Exploiting extension bias in polymerase chain reaction to improve primer specificity in ensembles of nearly identical DNA templates
We describe a semi‐empirical framework that combines thermodynamic models of primer hybridization with experimentally determined elongation biases introduced by 3′‐end mismatches for improving polymerase chain reaction (PCR)‐based sequence discrimination. The framework enables rational and automatic...
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| Published in: | Environmental microbiology Vol. 16; no. 5; pp. 1354 - 1365 |
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| Main Authors: | , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Oxford
Blackwell Science
01-05-2014
Blackwell Publishing Ltd Blackwell Wiley Subscription Services, Inc |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | We describe a semi‐empirical framework that combines thermodynamic models of primer hybridization with experimentally determined elongation biases introduced by 3′‐end mismatches for improving polymerase chain reaction (PCR)‐based sequence discrimination. The framework enables rational and automatic design of primers for optimal targeting of one or more sequences in ensembles of nearly identical DNA templates. In situations where optimal targeting is not feasible, the framework accurately predicts non‐target sequences that are difficult to distinguish with PCR alone. Based on the synergistic effects of disparate sources of PCR bias, we used our framework to robustly distinguish between two alleles that differ by a single base pair. To demonstrate the applicability to environmental microbiology, we designed primers specific to all recognized archaeal and bacterial genera in the Ribosomal Database Project, and have made these primers available online. We applied these primers experimentally to obtain genus‐specific amplification of 16S rRNA genes representing minor constituents of an environmental DNA sample. Our results demonstrate that inherent PCR biases can be reliably employed in an automatic fashion to maximize sequence discrimination and accurately identify potential cross‐amplifications. We have made our framework accessible online as a programme for designing primers targeting one group of sequences in a set with many other sequences (http://DECIPHER.cee.wisc.edu). |
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| Bibliography: | http://dx.doi.org/10.1111/1462-2920.12259 ark:/67375/WNG-Q013K6NZ-B Supplementary Methods. Detailed description of methods used for primer design.Fig. S1. Comparison of three different design strategies for targeting Ohtaekwangia sequences. Gel runs of PCR products before and after digestion with the restriction enzyme HinfI, which cuts near the centre of Ohtaekwangia amplicons. Lane 1 contains a 100 base pair ladder. Lanes 2 and 3 contain PCR products obtained with Ohtaekwangia primers designed with hybridization efficiency alone (strategy #1) before and after digestion respectively. The PCR products in lanes 4 and 5 were obtained with primers designed using elongation efficiency (strategy #2), and lanes 6 and 7 with primers designed using elongation efficiency and an induced mismatch in the 6th position from the 3′-end. Intensity profiles from the top to bottom of lanes 3, 5 and 7 are shown in (B), (C) and (D) respectively. The digested (shorter) target amplicon (Ohtaekwangia) is coloured in green, whereas undigested non-target amplicons are coloured in red. Note that the reverse primer's target site in strategy #3 (lanes 6 and 7) is shifted towards the forward primer by 31 nucleotides relative to the reverse primer's target site used for strategy #2 (lanes 4 and 5). This difference in amplicon sizes (Table S2) explains the shorter digested and undigested product lengths in lanes 6 and 7 relative to lanes 4 and 5 respectively.Fig. S2. Comparison of decreased hybridization efficiency with decreased elongation efficiency. Amplification curves for the Mycobacterium template. This figure illustrates (A) decreased hybridization efficiency and (B) decreased elongation efficiency. A. Equal initial concentrations of template were amplified with an annealing gradient from 50°C to 75°C. B. Equal initial concentrations of template were amplified using either perfect match forward (F) and reverse (R) primers (solid line), or one primer with a 3′-terminal mismatch (primer/template, dashed lines).Fig. S3. Flowchart of describing how to design a primer step-by-step using the online Design Primers web tool.Table S1. Terminal mismatched primers and observed elongation efficiencies.Table S2. Efficiency of elongation of 3′ non-terminal mismatches.Table S3. Results obtained with primers designed to discriminate alleles of the Human IDH2 gene using qPCR (n = 3).Table S4. Primer sets used to validate the primer design methodology.Table S5. Average efficiency of elongation of 3′ terminal mismatches separated by the penultimate primer nucleotide.Table S6. DNA templates and perfect match primer sequences used to determine relative elongation efficiency of 3′ terminal mismatches. ArticleID:EMI12259 Water Research Foundation - No. 4291 istex:C331F7213F1998609BF8F37821EB9F46712EB0FA ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 1462-2912 1462-2920 |
| DOI: | 10.1111/1462-2920.12259 |