Nucleic Acids: Structure and Function

A nucleotide contains a nitrogenous base, a pentose sugar and phosphate. A nucleoside contains the base and sugar without phosphate. The sugar is 2-prime-deoxyribose in DNA and ribose in RNA. Ribose has a hydroxyl group at its 2-prime carbon where deoxyribose has hydrogen.

Adenine and guanine are purines with two fused rings. Cytosine, thymine and uracil are pyrimidines with one ring. Ordinary DNA uses A, T, G and C; ordinary RNA uses A, U, G and C. Thymine is methylated uracil. Modified bases and damage products mean these basic lists are not descriptions of every possible chemical state.

In a strand, 3-prime–5-prime phosphodiester linkages join successive sugar units. During polymerisation, the existing 3-prime hydroxyl reacts with the incoming nucleotide triphosphate and pyrophosphate is released. Synthesis proceeds 5-prime to 3-prime; the numerical labels refer to sugar-carbon positions, not to different base types.

RNA’s 2-prime hydroxyl permits backbone cleavage under suitable conditions, particularly alkaline conditions. RNases also influence stability. DNA is often more persistent, but protected RNA can remain detectable in old dried stains. It is inaccurate to say that all unpreserved RNA disappears within hours.

Uracil-DNA glycosylase carryover prevention targets uracil-containing DNA products made by incorporating dUTP in an earlier amplification. It is not a general treatment that destroys RNA-derived contamination while leaving all intended DNA untouched. The assay must be designed and validated for that system.

In ordinary Watson–Crick pairing, A pairs with T and G with C. A–T has two hydrogen bonds and G–C has three. Base stacking, sequence context, salt, temperature and chemical conditions also contribute to duplex stability. Counting hydrogen bonds alone does not determine a primer’s melting temperature.

The common B-DNA helix is right-handed, with approximately 10.5 base pairs per turn under typical conditions. Other conformations can occur. The sugar-phosphate backbones lie outside the stacked bases and form strands with opposite orientations. The major and minor grooves offer different surfaces for molecular recognition.

For example, 5-prime-ATTGCAG-3-prime pairs with 3-prime-TAACGTC-5-prime. Writing that complementary strand in the conventional 5-prime to 3-prime direction gives CTGCAAT. Complementing and reversing are separate steps; omitting the reversal is a common error in primer exercises.

Mismatch and non-canonical pairing can occur. Hybridisation specificity depends on sequence and reaction conditions, so a short oligonucleotide is not guaranteed to bind only one target simply because a complementary sequence exists. Validated assay design considers off-target binding and primer interactions.

Chargaff’s equality of A with T and G with C describes ordinary paired double-stranded DNA across both strands. It is not a routine diagnostic test for contamination or degradation in a forensic extract. Measurement method and the actual nucleic-acid composition would have to be considered before interpreting a departure.

A human haploid chromosome set contains roughly three billion base pairs. Protein-coding sequence occupies a small part of it; other regions include introns, regulatory sequences and repeats. Non-coding does not mean functionless, and a non-coding marker must not be promised to reveal no information relevant to privacy.

In transcription, RNA polymerase reads a DNA template strand 3-prime to 5-prime while producing RNA 5-prime to 3-prime. Not every RNA is translated. For protein-coding genes, precursor RNA processing can include splicing and other modifications before mature messenger RNA is translated by a ribosome.

The ribosome reads mRNA codons, with transfer RNAs supplying amino acids. The near-universal genetic code has exceptions, including some mitochondrial assignments. Translation explains protein production; STR fragment comparison does not require translating a repeat into a protein.

Expression patterns can help assess which tissue or fluid contributed material. A semen-marker panel may include sperm-associated protamine transcripts and prostate-associated markers such as TGM4. TGM4 must not be described as transcribed only in sperm. Marker specificity, expression variability and the presence of mixtures need validation.

