Читать книгу Genotyping by Sequencing for Crop Improvement - Группа авторов - Страница 47

KASP assay is a fluorescence‐based genotyping variant of PCR. It utilized allele‐specific primer for amplification and generation fluorescence signals utilizing dsDNA template, two allele‐specific forward primers, common reverse primer where the forward primers are fluorescently labeled having sequences identical to that of 5′ tail of allele‐specific primers. The first round of PCR amplification is carried out using allele‐specific forward primer and common reverse primer. It allows the extension of only perfectly matched primes to result in an amplified product, while mismatched primers remain unamplified. In the third PCR cycle, fluorescence‐labeled forward primer and common reverse primer bind to PCR‐amplified products and extend the amplification reaction. During the primer annealing process quencher molecules present in complementary strands goes away and fluorescence starts emitting which are further detected by the detector.