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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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Effect of toehold length on SNP distinction by THP primers. Hairpin primers specific for the Mycobacterium tuberculosis wild-type (WT) katG allele were designed with 4, 5 or 6 nucleotide long toeholds (T4, T5 and T6, respectively) at their 3′-ends. The amplification efficiency of linear (Lin) and THP primers and their ability to discriminate between the WT and single nucleotide mismatch (SNP)-containing alleles of katG was compared by real-time qPCR analysis of cloned gene variants. The average Cq of amplification from triplicate experiments, (A) a representative set of qPCR amplification curves, (B) and Cqs of amplification from triplicate experiments for T4 WT and SNP primers for katG (C) and rpoB alleles (D) are depicted.
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Figure 2: Effect of toehold length on SNP distinction by THP primers. Hairpin primers specific for the Mycobacterium tuberculosis wild-type (WT) katG allele were designed with 4, 5 or 6 nucleotide long toeholds (T4, T5 and T6, respectively) at their 3′-ends. The amplification efficiency of linear (Lin) and THP primers and their ability to discriminate between the WT and single nucleotide mismatch (SNP)-containing alleles of katG was compared by real-time qPCR analysis of cloned gene variants. The average Cq of amplification from triplicate experiments, (A) a representative set of qPCR amplification curves, (B) and Cqs of amplification from triplicate experiments for T4 WT and SNP primers for katG (C) and rpoB alleles (D) are depicted.

Mentions: The linear primers (Lin) demonstrated relatively small Cq differences between matched and mismatched targets (Cq delay = 6.2). The THPs showed greater discrimination: the T6 primer gave a Cq delay of 8.7, the T5 primer gave a Cq delay of 15, while the T4 primer did not amplify the mismatched target (Figure 2A). The T4 primers reproducibly gave an average Cq of 32.5 for the WT template and showed no amplification through 45 cycles with the mutant template (Figure 2B). These results were in general concordance with the notion that mismatch discrimination by THPs was highly dependent upon the initial contact of the toehold with the template. It should also be noted that these results show much greater Cq delay values than previously published hairpin primers without allele-specific toeholds (16). For example, a hairpin primer with the toehold in the loop of the hairpin yielded a maximum difference in cycle number between a matched template and a single mismatch of 11.2 cycles, as opposed to the 15 or greater cycle differences that we routinely observe.


Exquisite allele discrimination by toehold hairpin primers.

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

Effect of toehold length on SNP distinction by THP primers. Hairpin primers specific for the Mycobacterium tuberculosis wild-type (WT) katG allele were designed with 4, 5 or 6 nucleotide long toeholds (T4, T5 and T6, respectively) at their 3′-ends. The amplification efficiency of linear (Lin) and THP primers and their ability to discriminate between the WT and single nucleotide mismatch (SNP)-containing alleles of katG was compared by real-time qPCR analysis of cloned gene variants. The average Cq of amplification from triplicate experiments, (A) a representative set of qPCR amplification curves, (B) and Cqs of amplification from triplicate experiments for T4 WT and SNP primers for katG (C) and rpoB alleles (D) are depicted.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Effect of toehold length on SNP distinction by THP primers. Hairpin primers specific for the Mycobacterium tuberculosis wild-type (WT) katG allele were designed with 4, 5 or 6 nucleotide long toeholds (T4, T5 and T6, respectively) at their 3′-ends. The amplification efficiency of linear (Lin) and THP primers and their ability to discriminate between the WT and single nucleotide mismatch (SNP)-containing alleles of katG was compared by real-time qPCR analysis of cloned gene variants. The average Cq of amplification from triplicate experiments, (A) a representative set of qPCR amplification curves, (B) and Cqs of amplification from triplicate experiments for T4 WT and SNP primers for katG (C) and rpoB alleles (D) are depicted.
Mentions: The linear primers (Lin) demonstrated relatively small Cq differences between matched and mismatched targets (Cq delay = 6.2). The THPs showed greater discrimination: the T6 primer gave a Cq delay of 8.7, the T5 primer gave a Cq delay of 15, while the T4 primer did not amplify the mismatched target (Figure 2A). The T4 primers reproducibly gave an average Cq of 32.5 for the WT template and showed no amplification through 45 cycles with the mutant template (Figure 2B). These results were in general concordance with the notion that mismatch discrimination by THPs was highly dependent upon the initial contact of the toehold with the template. It should also be noted that these results show much greater Cq delay values than previously published hairpin primers without allele-specific toeholds (16). For example, a hairpin primer with the toehold in the loop of the hairpin yielded a maximum difference in cycle number between a matched template and a single mismatch of 11.2 cycles, as opposed to the 15 or greater cycle differences that we routinely observe.

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