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Exquisite allele discrimination by toehold hairpin primers.

Byrom M, Bhadra S, Jiang YS, Ellington AD - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.

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Schematic of toehold-dependent hairpin (THP) primers for enhanced SNP distinction. Most of the target-binding sequence of the hairpin primer is sequestered by hybridization to the complementary sequence in the stem (shown in blue) leaving a short target-specific single stranded ‘toehold’ (red arrow) at the 3′-end. Binding of the toehold to its complementary target sequence initiates strand displacement leading to primer unfolding and stabilization of the primer:template duplex by extended hybridization. Concomitant initiation of polymerization from the 3′-end by the DNA polymerase leads to template amplification. The toehold hybridization efficiency (and the ensuing primer strand displacement) is designed to be significantly impaired by even a single mismatched nucleotide resulting in primer destabilization and significantly diminished amplification.
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Figure 1: Schematic of toehold-dependent hairpin (THP) primers for enhanced SNP distinction. Most of the target-binding sequence of the hairpin primer is sequestered by hybridization to the complementary sequence in the stem (shown in blue) leaving a short target-specific single stranded ‘toehold’ (red arrow) at the 3′-end. Binding of the toehold to its complementary target sequence initiates strand displacement leading to primer unfolding and stabilization of the primer:template duplex by extended hybridization. Concomitant initiation of polymerization from the 3′-end by the DNA polymerase leads to template amplification. The toehold hybridization efficiency (and the ensuing primer strand displacement) is designed to be significantly impaired by even a single mismatched nucleotide resulting in primer destabilization and significantly diminished amplification.

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).


Exquisite allele discrimination by toehold hairpin primers.

Byrom M, Bhadra S, Jiang YS, Ellington AD - Nucleic Acids Res. (2014)

Schematic of toehold-dependent hairpin (THP) primers for enhanced SNP distinction. Most of the target-binding sequence of the hairpin primer is sequestered by hybridization to the complementary sequence in the stem (shown in blue) leaving a short target-specific single stranded ‘toehold’ (red arrow) at the 3′-end. Binding of the toehold to its complementary target sequence initiates strand displacement leading to primer unfolding and stabilization of the primer:template duplex by extended hybridization. Concomitant initiation of polymerization from the 3′-end by the DNA polymerase leads to template amplification. The toehold hybridization efficiency (and the ensuing primer strand displacement) is designed to be significantly impaired by even a single mismatched nucleotide resulting in primer destabilization and significantly diminished amplification.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150758&req=5

Figure 1: Schematic of toehold-dependent hairpin (THP) primers for enhanced SNP distinction. Most of the target-binding sequence of the hairpin primer is sequestered by hybridization to the complementary sequence in the stem (shown in blue) leaving a short target-specific single stranded ‘toehold’ (red arrow) at the 3′-end. Binding of the toehold to its complementary target sequence initiates strand displacement leading to primer unfolding and stabilization of the primer:template duplex by extended hybridization. Concomitant initiation of polymerization from the 3′-end by the DNA polymerase leads to template amplification. The toehold hybridization efficiency (and the ensuing primer strand displacement) is designed to be significantly impaired by even a single mismatched nucleotide resulting in primer destabilization and significantly diminished amplification.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.

Show MeSH
Related in: MedlinePlus