PrimeQHelp@bibby-scientific.com | www.bibby-scientific.com +44(01785) 810433 1 Application Note A08-004A Discrimination of a single base pair mismatch using melting analysis Introduction Melting analysis is frequently used to characterise PCR products post-amplification. The melting temperature (T m ) of a PCR product is dependent on both the length and sequence of the DNA and can therefore be used to check for non-specific products of a reaction or to analyse DNA sequence variations such as insertions, deletions and single base pair changes. Melting analysis requires the use of an intercalating dye. Intercalating dyes bind to the minor groove of double-stranded DNA (dsDNA) producing up to a thousand-fold increase in fluorescence. Third generation fluorescent dsDNA dyes such as SYTO® 9 and EvaGreen® are able to be used at higher concentration in the reaction than traditional dyes due to their lower toxicity. This allows for greater saturation of the dsDNA leading to increased sensitivity, high fidelity and better resolution in melting curve profiles. In this application note we demonstrate that using melting analysis, PrimeQ was able to discriminate a single base pair change (G/A conversion) using various intercalating dyes. Methods Two artificial 100 base ssDNA templates were synthesized based on the genomic sequence of Enterobacteria phage lambda (Figure 1). The lambda DNA sequence normally has a G base at position 16404. One of the templates was synthesized with the G base in this position (“wild type”) and the second with an A in this position (“mutant”). The primers were designed to amplify a 76 base pair product within the templates, encompassing the base change. Selecting a short product ensures that the mismatch will have greatest effect on the overall T m of the product. Figure 1: Sequences of the lambda DNA templates and primers. The 100 base artificial templates were based on the sequence of bases 16361 to 16460 inclusive (accession number J02459.1). The “wild type” template had a G base at position 16404 and the “mutant” an A base. Primers were designed using NCBI Primer-BLAST and are shown in blue and green. Four different reagent kits were used for amplification and detection of the products (Table 1). Kit name Supplier Part code JumpStart™ Taq ReadyMix™ Sigma S4438 AccuMelt™ HRM SuperMix Quanta Biosciences™ 95103-250 Fast EvaGreen® qPCR Master Mix Biotium 31003 QuantiFast® PCR Kit Qiagen 204052 Table 1: Details of the reagent kits used. All kits were supplied as complete 2X master mixes. Approximately 5 x 10 5 copies of either template were used per reaction. Six replicates of either template were run for each kit plus two no template controls (NTC). The final primer concentration was 0.2µM and ROX™ passive
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