An effective method based on real time fluorescence quenching for single nucleotide polymorphism detection

AbstractIn the Human Genome Project, the most common type of these variations is single nucleotide polymorphisms (SNPs). A large number of different SNP typing technologies have been developed in recent years. Enhancement and innovation for genotyping technologies are currently in progress. We described a rapid and effective method based on real time fluorescence quenching for SNP detection. The new method, Quenching-PCR, offering a single base extension method fully integrated with PCR which used a probe with quencher to eliminate fluorophor of the terminal base according to dideoxy sequencing method. In this platform, dideoxy sequencing reaction and obtaining values of real-time fluorescence occur simultaneously. The assay was validated by 106 DNA templates comparing with Sanger's sequencing and TaqMan assay. Compared with the results of DNA sequencing, the results of Quenching-PCR showed a high concordance rate of 93.40%, while the results of TaqMan platform showed a concordance rate of 92.45%, indicating that Quenching PCR and TaqMan assay were similar in accuracy. Therefore, Quenching PCR will be easily applicable and greatly accelerate the role of SNP detection in physiological processes of human health