Discrimination of Primer 3′-Nucleotide Mismatch by Taq DNA Polymerase during Polymerase Chain Reaction

Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping

“…Mispriming of the allelespecific primers was another well-known obstacle of ASPCR [Yu et al, 2004]. Complicated modifications of primer sequences, mostly by additional intrinsic mismatches, were designed to minimize the problem but also limited its robustness [Ayyadevara et al, 2000]. Additionally, our data found crossover amplification, which might significantly obscure more downstream haplotypes, occurred in long-range PCR.…”

“…Although we did not include other SNPs, many reports had validated the high specificity of single-base extension reaction, irrespective of different polymorphisms, with assembly of the proofreading polymerases and phosphorothioate primers [Zhang and Li, 2003;Di Giusto and King, 2003]. Transition SNPs were reported to harbor lower allelic specificity than transversion SNPs in ASPCR, suggesting LALA would be also robust for other SNPs [Ayyadevara et al, 2000].…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…It appears that both the local sequence and the type of polymorphism affect the specificity of AS-PCR. 40 Furthermore, the nature of exponential amplification of AS-PCR makes the decay of discriminating power much quicker than that of linear amplification as seen in both SBE and ASE. For example, if the polymerase makes 1% error in at the polymorphic base or a discriminating factor of 100 to 1 for a homozygous sample, the accumulated error in the amplified population would reach B24% for AS-PCR (1-0.99 30 ) after 30 cycle amplification, but that for SBE and ASE remains at 1%.…”

“…Factors that contribute to the success rate of markers are the nature of polymorphisms, allele discrimination and detection methods. Some mismatches are known to be difficult for certain allele discrimination, for example, G/T mismatch is difficult to be distinguished by hybridization and ASE, 40 others may be difficult for a detection method, such the A/T polymorphism for MALDI-TOF MS detection. 82,112 Local sequences where the target polymorphisms located could limit the choices of primer design this could result in nonspecific reactions or failures.…”

Universal primers for the amplification of the plastid <i>trnK/matK</i> region in land plants

“…The primer heads of the reverse trn2RB primer and its derived MG1 primer span this region and cannot anneal properly. As a result, synthesis activity of polymerase activity is strongly impaired by mismatches affecting the 3'-primerhead (Thweatt & al., 1990;Liang & Pardee, 1992;Ayyadevara & al., 2000).…”