SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis

• Published in: Scientific reports 2020-03, Vol.10 (1), p.5476-5476, Article 5476
• Main Authors: Kondratova, V. N., Botezatu, I. V., Shelepov, V. P., Lichtenstein, A. V.
• Format: Article
• Language: English
• Subjects: 631/208; 631/67; 692/308; 692/4028; 692/53; Alleles; Antisense DNA; Colonic Neoplasms - genetics; Conformation; Deoxyribonucleic acid; DNA; DNA Mutational Analysis - methods; DNA Probes; Genetic analysis; Humanities and Social Sciences; Humans; Hybrids; Melting; multidisciplinary; Mutants; Mutation; Mutation - genetics; Mutation hot spots; Nucleic Acid Denaturation; Polymerase chain reaction; Polymerase Chain Reaction - methods; Probes; Proto-Oncogene Proteins p21(ras) - genetics; Scanning; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-62173-x

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 ( S tem- L oop AM plicon M utation S canning) 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.