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

Polymorphism analysis and gene detection by minisequencing on an array of gel-immobilized primers

“…The melting temperature (T m ) of primers calculated using different formulae (21,22) did not correlate well with the experimental values. In addition, as shown earlier, T m was different for duplexes formed on a microchip and in solution (23).…”

Easy-to-Use InDel Markers for Genetic Mapping between Col-0 and Ler-0 Accessions of Arabidopsis thaliana

“…To prevent mispriming at GC-rich sequences and smearing of DNA bands in the gel, A or T was placed at the second position from the 3′ end and inclusion of more than 3 consecutive G/Cs near the With only a few exceptions, we designed PCR primers to be 20 nucleotides in length with the GC content of 9 to 11 nucleotides and the 3 ending with G or C to promote specific binding (Table 1). To prevent mispriming at GC-rich sequences and smearing of DNA bands in the gel, A or T was placed at the second position from the 3 end and inclusion of more than 3 consecutive G/Cs near the 3 end was avoided [16]. The length of amplified DNA fragments was generally set ranging from 300 bp to 1000 bp.…”

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes

“…The typical design rules for PCR primers and probes recommend that the range of hybridization temperatures in a given set should be narrow, of the order of 10 • C [25,43]. Several authors even suggest that the range for primer pair should be no more than 5 • C [22,44] or even less than 1 • C [45].…”