PCR-Based Detection Methods for Single-Nucleotide Polymorphism or Mutation: Real-Time PCR and Its Substantial Contribution Toward Technological Refinement

• Published in: Advances in clinical chemistry Vol. 80; pp. 45 - 72
• Main Author: Matsuda, Kazuyuki
• Format: Journal Article
• Language: English
• Published: United States 2017
• Subjects: DNA - genetics; Genetic Markers; Humans; Mutation; Polymorphism, Single Nucleotide; Real-Time Polymerase Chain Reaction - methods; Polymerase chain reaction; Allele-specific PCR; Real-time PCR; COLD-PCR; Digital PCR; Single-nucleotide polymorphism; Mutation; High-resolution melting analysis
• ISSN: 0065-2423
• DOI: 10.1016/bs.acc.2016.11.002

Single-nucleotide polymorphisms (SNPs) and single-nucleotide mutations result from the substitution of only a single base. The SNP or mutation can be relevant to disease susceptibility, pathogenesis of disease, and efficacy of specific drugs. It is important to detect SNPs or mutations clinically. Methods to distinguish/detect SNPs or mutations should be highly specific and sensitive. In this regard, polymerase chain reaction (PCR) has provided the necessary analytical performance for many molecular analyses. PCR-based methods for SNP/mutation detection are broadly categorized into two types-(1) polymorphic or mutant allele-directed specific analysis using primers matched with substituted nucleotide or using oligonucleotides to block or clamp the nontargeted template, and (2) melting curve analysis, which is combined with the real-time PCR techniques using hydrolysis probes, hybridization probes, or double-stranded DNA-binding fluorescent dyes. Innovative and novel approaches as well as technical improvements have made SNP- or mutation-detection methods increasingly more sophisticated. These advances include DNA/RNA preparation and subsequent amplification steps, and miniaturization of PCR instruments such that testing may be performed with relative ease in clinical laboratories or as a point-of-care test in clinical settings.