PCRrelated

PCR-related refers to techniques, methods, and applications that rely on the polymerase chain reaction (PCR). Developed in the 1980s, PCR enables exponential amplification of a specific DNA sequence by cycling through denaturation, primer annealing, and strand extension using a thermostable DNA polymerase.

End-point PCR amplifies DNA with final detection by gel electrophoresis. Real-time PCR (qPCR) monitors amplification via fluorescence and provides Ct values. RT-PCR uses reverse transcription to convert RNA to cDNA before amplification. Digital PCR (ddPCR) partitions the sample for absolute quantification. Multiplex PCR amplifies multiple targets; nested PCR and hot-start PCR improve specificity.

Core reagents include template DNA, primers, thermostable DNA polymerase, dNTPs, buffer, and Mg2+. Primer design requires 18–25 nucleotide length, balanced GC content, specificity to the target, and minimal secondary structure. Detection methods include gel electrophoresis for size and presence, and fluorescence-based readouts in qPCR, with Ct values and melting-curve analysis used to assess product specificity.

Applications span clinical diagnostics, infectious disease testing, genetics and oncology, microbiology, forensics, paternity testing, and food safety. Limitations include contamination leading to false positives, inhibitors causing false negatives, and amplification biases. Proper controls, validation, and standardized protocols help mitigate these issues.

Advances include digital PCR variants and multiplexing, while isothermal amplification represents a related approach that operates at a constant temperature. Quality standards such as good laboratory practice, accreditation, and assay validation ensure accuracy and traceability in PCR-related work.