Abstract
DNA Melting Analysis (DMA) with a TaqMan probe covering the mutation “hot spot” is a simple, sensitive, and “closed tube” method of mutation detection. However, DMA requires asymmetric PCR to produce single-stranded amplicons capable of interacting with TaqMan probes. This makes quantitative analysis impossible owing to low amplification efficiency. Moreover, bi-strand mutation detection necessitates two independent PCRs. The SLAM-MS (Stem-Loop AMplicon Mutation Scanning) assay, in which symmetric PCR is performed using primers with 5'-universal primer sequence (UPS), has been developed to detect KRAS mutations. Some of the resulting amplicons, sense and antisense, adopt single-stranded stem-loop conformation and become unable to renature, but able to hybridize with TaqMan probes. Hybrids of stem-loops and complementary TaqMan probes are suitable for melting analysis and simultaneous bi-strand mutation scanning. In addition, the areas under the melting peaks are determined by the PeakFit software, a non-linear iterative curve fitting program, to evaluate the wild-type/mutant allele ratio. Thus, the SLAM-MS assay permits quantification of both the number of copies of the target sequence and the percentage of mutant alleles. For mutant enrichment, the SLAM-MS assay uses TaqMan probes as PCR blocking agents allowing an ~10 times higher mutation detection sensitivity than High Resolution Melting (HRM) assay.
Highlights
In basic cancer research, priority is given to large-scale generation sequencing (NGS) methods, which give a panoramic picture of defects in the cancer genome
A disadvantage of this approach is that the quantitative real time PCR is symmetric, while DNA Melting Analysis (DMA) with TaqMan probes requires an asymmetric PCR; two independent specific reactions are necessary
A detailed study of this phenomenon was not the aim of this work, we found in preliminary experiments that either increasing or decreasing the loop size diminished the DMA signal, probably due to steric hindrances of the probe fluorophore and/or quencher
Summary
Priority is given to large-scale (genome- and exome-wide) generation sequencing (NGS) methods, which give a panoramic picture of defects in the cancer genome. For clinical diagnostics, a “targeted approach” is more often required, i.e. screening of previously known clinically important mutations, in particular in the KRAS, NRAS, BRAF, EGFR, and PIK3CA genes[1,2,3,4,5,6,7] In this case, the priorities are the feasibility of the method in a clinical laboratory, and its simplicity, performance, and sensitivity. The priorities are the feasibility of the method in a clinical laboratory, and its simplicity, performance, and sensitivity These requirements are largely met by asymmetric PCR with a TaqMan probe covering the mutation “hot spot” and subsequent DMA (DNA Melting Analysis), which is one of the most simple, fast, and least costly methods to detect DNA mutations. This is one of the most clinically significant genes, exhibiting mutations in ~40% of colon cancer cases[11]
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