DNAprimer

DNA primer is a short single-stranded oligonucleotide that serves as a starting point for DNA synthesis by DNA polymerases. In laboratory methods such as polymerase chain reaction (PCR) and Sanger sequencing, primers are synthetically produced DNA sequences typically 18 to 30 nucleotides long. They bind to complementary regions on the template DNA, defining the boundaries of the segment to be copied and providing a free 3' hydroxyl group for chain elongation.

Primer design is central to assay success. Ideal primers have balanced GC content (roughly 40–60%), moderate length, and similar melting temperatures for paired primers. They should avoid strong secondary structures, significant self-complementarity, or cross-dimer formation with the partner primer. The 3' end should be stable and specific to minimize non-specific amplification. In some protocols, primers include additional sequences such as restriction sites or sequencing adapters.

Applications include amplifying DNA for cloning, genotyping, and library preparation, sequencing primers for read initiation, and primers used in mutagenesis or next-generation sequencing workflows. Degenerate primers allow amplification when target sequence is variable, while nested or nested PCR uses two sets of primers to increase specificity.

Limitations include potential off-target binding, primer-dimer artifacts, and biases that can affect amplification efficiency. Primer design often relies on software tools that model thermodynamics and sequence complexity. The concept of DNA primers emerged with the development of DNA polymerases and sequencing methods in the late 20th century, most notably during PCR and Sanger sequencing breakthroughs.