marker MCQs you will see.

Why autosomal STR became the global standard. STRs are short (under 400 bp amplicons), which means a partially degraded sample still gives a typeable signal. They are highly polymorphic; a single locus can have 10 to 30 alleles in a population, so combining 15 to 20 loci yields random match probabilities below 10⁻¹⁵. They multiplex cleanly in a single PCR tube. The CODIS core (originally 13 loci, expanded to 20 in January 2017) and the European ESS set anchor the international convention. Indian labs run AmpFISTR Identifiler Plus (15 STRs plus amelogenin) and increasingly the 21-locus GlobalFiler kit on ABI 3500 platforms.

Y-STRs are essential for male-female mixtures from sexual-assault evidence because they ignore the female component. They cannot distinguish patrilineal relatives (father, son, brother, paternal uncle all share the same Y haplotype barring mutation), which is a known MCQ trap.

Mitochondrial DNA wins when nuclear DNA has failed. Hundreds to thousands of mtDNA copies per cell mean a hair shaft, a tooth pulp from a 30-year-old case, or charred remains can still type. The high control-region mutation rate gives discrimination, but mtDNA only identifies a maternal lineage, not an individual.

SNPs and SNaPshot assays are used for ancestry, phenotype prediction (eye, hair, skin colour) and for highly degraded DNA where even mini-STRs fail.

Extraction has to do three things: lyse the cell, separate DNA from protein and lipid, and recover the DNA in a buffer compatible with downstream PCR. Sample integrity from the scene through the lab depends on a documented chain of custody; a contaminated swab cannot be unsplit by good chemistry. NTA tests the four method names, their pros and cons, and the special case of differential extraction.

Differential extraction is the one method you must name in any sexual-assault evidence question. The vaginal/anal swab carries epithelial cells from the victim and sperm cells from the male contributor. Sperm heads are extraordinarily resistant to lysis because their DNA is packed with disulfide-bonded protamines. The trick:

1. First lysis with SDS and proteinase K dissolves the epithelial cells. Centrifuge, save the supernatant as the female fraction.
2. Wash the pellet, then add dithiothreitol (DTT) to reduce the protamine disulfides. Re-lyse with SDS and proteinase K to release the sperm DNA, the male fraction.

Run the two fractions through separate PCR and CE injections. The male profile becomes interpretable without being drowned out by the victim's DNA.

PCR principle. A target sequence is amplified exponentially over 28 to 32 thermal cycles. Each cycle has three steps: denaturation at ~94 °C (strand separation), annealing at ~55 to 65 °C (primer binding), extension at ~72 °C (Taq polymerase synthesises the complementary strand). Yield doubles per cycle, so 30 cycles can take 1 pg of template to nanogram quantities of amplicon. Kary Mullis published the method in 1983 and won the 1993 Nobel Prize in Chemistry.

Multiplex STR kits put 15 to 24 primer pairs into a single PCR. Each forward primer carries one of four to six fluorescent dyes (FAM, VIC, NED, PET, LIZ are the AmpFISTR set). Amplicon size + dye colour together resolve each locus on a single capillary injection. Indian labs typically run AmpFISTR Identifiler Plus (15 autosomal STRs plus amelogenin), PowerPlex 21 or GlobalFiler (21+ loci including CODIS 20). The amelogenin marker is the conventional sex-typing locus: the X amplicon is 6 bp shorter than the Y amplicon, so a male sample shows two peaks and a female shows one.

Capillary electrophoresis is the workhorse separation step. The classic Indian setup is an Applied Biosystems 3500 Genetic Analyser, an 8-capillary instrument loaded with POP-4 polymer. Fluorescently labelled amplicons electrokinetically inject into each capillary, separate by size as they migrate to the detector, and emit at different wavelengths as the laser excites them. The internal LIZ-labelled size standard runs in the same capillary so allele sizing is calibrated injection by injection. CE is fundamentally the high-resolution descendant of slab-gel methods covered in the electrophoresis topic; the principle is identical, the format is single-capillary microfluidic rather than vertical slab.

