Technical report: Part 2. Basic requirements for designing optimal PCR primers

Designing optimal polymerase chain reaction (PCR) primer sequences is one of the critical factors for successful PCR with sensitive, specific, and assay-to-assay reproducible results. In this review, all the requirements of PCR primer sequences are summarized, such as location, size of amplicon, length of primers, nucleotide composition, Tm, 3' terminal hybridization strength and frequency, hairpin formation energy, primer-to-primer interaction, specificity, and location of mismatches to sequences of cross-hybridization. The report also discusses how to explore these various types of information for more advanced PCR applications, which include nested PCR, multiplex PCR, competitive PCR, long PCR, point mutation detection, degenerate primers, and PCR cloning.