Exquisite allele discrimination by toehold hairpin primers.
Bottom Line: We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex.However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur.During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a 'yes/no' answer.
Affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.Show MeSH
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Mentions: For the remaining assays, we noted that at least one primer in each set might have more weakly annealed to the template than its partner. For example, in the mepA S33 assay we noted that while the common reverse linear primer had 50% GC content and a Tm of 63.9°C, its 3′ end contained two adenosines. The efficiency of both the mepA S33 SNP and WT primers was improved by developing a new common reverse primer. This primer was identified by allowing a 5°C difference between the forward and reverse primers, rather than the 3°C default difference set by the NCBI Primer Blast program. A reverse primer (Table 2) predicted to have a Tm of 68.3°C under our assay conditions and which yielded a shorter product length (115 versus 190 bp) was identified. In a three-step PCR, the new primer yielded Cq values of 44 and 32 for the SNP- and WT-detecting primers on their respective target genomes (Table 3). This primer could also be extended to a 5 nt toehold, terminating in a 3′ G, which further resulted in Cq values of 42 for the SNP and 28 for the WT primers. No amplification was detected for either toehold length on mismatched target sequences. In contrast, the linear ARMs primer with a 3′ SNP-specific base had a Cq delay of only 6 cycles between matched and unmatched targets (Table 3, Figure 5).
Affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.