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: In attempting to identify SNPs during real-time PCR amplification, the ideal result would be that a SNP-specific primer would perfectly bind its matched template and not react at all with the mismatched template. While this is energetically impossible, it may nonetheless be possible to create situations where the initial discrimination between matched and mismatched primers leads to much more productive amplification of only the matched sets. By manipulating the DNA toehold strand displacement designs originally described in the field of DNA computing, we have come up with a model for mismatch discrimination that relies on equilibration of a very small sequence ‘seed’, rather than equilibration of a much larger primer. In this model, the initial binding of the seed leads to two processes, which may occur in parallel: first, strand displacement that leads to additional primer-binding and second, strand extension (Figure 1).
Affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.