Читать книгу Genotyping by Sequencing for Crop Improvement - Группа авторов - Страница 48

rhAmp SNP genotyping is a PCR assay that uses a unique two‐enzyme system coupled with RNA–DNA hybrid primers to detect the target SNPs. It required a double‐stranded DNA template, two allele‐specific forward primers containing RNA base and blocking moiety at 3′ end, common reverse primer containing RNA base and blocking moiety at 3′ end, RNase H2 enzyme, universal forward primer and universal probe having fluorescence‐labeled at 5′ end and quencher molecule at 3′ end. It involves an allele‐specific amplification process, where allele 1 specific forward primer 1 perfectly binds with SNP site and common reverse primer binds at the 5′ end of the complementary strand. RNase H2 binds cleaves at the RNA base of primer and removes blocking moiety, which allows extension of primers and amplification of both the strand. In the subsequent amplification cycle, the universal primer and universal probe 1 site merged into an amplified product. In PCR cycle 3, universal probe 1 and universal primer 1 bind to the PCR cycle 2 amplified product. Universal forward primer start extension, 5′ to 3′ exonuclease activity of Taq polymerase cleaves the probe and removes fluorescence which was detected by the detector. When allele 2 specific primer 2 binds to the template DNA which generates a universal prob 2‐binding sites labeled with different fluorescence molecules. In this way, different genotypes were clustered based on fluorescence signals detected.