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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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Comparison of the Cq delay in qPCR amplification of mismatched Escherichia coli genomic alleles using THP versus linear primers. Triplicate qPCR assays were used to determine the Cq delay in amplification of crosspaired E. coli genomic targets using linear (blue bars) or THP (red bars) primers specific for WT (REL606) or SNP (CZB154) alleles. Primers that did not generate significant fluorescent signals from their mismatched target genome at the end of 60 amplification cycles are tagged with an asterisk.
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Figure 5: Comparison of the Cq delay in qPCR amplification of mismatched Escherichia coli genomic alleles using THP versus linear primers. Triplicate qPCR assays were used to determine the Cq delay in amplification of crosspaired E. coli genomic targets using linear (blue bars) or THP (red bars) primers specific for WT (REL606) or SNP (CZB154) alleles. Primers that did not generate significant fluorescent signals from their mismatched target genome at the end of 60 amplification cycles are tagged with an asterisk.

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).


Exquisite allele discrimination by toehold hairpin primers.

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

Comparison of the Cq delay in qPCR amplification of mismatched Escherichia coli genomic alleles using THP versus linear primers. Triplicate qPCR assays were used to determine the Cq delay in amplification of crosspaired E. coli genomic targets using linear (blue bars) or THP (red bars) primers specific for WT (REL606) or SNP (CZB154) alleles. Primers that did not generate significant fluorescent signals from their mismatched target genome at the end of 60 amplification cycles are tagged with an asterisk.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Comparison of the Cq delay in qPCR amplification of mismatched Escherichia coli genomic alleles using THP versus linear primers. Triplicate qPCR assays were used to determine the Cq delay in amplification of crosspaired E. coli genomic targets using linear (blue bars) or THP (red bars) primers specific for WT (REL606) or SNP (CZB154) alleles. Primers that did not generate significant fluorescent signals from their mismatched target genome at the end of 60 amplification cycles are tagged with an asterisk.
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).

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