Gene technology: PCR, recombinant DNA and genetic fingerprinting

Polymerase chain reaction (PCR)

• 95 °C: hydrogen bonds between strands break, separating the DNA.
• About 55 °C: primers anneal to complementary sequences at the ends of each strand.
• 72 °C: Taq polymerase (from a thermophilic bacterium, so not denatured) adds nucleotides to build new strands.
• Each cycle doubles the DNA, so n cycles give 2ⁿ copies of each original molecule.

Making recombinant DNA

The gene can be obtained with reverse transcriptase from mRNA (giving cDNA with no introns), with restriction endonucleases that cut at specific recognition sequences, or with a gene machine. The same restriction enzyme cuts a plasmid vector, leaving complementary sticky ends. DNA ligase joins the sugar-phosphate backbones. Promoter and terminator regions are added so the gene is transcribed.

Identifying transformed cells

Only a small proportion of host cells take up the plasmid. Marker genes — for antibiotic resistance, fluorescence or an enzyme producing a colour change — are included so transformed cells can be identified. Replica plating uses a gene inserted into an antibiotic-resistance gene to distinguish cells with recombinant plasmids.

Genetic fingerprinting

Non-coding DNA contains variable number tandem repeats (VNTRs) that differ between individuals. DNA is extracted, amplified by PCR, cut with restriction enzymes and separated by gel electrophoresis — smaller fragments travel further towards the positive electrode. The resulting band pattern is compared between samples in forensic science, paternity testing and measuring genetic diversity in breeding programmes.