Читать книгу Genotyping by Sequencing for Crop Improvement - Группа авторов - Страница 46

TaqMan SNP genotyping assay utilizes PCR amplification principle for discriminating the samples based on the presence of the SNPs where forward and reverse primers along with Taq polymerase and two SNP specific fluorescent‐labeled probes are used for the amplification. The first probe, specific to allele 1 is labeled with VIC green fluorescent dye, while the second probe is specific to allele 2 labeled with FAM blue fluorescent dye. During the amplification process, probe specific to allele hybridize with template DNA and 5′ to 3′ exonuclease activity of Taq polymerase cleave the probe and remove the fluorescent dye. The dye was further detected by the detector which gives allele‐specific signals. If the sample is homozygous for allele 1, green fluorescence signals from VIC dye are detected by the detector, while the sample is homozygous for allele 2, blue fluorescence signals from FAM dye are detected by the detector. Under the heterozygous condition, there should be a roughly equal signal from both the dyes that produce cyan color signals.