DNA, RNA and the Central Dogma of Molecular Biology

DNA replication is semi-conservative: each daughter molecule retains one parental strand and synthesises one new strand. The process begins at defined sites on each chromosome called origins of replication. In humans there are tens of thousands of such origins, allowing simultaneous initiation across the genome so that cell division can proceed in hours rather than months.

The enzymatic machinery begins with helicase unwinding the double helix at the replication fork, separating the strands. Single-strand DNA-binding proteins (SSBPs) stabilise the separated strands. Primase lays down a short RNA primer, providing the free 3'-OH that DNA polymerase III requires to start synthesis. DNA polymerase then extends in the 5'-to-3' direction on each strand template. On the leading strand this proceeds continuously in the same direction as fork movement. On the lagging strand, which runs antiparallel to fork movement, synthesis is discontinuous, producing Okazaki fragments that are later joined by DNA ligase after RNA primers are replaced.

Two enzymes matter most to the forensic context. Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, is the workhorse of PCR. Like all DNA polymerases it can only extend an existing primer and only in the 5'-to-3' direction, but it is stable at the 95°C denaturation temperatures a PCR cycle demands, which is why it replaced the earlier, heat-labile polymerases. In forensic STR typing the PCR cycle temperature programme, typically 95°C denaturation, 59-60°C annealing, and 72°C extension, is a direct translation of the enzymatic logic of natural replication into a controlled laboratory reaction. Hot-start Taq variants (antibody-mediated or aptamer-based inhibition released at high temperature) reduce non-specific amplification in degraded-sample workflows.

In India, the CFSL (Central Forensic Science Laboratory) laboratories under the Directorate of Forensic Science Services use validated PCR protocols whose cycling conditions are documented in their internal SOPs and reviewed under NABL audit. In the US, the FBI's Quality Assurance Standards specify that each laboratory must document the cycling conditions for each kit it uses, a direct reference to the enzymatic parameters described here. In the UK the Forensic Science Regulator's Guidance on DNA Mixture Interpretation implicitly requires this: a laboratory cannot interpret a result it cannot explain at the molecular level.

The central dogma of molecular biology, articulated by Francis Crick in 1958, describes the directional flow of biological information: DNA is transcribed into messenger RNA, and mRNA is translated into protein. The flow is directional in the sense that information travels from nucleic acid to protein but not back from protein to nucleic acid in normal cellular biology (reverse transcriptase in retroviruses is the classical exception, not the rule in somatic cells).

Transcription begins when RNA polymerase binds to a promoter sequence upstream of a gene. The polymerase unwinds the double helix locally and reads the template strand in the 3'-to-5' direction, synthesising a complementary RNA molecule in the 5'-to-3' direction. The resulting pre-mRNA undergoes processing: a 5' cap is added, a poly-A tail is added at the 3' end, and non-coding intervening sequences (introns) are spliced out by the spliceosome. The mature mRNA that emerges carries only the exonic sequence. This detail matters in one forensic application: forensic STR primers flank introns, not exons, because the non-coding regions of the genome carry the polymorphic repeats used for identification, while the protein-coding regions are far too conserved across individuals to distinguish one person from another.

Translation occurs on ribosomes, which read mRNA codons (triplets of bases) and recruit transfer RNA molecules that carry the corresponding amino acids. The genetic code is degenerate (multiple codons specify the same amino acid) but not ambiguous (each codon specifies only one amino acid or a stop signal). The ribosome assembles the polypeptide chain from the N-terminus to the C-terminus.

The forensic application of the transcription step is more direct than it might appear. Forensic body-fluid identification using messenger RNA profiling, developed by Rolf Zubakov and colleagues and now commercially available as the RNA Body Fluid ID kit, reads tissue-specific mRNA transcripts to identify which body fluid is present in a mixed stain. Blood, semen, saliva, vaginal secretions, and menstrual blood each carry a signature mRNA profile. This method reached UK casework practice through the FSR validation reports, and US laboratories have begun validation under SWGDAM guidelines. Because mRNA is inherently less stable than DNA, the technique is limited to relatively fresh stains, but it provides a non-presumptive identification at the cellular level.

DNA at a crime scene is subject to hydrolytic, oxidative, and enzymatic attack the moment a cell dies. Hydrolysis of the phosphodiester backbone produces strand breaks. Depurination (loss of purine bases, especially from adenine and guanine) introduces abasic sites that block polymerase extension. Oxidative damage, particularly 8-oxoguanine formation, causes miscoding and can shift allele balance in a mixed profile. UV irradiation forms cyclobutane pyrimidine dimers that also stall polymerases. The practical outcome of all of this is fragmentation: high-molecular-weight DNA becomes a distribution of shorter fragments, some as short as 50-100 base pairs, some longer.

