Essence of DNA Amplification PCR, or polymerase chain reaction, is an amplification technique that multiplies specific DNA segments exponentially. It utilizes DNA polymerase to extend primers and synthesize new strands. This process acts like a molecular Xerox machine, producing millions of copies from a single template.
Essential Components and Reagents A thermal cycler is used for precise temperature regulation during the reaction. Template DNA, short oligonucleotide primers (20 to 30 nucleotides) that bind to target sequences, and deoxynucleotide triphosphates form the core ingredients. Taq DNA polymerase from thermus aquaticus, capable of withstanding high temperatures, ensures efficient DNA synthesis.
Core PCR Mechanism and Cycles The process involves denaturation at 95°C to separate DNA strands, annealing at around 56°C for primer binding, and extension at 70–72°C where new DNA strands are synthesized. This cycle repeats 25 to 30 times, incrementally doubling the number of DNA molecules. The cycle’s repetition leads to exponential amplification of the target DNA.
Diverse Applications in Science and Medicine PCR is instrumental in molecular biology, recombinant DNA technology, and forensic science for tasks such as constructing gene libraries and DNA fingerprinting. It aids in prenatal diagnosis and detecting inborn errors of metabolism. Additionally, PCR is critical in identifying infections and certain cancers by amplifying specific DNA markers.
Quantitative PCR in COVID-19 Detection For viral detection, particularly SARS-CoV-2, the technique incorporates reverse transcription to convert RNA into complementary DNA. Real-time PCR uses fluorescent probes that separate from quencher molecules during DNA synthesis, allowing quantification of the viral load. The fluorescent signal increases with each amplification cycle, providing a rapid and sensitive means of diagnosing infections.