Nucleic Acids: Structure and Function

Every nucleic acid strand is a polymer of nucleotides. Each nucleotide has three parts: a five-carbon (pentose) sugar, a phosphate group attached to the 5' carbon of the sugar, and a nitrogenous base attached to the 1' carbon. In DNA the sugar is 2'-deoxyribose, lacking a hydroxyl group at the 2' position. In RNA the sugar is ribose, which has a hydroxyl group at the 2' position. That single chemical difference has large consequences for stability: the 2'-OH in ribose makes RNA far more susceptible to hydrolysis than DNA, explaining why RNA degrades in biological evidence far faster than DNA and why forensic workflows for RNA must be more stringent.

The nitrogenous bases fall into two chemical classes. Purines have a double-ring structure: adenine (A) and guanine (G). Pyrimidines have a single ring: cytosine (C), thymine (T, in DNA), and uracil (U, in RNA, replacing thymine). The substitution of uracil for thymine in RNA is a biosynthetic simplification; thymine is essentially uracil with a methyl group at the 5 position, and that methyl group makes thymine slightly more resistant to certain mutational processes. In the forensic laboratory, the presence of uracil in RNA is exploited by uracil-DNA glycosylase (UNG) carryover prevention in some PCR setups, which destroys RNA-derived contamination but not DNA targets.

Nucleotides are joined by phosphodiester bonds. The 3'-OH of one sugar reacts with the 5'-phosphate of the next nucleotide, releasing pyrophosphate and forming the backbone. This condensation reaction requires energy (the incoming nucleotide arrives as a triphosphate) and is catalysed by DNA polymerase during replication or by RNA polymerase during transcription. The directionality of this reaction means that a strand always grows in the 5' to 3' direction, a constraint that shapes primer design and the mechanism of PCR.

The two polynucleotide strands of DNA wind around a common axis in a right-handed helix. The phosphate-sugar backbones run along the outside, where they are exposed to solvent and accessible to nucleases. The bases stack on the inside, where hydrophobic stacking interactions between adjacent base pairs contribute to helix stability alongside the inter-strand hydrogen bonds. The helix completes one full turn every approximately 10.4 base pairs in its B-form (the predominant form under physiological conditions), creating a major groove and a minor groove. Proteins that bind DNA to regulate gene expression or repair damage typically contact the base sequence through the major groove.

Watson-Crick base pairing is strict: A pairs with T via two hydrogen bonds, G pairs with C via three hydrogen bonds. These rules follow from the complementary shapes and hydrogen-bond donor/acceptor patterns of the bases. No other pairing is geometrically and energetically favourable enough to maintain the helix. As a practical consequence, if you know the sequence of one strand, you can write the complementary sequence of the other. A sequence such as 5'-ATTGCAG-3' on one strand is paired with 3'-TAACGTC-5' (written 5'-CTGCAAT-3') on the complementary strand. This property underlies all primer-based amplification: a short synthetic oligonucleotide will bind specifically and stably to a complementary target sequence.

Chargaff's rules, established empirically in the 1940s before the structure of DNA was known, state that in any double-stranded DNA sample, the molar amount of A equals T, and the molar amount of G equals C. This is now understood as a direct consequence of Watson-Crick base pairing: because every A is paired with a T and every G with a C, the total must be equal. Deviations from Chargaff's rules in a sample can indicate degradation, contamination with single-stranded RNA, or measurement error.

The information content of DNA lies entirely in the linear sequence of its bases. The human genome contains approximately 3.2 billion base pairs per haploid set, distributed across 23 chromosomes. Of that total, roughly 1.5 percent codes for protein (exons). The remainder includes regulatory elements, introns, repetitive sequences, and large stretches with no yet-characterised function. Forensic DNA profiling works precisely in the non-coding regions: the short tandem repeat (STR) loci targeted by systems such as CODIS in the United States, the National DNA Database in the United Kingdom, and equivalent systems in the European Union and India do not fall within protein-coding sequences and are not associated with identifiable phenotypic traits.

Information flows from DNA to RNA to protein through two steps. In transcription, an RNA polymerase reads the template strand of a gene in the 3' to 5' direction and synthesises a complementary messenger RNA (mRNA) strand in the 5' to 3' direction. In translation, ribosomes read the mRNA in triplets of bases (codons), each specifying an amino acid or a stop signal. The genetic code is nearly universal across all life, which is why a DNA sequence from any human sample anywhere in the world can be read and interpreted using the same rules.

The forensic relevance of transcription is not only theoretical. Certain mRNA species are tissue-specific: genes active in spermatozoa, saliva glands, or vaginal epithelium produce characteristic mRNA transcripts. Body-fluid identification methods based on mRNA expression profiling exploit this specificity. A stain can be identified as semen, not just as containing DNA, if it expresses genes such as PRM1 or TGM4, which are transcribed only in spermatozoa. The UK Forensic Science Regulator's codes of practice and the recommendations of the European Network of Forensic Science Institutes (ENFSI) both recognise mRNA profiling as a validated approach for body-fluid characterisation.

Although RNA is far less stable than DNA, several classes of RNA are forensically relevant. Messenger RNA (mRNA) carries protein-coding information from nucleus to ribosome and is present transiently. Ribosomal RNA (rRNA) is the structural and catalytic component of ribosomes, comprising more than 80 percent of total cellular RNA and more stable than mRNA. Transfer RNA (tRNA) carries amino acids to the ribosome. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression; they are more stable than mRNA and are being investigated as biomarkers for body-fluid and tissue identification in forensic research.

