Detection of rare point mutation via allele-specific amplification in emulsion PCR

Abstract

It is essential to analyze rare mutations in many fields ofbiomedical research. However, the detection of rare mutationsis usually failed due to the interference of predominantwild-type DNA surrounded. Herein we describe a sensitiveand facile method of detecting rare point mutation on the basisof allele-specific amplification in emulsion PCR. The identificationand selective amplification of rare mutation are accomplishedin one-pot reaction. The allele-specific primers coupledon magnetic beads allow the exclusive amplification andenrichment of the mutant amplicons. The productive beadsbearing mutant amplicons are subsequently stained with thefluorescent dyes. Thus, the rare point mutations with a percentageas low as 0.1%, can be detected by fluorescent analysis.The relative percentages of mutation among different samplescan be roughly accessed by counting the fraction of fluorescentpositive beads through flow cytometry. [BMB Reports2013; 46(5): 270-275]

Keywords

• Allele-specific amplification
• Emulsion PCR
• Rare mutation
• TP53 gene