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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 amplification efficiency, reproducibility and limit of detecting Escherichia coli yaaH WT (N174) and SNP (N174T) genomic alleles by qPCR using allele-specific linear or THP primers. (A) A representative set of qPCR amplification curves generated by amplification of 107 to 102 wild-type E. coli genomes using the yaaH N174 WT-specific T5 THP primers. (B) Comparison of the amplification efficiencies of linear versus T5 THP primers specific for E. coli yaaH N174 WT or the N174T SNP genomic alleles. Standard curves were generated from triplicate qPCR amplifications of 107 to 102 copies of target genomes mixed with 107 copies of non-specific genomes. (C) Comparison of linear versus THP primer-based qPCR assay reproducibility. Absolute quantification of E. coli yaaH WT and N174T SNP genomic alleles from replicate assays using specific linear versus T5 THP primers is depicted. The lowest reproducibly detected template concentration is reported as the limit of detection for each primer.
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Figure 4: Comparison of amplification efficiency, reproducibility and limit of detecting Escherichia coli yaaH WT (N174) and SNP (N174T) genomic alleles by qPCR using allele-specific linear or THP primers. (A) A representative set of qPCR amplification curves generated by amplification of 107 to 102 wild-type E. coli genomes using the yaaH N174 WT-specific T5 THP primers. (B) Comparison of the amplification efficiencies of linear versus T5 THP primers specific for E. coli yaaH N174 WT or the N174T SNP genomic alleles. Standard curves were generated from triplicate qPCR amplifications of 107 to 102 copies of target genomes mixed with 107 copies of non-specific genomes. (C) Comparison of linear versus THP primer-based qPCR assay reproducibility. Absolute quantification of E. coli yaaH WT and N174T SNP genomic alleles from replicate assays using specific linear versus T5 THP primers is depicted. The lowest reproducibly detected template concentration is reported as the limit of detection for each primer.

Mentions: While the THPs showed excellent discrimination between alleles, they were less efficient than their linear counterparts. This could potentially limit their applicability for the detection of small amounts of template. We therefore carried out real-time PCR assays with the katG and rpoB THPs at different template concentrations (between 50 pg/μl (2.3 × 108 targets) and 50 fg/μl (2.3 × 105 targets) to better establish their efficiencies and limits of detection (Figure 3A and C). Perfectly optimized real-time PCR primers should exhibit an efficiency of 2, indicating a doubling of the target sequence at each cycle. KatG linear primer efficiencies averaged 1.9, while comparable T4 primer efficiencies were 1.3. efficiencies for the rpoB linear primers averaged 1.6, while the THPs averaged 1.4 (Figure 3B). Efficiencies for the E. coli yaaH N174 T5 THPs were 1.6 for both WT- and SNP-detecting primers, while their linear counterparts were 1.9 and 2, respectively. Even so, the THPs could detect down to 1000 copies of relevant E. coli genomes relative to no template controls (Figure 4B and C). It is possible that even smaller amounts of template would not be amplified by THPs, but this could be readily overcome by using nested PCR amplification with linear primers specific for extensions embedded within the THPs.


Exquisite allele discrimination by toehold hairpin primers.

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

Comparison of amplification efficiency, reproducibility and limit of detecting Escherichia coli yaaH WT (N174) and SNP (N174T) genomic alleles by qPCR using allele-specific linear or THP primers. (A) A representative set of qPCR amplification curves generated by amplification of 107 to 102 wild-type E. coli genomes using the yaaH N174 WT-specific T5 THP primers. (B) Comparison of the amplification efficiencies of linear versus T5 THP primers specific for E. coli yaaH N174 WT or the N174T SNP genomic alleles. Standard curves were generated from triplicate qPCR amplifications of 107 to 102 copies of target genomes mixed with 107 copies of non-specific genomes. (C) Comparison of linear versus THP primer-based qPCR assay reproducibility. Absolute quantification of E. coli yaaH WT and N174T SNP genomic alleles from replicate assays using specific linear versus T5 THP primers is depicted. The lowest reproducibly detected template concentration is reported as the limit of detection for each primer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Comparison of amplification efficiency, reproducibility and limit of detecting Escherichia coli yaaH WT (N174) and SNP (N174T) genomic alleles by qPCR using allele-specific linear or THP primers. (A) A representative set of qPCR amplification curves generated by amplification of 107 to 102 wild-type E. coli genomes using the yaaH N174 WT-specific T5 THP primers. (B) Comparison of the amplification efficiencies of linear versus T5 THP primers specific for E. coli yaaH N174 WT or the N174T SNP genomic alleles. Standard curves were generated from triplicate qPCR amplifications of 107 to 102 copies of target genomes mixed with 107 copies of non-specific genomes. (C) Comparison of linear versus THP primer-based qPCR assay reproducibility. Absolute quantification of E. coli yaaH WT and N174T SNP genomic alleles from replicate assays using specific linear versus T5 THP primers is depicted. The lowest reproducibly detected template concentration is reported as the limit of detection for each primer.
Mentions: While the THPs showed excellent discrimination between alleles, they were less efficient than their linear counterparts. This could potentially limit their applicability for the detection of small amounts of template. We therefore carried out real-time PCR assays with the katG and rpoB THPs at different template concentrations (between 50 pg/μl (2.3 × 108 targets) and 50 fg/μl (2.3 × 105 targets) to better establish their efficiencies and limits of detection (Figure 3A and C). Perfectly optimized real-time PCR primers should exhibit an efficiency of 2, indicating a doubling of the target sequence at each cycle. KatG linear primer efficiencies averaged 1.9, while comparable T4 primer efficiencies were 1.3. efficiencies for the rpoB linear primers averaged 1.6, while the THPs averaged 1.4 (Figure 3B). Efficiencies for the E. coli yaaH N174 T5 THPs were 1.6 for both WT- and SNP-detecting primers, while their linear counterparts were 1.9 and 2, respectively. Even so, the THPs could detect down to 1000 copies of relevant E. coli genomes relative to no template controls (Figure 4B and C). It is possible that even smaller amounts of template would not be amplified by THPs, but this could be readily overcome by using nested PCR amplification with linear primers specific for extensions embedded within the THPs.

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