The instability of RNA in forensic samples is a serious practical constraint. RNases, the enzymes that degrade RNA, are ubiquitous in the environment and on skin surfaces. An unpreserved biological stain will lose most of its RNA within hours at room temperature. To conduct mRNA profiling, samples must be collected into RNA stabilisation reagent (such as RNAlater) or processed within a very short time window. Extraction protocols for RNA use RNase-inhibiting conditions and separate equipment from DNA workflows to prevent cross-contamination and enzymatic degradation.

In mitochondrial DNA (mtDNA) analysis, which is used when nuclear DNA is absent or too degraded to profile, the relevant molecule is still DNA rather than RNA. Mitochondria have their own small circular genome of approximately 16,569 base pairs in humans. Because each cell contains hundreds to thousands of mitochondria, each with multiple copies of mtDNA, the mitochondrial genome is far more abundant than nuclear DNA in highly degraded samples such as old bones, shed hair without roots, and ancient remains. Forensic applications of mtDNA include the identification of missing persons, disaster victims, and archaeological remains across multiple jurisdictions.

DNA in biological evidence degrades through several concurrent mechanisms. Hydrolytic damage cleaves phosphodiester bonds in the backbone and removes bases, particularly purines, through depurination. Oxidative damage from reactive oxygen species modifies bases and can block polymerase progression. Ultraviolet radiation from sunlight causes thymine dimers that distort the helix and impede replication. Heat and humidity accelerate all chemical reactions. Microbial and endogenous nucleases from the cells themselves continue to act until the sample dries or is frozen. The combined effect is fragmentation of the DNA into progressively shorter pieces.

The practical consequence of fragmentation is that PCR amplification can only succeed if the target sequence is intact in at least some DNA molecules in the extract. STR alleles amplified by standard forensic kits have amplicon sizes ranging from approximately 100 to 450 base pairs. When DNA is extensively degraded, longer amplicons fail first. This pattern, called allele drop-out, means that a partial profile, with some loci producing no result, is often the first sign of degradation. Miniaturised STR kits designed for degraded samples use primers placed closer to the repeat region, reducing amplicon size and improving the chance of success.

Environmental conditions strongly influence the rate of degradation. Dry, cool, dark, and low-oxygen conditions preserve DNA best. Permafrost has preserved ancient human DNA for tens of thousands of years. A bloodstain dried and stored at room temperature in a dark folder may yield full profiles after decades. The same stain left wet in a warm, humid environment with microbial access may be unprofilable within days. Collection and preservation decisions made at the crime scene therefore directly determine what is possible in the laboratory.

Forensic biology laboratories in multiple jurisdictions, including those accredited under ISO 17025 in the European Union, the UK Forensic Science Regulator's codes, and the FBI QAS (Quality Assurance Standards) in the United States, require quantification of recovered DNA before profiling. Real-time quantitative PCR (qPCR) assays that measure both total human DNA and degraded human DNA provide a degradation index that guides the choice of amplification protocol. Under Indian law, the Bharatiya Sakshya Adhiniyam 2023 (which replaced the Indian Evidence Act 1872) governs admissibility of scientific evidence, and courts have accepted DNA profiles where proper chain of custody and validated methods are documented.

Every major technique in forensic DNA analysis is a direct application of nucleic acid properties. PCR (polymerase chain reaction) exploits three properties: the ability to denature double-stranded DNA by heating, the specificity of complementary base pairing between short synthetic primers and a target sequence, and the 5' to 3' directionality of DNA polymerase synthesis. Repeated cycles of denaturation, primer annealing, and extension exponentially amplify the target region, producing millions of copies from as little as a single template molecule.

Capillary electrophoresis (CE) separates DNA fragments by size as they migrate through a polymer matrix under an electric field. The size differences between STR alleles at a given locus are small, typically four base pairs per repeat unit, so the separation must be highly precise. Fluorescent labels on PCR primers allow the instrument to detect each fragment as it passes a laser, generating a profile of peaks at known size positions. The fragment sizes are compared against an internal size standard in every sample, providing a calibrated, reproducible measurement independent of minor variations in gel concentration or temperature.

Next-generation sequencing (NGS), increasingly used in forensic reference laboratories, sequences every nucleotide in a fragment rather than just measuring its length. This allows discrimination between alleles that are the same length but differ in internal sequence, a phenomenon called microvariant or isoallele. It also allows simultaneous analysis of nuclear STRs, mitochondrial sequence, and single nucleotide polymorphisms (SNPs) associated with externally visible characteristics or biogeographic ancestry, in a single workflow. Forensic science institutes in the Netherlands (NFI), Germany (BKA), and the United States (FBI) have each published validation studies for NGS-based forensic typing. The forensic biotechnology subject covers sequencing platforms and their validation requirements in detail.

Biological evidence categories relevant to nucleic acid analysis span a wide range of substrates. Blood, semen, and saliva are the most common and typically yield high-quality nuclear DNA. Touch DNA from skin cells deposited on surfaces, hair without roots (mitochondrial DNA only), aged bone and teeth (used for disaster victim identification and historical cases), and environmental samples all require specialised extraction and quantification approaches. The scope of biological evidence topic maps these categories and their associated collection protocols.