3.3 DNA Amplification, Profiling and Sequencing

The polymerase chain reaction produces many copies of a selected DNA region. A reaction contains template DNA, free dNTPs, two primers, a heat-stable DNA polymerase and a suitable buffer with required ions. The primers bind to opposite strands on either side of the target. Their sequences determine the endpoints and orientation of amplification; polymerase amplifies what lies between correctly bound primers rather than choosing a gene by its biological function.

Each cycle has three conceptual stages. High temperature denatures the duplex by disrupting hydrogen bonds. Cooling allows primers to anneal to complementary target sequences. At an intermediate high temperature, Taq polymerase extends the primers 5′→3′. Taq comes from a thermophilic bacterium and remains functional after repeated heating that would denature many ordinary polymerases. A thermocycler repeats the temperature program automatically.

Under ideal conditions, every target molecule gives two target molecules per cycle, producing exponential growth. Real reactions eventually depart from this ideal because primers and dNTPs become limiting, products re-anneal, polymerase loses activity and inhibitory substances may be present. Contamination is particularly dangerous: DNA introduced before amplification may be copied along with the intended sample, turning a trace handling error into a strong signal.

DNA has a net negative charge because of phosphate groups in its backbone. When voltage is applied across a gel, fragments migrate from wells near the negative electrode toward the positive electrode. The porous gel resists movement. Small fragments pass through the matrix more easily and travel farther in a fixed time than large fragments. A DNA ladder containing fragments of known sizes provides a reference for estimating unknown fragment lengths.

A visible band represents many DNA fragments of similar length, not one molecule and not necessarily one gene. In profiling, variable-number or short tandem-repeat loci are amplified. Individuals can differ in repeat number and therefore fragment length at each locus. Examining many independent loci makes a coincidental match between unrelated individuals increasingly unlikely, while relatives are expected to share more alleles.

For a paternity comparison, every band or allele in a child's profile must be explainable by inheritance from one biological parent or the other. For a crime-scene comparison, a mismatch can exclude a source under appropriate sampling assumptions, but a match does not by itself prove when or how DNA arrived. Mixed samples, contamination, laboratory error, identical twins and statistical interpretation all limit what a profile can establish.

DNA sequencing determines the order of bases in a selected region, chromosome or genome. A DNA profile instead compares patterns at chosen variable loci and does not normally reveal an individual's complete base sequence. Sequencing can identify an allele, discover a mutation, compare conserved regions or assemble a genome; profiling is optimized for identity and relatedness. Treating the two as synonyms overstates the information in a forensic profile.

Sequence-specific primers allow PCR to test whether a sample contains a pathogen-associated or altered DNA region. RNA viruses require reverse transcription to make DNA before PCR amplification. A positive signal supports the presence of the targeted sequence, while a negative result may mean absence, poor sampling, degradation, inhibitors or sequence variation at primer sites. Controls are needed to distinguish a true negative from a failed reaction.

DNA databases create a further distinction between technical reliability and acceptable use. Retaining profiles can help connect cases or identify missing people, but access controls, consent, retention periods, familial searching and the treatment of people never convicted all raise ethical questions. A profile is intensely identifying even when it contains selected repeat markers rather than a complete genome. Sound policy must consider both investigative value and the consequences of error, misuse or unequal representation.