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| Figure 7: TaqMan assay is used to distinguish between the C and T allele in rs2943641. The DNA was extracted to carry out the experiment, panel 1 presents the component of the assay: forward and reverse primers for the flanking sequence of the SNP. Two probes were used with two different reported dyes, VIC dye labelled the probe which was used to detect the C allele and FAM dye labelled the probe which was used to detect the T allele. Panel 2 presents the hybridizing and the extension: after denaturation the template, the probes and the primers anneal in the single strand template. The probes have annealed before the primer due to the melting temperature for the probes being higher by 10°C than the melting temperature of the primers. The 5’-3’ exonuclease activity of Taq polymerase degrades the matching probes which hybridize to the template. Degradation leads the florescent being released from the probes. Panel 3 shows the dye emission and produce signalling after the cleavage by exonuclease activity of the Taq polymerase in the matching probe. However, in the mismatch the probe will be displaced without producing signalling due to no cleavage occurring [24]. |
