PCRPrimer

PCRPrimer is the short single-stranded DNA oligonucleotide used to initiate DNA synthesis in polymerase chain reaction (PCR). In standard PCR, two primers bind to complementary sequences on opposite strands of the target DNA, flanking the region to be amplified. The DNA polymerase extends from the primers, copying the template and producing new double-stranded DNA that serves as a template for subsequent cycles.

Most PCR primers are about 18 to 25 nucleotides long, with melting temperatures typically in a range that supports specific annealing, and GC content around 40 to 60 percent. They should be specific to the target sequence, minimize secondary structures and primer-dimer formation, and have similar melting temperatures for the paired forward and reverse primers to enable efficient amplification. The chosen amplicon length can vary, but is generally balanced to maintain specificity and amplification efficiency.

Applications of PCR primers span conventional end-point PCR, quantitative real-time PCR (qPCR), and various cloning, sequencing, and genotyping workflows. In multiplex PCR, multiple primer pairs are used in a single reaction to amplify several targets simultaneously. Primers can also be modified with additional features for downstream use, such as fluorescent labels for detection or tails that introduce restriction sites or sequencing adapters.

Design and synthesis: Primers are chemically synthesized oligonucleotides. Their performance depends on target sequence, primer design, and reaction conditions, and may be influenced by template quality and the presence of inhibitors.