Universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR) for
SNP genotyping
Abstract
The detection and identification of single nucleotide polymorphism (SNP)
is an important basis for the evaluation of individual medicine and the
judgement of disease susceptibility. At present, SNP genotyping
technology includes the Sanger sequencing, TaqMan probe quantitative
polymerase chain reaction (qPCR), amplification-refractory mutation
system (ARMS)-PCR, Kompetitive allele specific PCR (KASP), and
next-generation sequencing (NGS), etc. However, there are some
disadvantages such as high cost of development and detection, long
detection period and easily occurring false-positive results to these
technologies. Focusing on these limitations, we proposed a new SNP
detection method named as universal probe-based intermediate
primer-triggered qPCR (UPIP-qPCR). In this method, only two types of
fluorescence-labeled probes were used for all SNP genotyping, thus,
greatly reducing the cost of development and detection for SNP
genotyping. In the amplification process, unlabeled intermediate primers
with template specific recognition function were able to trigger probe
hydrolysis and specific signal release. The sensitivity of UPIP-qPCR in
SNP genotyping was 0.01 ng, the call rate was more than 99.1%, and the
accuracy was more than 99.9%. This novel technology gave rise to the
low cost and high accuracy of SNP genotyping, and provided a new and
reliable SNP genotyping method for the development of precision
medicine.