Combination of a modified block PCR and endonuclease IV-based signal amplification system for ultra-sensitive detection of low-abundance point mutations

Abstract

By combination of a modified block PCR and endonuclease IV-based signal amplification system, we have developed a novel approach for ultra-sensitive detection of point mutations. The method can effectively identify mutant target sequence immersed in a large background of wild-type sequences with abundance down to 0.03% (for C→A) and 0.005% (for C→G). This sensitivity is among the highest in comparison with other existing approaches and the operating procedures are simple and time saving. The method holds great potential for future application in clinical diagnosis and biomedical research.

Keywords: AP; AS-PCR; Abasic site-containing DNA fluorescent probe; COLD-PCR; Endo IV; Endo IV-based signal amplification system; HRM; LOD; MT; Modified block PCR; PCR; Point mutation detection; UDG; Uracil-DNA Glycosylase; WT; allele specific PCR; apurinic/apyrimidinic; co-amplification at lower denaturation temperature-PCR; dNTP; deoxyribonucleoside triphosphate; endonuclease IV; high resolution melting; lambda exonuclease; limit of detection; mutant; polymerase chain reaction; wild type; λexo.