The forensic implication is direct. The STR loci targeted by commercial multiplex kits have amplicon sizes typically ranging from about 100 to 400 base pairs. When DNA is heavily degraded, the larger amplicons fail to amplify first, producing a partial profile with drop-out of the higher-molecular-weight alleles. This is called interlocus drop-out, and it results in a degraded profile that may include only the smaller loci in the multiplex. Interpretive guidelines in the UK (FSR guidance), the US (SWGDAM Interpretation Guidelines 2017), and Australia (National Institute of Forensic Science guidelines) all address how degraded profiles are interpreted and what weight they carry in a likelihood-ratio calculation.

Miniaturised STR systems and reduced-size (mini-STR) kits, such as the GlobalFiler Express or the Investigator 24plex QS, were developed specifically to target degraded DNA: they use primers that sit very close to the STR repeat, generating amplicons in the 70-170 base-pair range rather than the 100-400 base-pair range of conventional multiplex kits. The same principle drives mitochondrial DNA analysis for samples like hair shafts and ancient bone: the mitochondrial genome is smaller, present in hundreds to thousands of copies per cell (versus the two nuclear copies), and survives longer in harsh conditions. Module 6 of this subject covers mitochondrial sequencing in detail.

The storage and transportation conditions of a sample govern its molecular integrity long before the laboratory opens a tube. In Kunhiraman v. Manoj (Supreme Court of India, 1991), the court addressed the admissibility of biological evidence and chain of custody for samples held under sub-optimal storage. In the US, FBI Quality Assurance Standards specify that evidence samples must be stored in conditions that minimise degradation. The European Network of Forensic Science Institutes (ENFSI) DNA Working Group guidelines similarly require documented sample-handling procedures. Every step from collection to extraction is a molecular intervention that either preserves or accelerates the degradation chemistry described above.

In the US, the admissibility of DNA evidence evolved rapidly from the late 1980s. The initial Frye general-acceptance standard, from Frye v. United States (DC Circuit, 1923), required that a scientific technique be generally accepted in the relevant scientific community before courts would hear expert testimony based on it. The more demanding Daubert standard, from Daubert v. Merrell Dow Pharmaceuticals (US Supreme Court, 1993), requires trial judges to act as gatekeepers assessing whether the methodology is scientifically valid, whether it has been tested, whether it has a known error rate, and whether it is generally accepted. Both standards have been applied to DNA evidence, most prominently in People v. Castro (New York, 1989), where the court found that while DNA typing was theoretically sound, the specific testing performed by Lifecodes Corporation had not followed adequate scientific procedures, and the prosecution's DNA result was excluded.

In the UK, the Court of Appeal's reasoning in R v. Doheny and Adams (1996) established the standard framework for how DNA statistics should be presented to a jury: the expert witnesses' role is to give the random-match probability, the jury's role is to weigh it against the other evidence in the case, and conflating the two produces the prosecutor's fallacy. The National DNA Database (NDNAD), which now holds profiles for over 6.6 million individuals, operates under the Police and Criminal Evidence Act 1984 as amended by the Protection of Freedoms Act 2012, which specifies retention and deletion rules keyed to conviction status.

In India, the Bharatiya Sakshya Adhiniyam 2023 (BSA), which replaced the Indian Evidence Act 1872, governs the admissibility of expert opinion evidence under Section 39 (formerly Section 45 of IEA). DNA evidence has been admitted in Indian courts in a growing body of cases. The DNA Technology (Use and Application) Regulation Bill 2019, though not yet enacted, would create a statutory framework for DNA databases and quality standards for DNA testing laboratories. Until that Bill passes, quality is ensured through NABL accreditation and CFSL internal SOPs.

In the European Union, the Prüm Convention (2005, incorporated into EU law in 2008) created a framework for cross-border exchange of DNA profiles between national databases, requiring that contributing profiles meet minimum quality standards aligned with the European Standard Set of STR loci. The ENFSI DNA Working Group publishes guidelines that participating national laboratories are expected to follow.

All of this admissibility architecture rests on the molecular biology in the preceding sections. When a defence expert in a Daubert hearing challenges the annealing temperature used in a PCR protocol, they are asking a question about the base-pairing thermodynamics described in Section 1. When a UK expert witness explains to a jury why a partial profile carries a higher match probability uncertainty than a full 17-locus profile, they are explaining the consequences of the interlocus drop-out described in Section 4.