The DNA Double Helix and Base Pairing

Each DNA strand contains deoxyribonucleotides joined by phosphodiester bonds. The sugar-phosphate backbone faces the surrounding solution, while the bases stack inside the duplex. At ordinary biological pH, backbone phosphates contribute negative charge, helping explain DNA movement toward the positive electrode during electrophoresis.

Watson and Crick’s 1953 model drew on experimental work including the X-ray studies of Rosalind Franklin and Maurice Wilkins and base-composition findings. The common B form is right-handed, approximately two nanometres wide and around 10.5 base pairs per turn. Its rise per base pair is about 0.34 nanometres. These are approximate structural values, not invariant dimensions for every sequence and condition.

Major and minor grooves expose different chemical surfaces. Proteins may recognise sequence or shape through these surfaces and other contacts. It is too simple to say that every DNA-interacting protein, including a polymerase, reads only the major groove.

Purine–pyrimidine pairing gives the ordinary A–T and G–C pairs similar overall dimensions. Purines are adenine and guanine; pyrimidines include cytosine and thymine. Hydrogen bonding and stacking contribute to duplex stability. Alternative conformations and non-canonical pairs exist, so geometric explanations should not claim that every other pairing is impossible.

The prime labels refer to carbon positions in the sugar. Successive nucleotides are linked through 3-prime–5-prime phosphodiester bonds. A free 3-prime hydroxyl provides the ordinary extension site for DNA polymerase. A 5-prime end may carry a phosphate or hydroxyl depending on how the molecule was made or treated; a synthetic primer need not have a free 5-prime phosphate.

Sequences are conventionally written 5-prime to 3-prime. If one strand is 5-prime-ATGCGT-3-prime, its aligned partner is 3-prime-TACGCA-5-prime. Written 5-prime to 3-prime, the partner is ACGCAT. This reverse-complement operation is essential when describing opposing primer sites.

In PCR, two primers bind opposite template strands with their extendable 3-prime ends directed toward the intervening target. A primer does not have to bind at the physical end of an entire chromosome. Incorrect orientation, off-target binding or interactions between primers can prevent the intended amplification even when complementary sequence is present.

In standard A–T pairing, adenine’s N6 amino group donates to thymine’s O4, while thymine’s N3–H donates to adenine’s N1. In G–C pairing, guanine’s N1–H donates to cytosine’s N3, cytosine’s N4 amino group donates to guanine’s O6, and guanine’s N2 amino group donates to cytosine’s O2. Thus A–T has two standard hydrogen bonds and G–C has three.

These interactions are non-covalent; the backbone bonds within each strand are covalent. Heating an ordinary PCR mixture separates the strands without intentionally breaking their backbones. Cooling under suitable conditions permits complementary sequences to hybridise again, although successful reannealing depends on concentration, time and reaction conditions.

Stacking between neighbouring bases also contributes strongly to stability. Sequence context, salt, length, concentration and mismatches influence duplex melting. GC-rich sequences often have higher melting temperatures in comparable conditions, but one cannot calculate an assay temperature from hydrogen-bond count alone.

A mismatch can reduce binding or extension, especially near a primer’s 3-prime end, but does not always prevent amplification. Specificity comes from the complete validated assay, not an absolute physical rule that primers can bind only perfectly complementary DNA. Primer-site variation can also cause an apparent missing allele.

DNA lacks RNA’s reactive 2-prime hydroxyl and often survives conditions under which some RNA targets are less recoverable. That comparison is conditional: protected RNA can survive in dried stains, and some structural proteins persist after DNA becomes uninformative. There is no universal rule that every protein denatures in hours or every RNA target disappears in minutes.

Complementary strands can preserve information when one strand is damaged, but a forensic extract does not automatically repair itself. An assay still needs suitable template across its target. Some nucleases act readily on double-stranded DNA; bases inside a helix are not a universal shield against nuclease cleavage of the backbone.

Chromatin, tissue structure and mineral association can influence preservation. Bone and teeth may provide useful targets after soft tissue is lost, but their condition and sampling location matter. Dry bloodstains can also retain useful DNA for long periods. No substrate is always best, and appearance does not establish amplifiability.

Light, heat, moisture and microbial exposure affect specimens through interacting processes. Germicidal ultraviolet-C laboratory irradiation must not be treated as equivalent to sunlight reaching an outdoor scene. Exposure duration alone cannot predict the fragment size or guarantee that a stain will become unprofilable within a set number of hours.

Damage can change bases, remove them or sever the backbone. Hydrolytic depurination creates abasic sites; oxidation can modify bases such as guanine; ultraviolet exposure can produce pyrimidine photoproducts. Nuclease cleavage and subsequent chemical reactions can fragment molecules. Some lesions block copying, some cause miscoding and some have limited effect on a particular target.

Longer targets often recover less well in fragmented samples. Short-target assays can improve the chance of copying surviving spans, but no universal average-fragment threshold dictates one mandatory technique. A one-allele result can be a true homozygote or reflect dropout or another analytical issue.

Use appropriate quantification, inhibition controls, method validation and profile review to assess the actual result. A qPCR degradation index describes recovery from particular targets, not a complete map of damage. Sequence damage is not automatically expressed as a new size peak in a capillary STR profile.

Appropriate dry, temperature-controlled storage or specimen-specific cooling can slow further change. Freezing does not preserve every kind of evidence indefinitely, and all specimens should not be placed in paper or frozen by default. Record the specimen, container, conditions and preservation rationale.

PCR uses strand separation, primer hybridisation and template-directed extension to copy selected regions. STR assays examine short tandem repeats, often using fluorescent products and capillary sizing. Sequence-based methods can examine additional variation, including same-length alleles with different internal sequences.

A genetic comparison needs the observed alleles or sequence, appropriate population information and stated propositions. Agreement at selected markers is not a probability of guilt. Nor does it establish the fluid, deposition route or time. The whole case question matters as much as the copying reaction.

A database’s marker specification is distinct from the physical structure of DNA. Different systems can use different loci and eligibility rules; the 20 US CODIS core loci are not a worldwide definition of an STR assay.

Collection authority, database use and reporting requirements vary by jurisdiction. They should be addressed where relevant, without putting country-specific legal assertions into the explanation of a universal molecular mechanism. The chemistry supports a test; validated implementation and justified interpretation support its forensic use.