A suitable panel and decision rules are more defensible than declaring a fluid identified from any one transcript. Mature sperm are generally transcriptionally inactive but can retain RNA; detecting a transcript does not establish that a cell was alive or expressing the gene when collected.

Messenger RNA carries protein-coding messages. Ribosomal RNA contributes to ribosome structure and catalytic function; transfer RNA acts in translation. MicroRNAs are short regulatory RNAs. Their abundance, protection and degradation differ, so no universal ranking predicts which RNA class will survive every forensic condition.

Many expression assays first reverse-transcribe RNA into complementary DNA, then amplify selected targets. Controls may include a no-reverse-transcriptase reaction to assess DNA-derived signal, an amplification negative control, positive material and suitable indicators of RNA recovery or quality. The exact combination depends on the assay.

A negative marker result has several possible explanations: absence or low expression of the target, degradation, inhibition, inefficient extraction or sampling of the wrong material. A positive human-DNA result does not resolve these alternatives. Controls and a validated interpretation scheme are needed before a fluid conclusion is drawn.

Use RNase-controlled handling and reagents suitable for the chosen method. A stabilising reagent may be appropriate, but immediate immersion in one commercial product is not a universal prerequisite for every forensic RNA stain. Collection and preservation must also remain compatible with other examinations.

Mitochondrial DNA is DNA, not a type of RNA marker. Its variable copy number and usual maternal inheritance give it a different forensic role. Do not infer mitochondrial quantity from a nuclear-DNA quantification assay unless a relevant mitochondrial target is actually measured.

Hydrolysis can remove bases and contribute to strand damage; oxidation can modify bases or the backbone. Ultraviolet light can form pyrimidine photoproducts that interfere with copying. Nucleases also fragment nucleic acids. These effects cannot all be reduced to a single mechanism such as UV breaking phosphodiester bonds.

A PCR target needs suitable surviving template spanning its primer-binding sites and intervening sequence. Fragmentation often reduces recovery of longer targets, producing a downward size trend or loss of some results. Low starting quantity, inhibition and assay problems can cause overlapping patterns. A partial profile does not diagnose degradation by itself.

Some human-DNA quantification systems compare short and longer targets to produce a degradation index. This is an assay-derived indicator, not a direct count of every damaged molecule or an exact measurement of the longest surviving fragment. An internal amplification control may help assess inhibition.

Shorter-amplicon designs can improve recovery from fragmented material by reducing the required intact span. They do not restore missing sequence, and not every locus or allele can be made equally short. More PCR cycles can increase signal and artefacts; any altered conditions require appropriate validation rather than ad hoc compensation for degradation.

Dry, protected storage can preserve useful targets, while warmth and moisture often promote damage. There is no universal conversion from days of exposure to fragment length, DNA concentration or a predicted match statistic. Preserve the actual observations and distinguish them from explanations proposed for them.

PCR cycles through strand separation, primer annealing and extension. The two primers must flank the target in suitable orientations, with their extendable 3-prime ends directed toward the region to be copied. Polymerase uses available nucleotide triphosphates to extend the new strands. Reaction conditions follow the validated assay.

Capillary electrophoresis separates labelled amplification products through a polymer under an electric field. An internal size standard supports fragment sizing; an allelic ladder and validated calling rules support STR allele assignment. Instrument settings, sequence-dependent mobility and analytical quality still matter—sizing is not independent of all run conditions.

Sequence-based methods can distinguish same-length alleles with different sequences, often called sequence isoalleles. A length microvariant and a sequence isoallele are not interchangeable terms. Which STRs, SNPs or mitochondrial regions are examined depends on the assay; one sequencing workflow does not automatically measure every forensic target.

Biological interpretation must follow the measured information. An inherited DNA comparison addresses potential donors under stated propositions. RNA-marker testing may inform a fluid question. Neither establishes the time, mechanism or legality of contact without additional evidence. Applicable collection, retention and reporting requirements vary by jurisdiction.