PCRs

PCR, or polymerase chain reaction, is a laboratory technique used to amplify a specific DNA sequence, producing millions of copies from a small initial amount. Since its development in the 1980s, PCR has become a foundational tool in molecular biology, medicine, and forensic science.

A standard PCR reaction contains a DNA template with the target sequence, two primers that flank the region to be amplified, a thermostable DNA polymerase, deoxynucleotide triphosphates, buffer, and sometimes salts or enhancers. The protocol uses cycles of heating and cooling: denaturation around 95 C to separate DNA strands, annealing at a lower temperature for primers to bind, and extension around 72 C when the polymerase adds nucleotides to synthesize new DNA. With each cycle, the amount of target DNA ideally doubles, leading to exponential amplification.

Variants of PCR expand its capabilities. Reverse transcription PCR (RT-PCR) converts RNA into DNA before amplification, enabling analysis of gene expression and RNA pathogens. Quantitative PCR (qPCR) monitors amplification in real time, using fluorescence to quantify the starting material. Digital PCR partitions the reaction to count DNA molecules more precisely. Multiplex PCR uses multiple primer pairs to amplify several targets in a single reaction, and nested PCR employs two rounds of amplification to increase sensitivity.

Applications span basic research, clinical diagnostics (including pathogen detection and genetic testing), forensic analysis, cloning, and sequencing. Limitations include the risk of contamination causing false positives, the need for careful primer design, inhibition by sample impurities, and amplification biases that can affect quantitative results.