The output is an electropherogram: dye-coloured peaks plotted on a size axis. Each STR locus shows one peak (homozygote) or two peaks (heterozygote), and the GeneMapper ID-X software calls the allele numbers against an allelic ladder.

A clean single-source profile from a high-template sample is straightforward: read the alleles, compare to the reference, declare match or exclusion. The interesting work, and the part NTA likes to probe, is in the artefacts and the statistics.

Peak height and stochastic effects. Each peak has a relative fluorescence unit (RFU) value. A clean profile has all peaks above the analytical threshold (typically 50 to 100 RFU) and heterozygote balance above 60 percent. Below the stochastic threshold, one of two alleles at a heterozygous locus may drop out, simulating a false homozygote.

Stutter peaks appear one repeat unit shorter than the true allele (n-4 stutter) at roughly 5 to 15 percent of the parent peak height. They arise from strand slippage during PCR extension. Stutter is a major problem when interpreting mixtures because it can mimic a minor contributor.

Allelic dropout, drop-in, and degradation. Low-template DNA shows allelic dropout (locus appears homozygous when it is actually heterozygous), allelic drop-in (sporadic contamination peaks), and a characteristic ski-slope electropherogram (taller peaks at small loci, shorter at large loci) when the sample is degraded.

Mixture interpretation is the hardest part of casework. Analysts determine the number of contributors, estimate the mixture proportion from peak-height ratios, and either deconvolve or evaluate likelihoods using probabilistic genotyping software like STRmix or EuroForMix. The output is a Likelihood Ratio: the probability of the evidence under the prosecution hypothesis (the suspect is a contributor) divided by the probability under the defence hypothesis (an unknown unrelated person is a contributor). An LR of 10⁹ means the evidence is a billion times more probable under the prosecution hypothesis than under the defence hypothesis. Modern Indian DNA reports increasingly cite LRs rather than simple match probabilities.

The Indian DNA ecosystem is centred on a few apex institutions that NTA names directly. The Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad is the autonomous institute under the Department of Biotechnology that pioneered Indian DNA fingerprinting under Dr Lalji Singh in the late 1980s. The CFSL Hyderabad DNA division is the central forensic lab DNA reference under DFSS. Other CFSLs at Kolkata, Chandigarh and Delhi run their own DNA units, and most major states have a DNA division in their SFSL. The National Forensic Sciences University (NFSU), Gandhinagar trains the next generation of DNA analysts.

The DNA Data Bank envisaged under DFSS is the Indian equivalent of CODIS, intended to hold convicted-offender profiles, crime-scene profiles, missing-persons profiles and unidentified-deceased profiles. Operational rollout depends on the legislation discussed below.

Statutory framework.

• The Bharatiya Sakshya Adhiniyam 2023 (BSA, replacing the Indian Evidence Act 1872) governs admissibility. Section 39 makes expert opinion (including DNA) admissible; Section 63 deals with electronic records, which now covers DNA electropherograms generated and stored digitally.
• The Bharatiya Nagarik Suraksha Sanhita 2023 (BNSS, replacing the CrPC 1973) mandates collection of fingerprint, footprint, palm-print, photograph and biological specimens from arrested and convicted persons. Section 349 BNSS (read with the Identification of Prisoners Act 1920 as amended by the Criminal Procedure (Identification) Act 2022) is the statutory hook for compelled sample collection.
• The DNA Technology (Use and Application) Regulation Bill was introduced in 2019 to create a National DNA Data Bank, Regional DNA Data Banks, a DNA Regulatory Board and a scheme of accreditation for DNA labs. The Bill was withdrawn in 2023 and has not been re-enacted as of this writing; the policy intent, however, is what NTA tests.

Three case-law anchors worth knowing: Selvi v. State of Karnataka (2010) on involuntary narco/polygraph/BEAP (DNA sample collection is not a Selvi-protected category once BNSS mandate exists), Nandlal Wasudeo Badwaik v. Lata Nandlal Badwaik (2014) where the Supreme Court held that DNA tests prevail over the presumption of legitimacy under the Evidence Act, and Inayath Ali v. State of Telangana (2022) on consent in paternity-disputed DNA orders.