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Figure 3 | BMC Genomics

Figure 3

From: Temperature Switch PCR (TSP): Robust assay design for reliable amplification and genotyping of SNPs

Figure 3

The effect of nested locus-specific primer melting temperature on TSP genotyping efficiency. Genomic DNA of known zygosity (homozygous wildtype or homozygous mutant) was used to test the genotyping efficiency of nested locus-specific primers with varying core melting temperatures ranging from 35°C to 50°C in TSP assays configured for allele-specific PCR. In these representative examples that target four SNP loci, an NLS primer pair with a core melting temperature of 50°C (LS forward 5'-GCCCATTCGTTTGATCAGGG and reverse 5'-CCTTTTCTTGGCGGTGATGC with NLS forward 5'-GCGCAAAATTTTAGTGTAACT and reverse 5'-CGTGATACCTGCAATGAAGT), 45°C (LS forward 5'-AGCTCCCATCGAGCTTGTGC and reverse 5'-GTTCAGCGACAGCCAACGAA with NLS forward 5'-TCGTCGAGAAGTTCCA and reverse 5'-AAAATTTGCAGGAAGTG), 40°C (LS forward 5'-GGGAGGAACAGTGCCTGCAA and reverse 5'-CCAGTCCTGGCACAACCACA with NLS forward 5'-TAGTACTGTTGCTATTGAT and reverse 5'-CTCCCACAGATGTATG) and 35°C (LS forward 5'-AGGCACTGCTGTCATGCTGG and reverse 5'-TTTTCAATCGGGCGTCTTCC with NLS forward 5'-GCATCTACAGTACCTTA and reverse 5'-TCTTCCACGGTATT) is shown (Figure 3A-D, respectively). The presence of the allele-specific genotyping product corresponds to the presence of the SNP allele. However, the presence of the alternate allele PCR product in each of these DNA samples also indicates the presence of the alternative allele, erroneously suggesting a heterozygous state. These results suggest the melting temperature of the NLS forward primer is too low to out-compete the participation of the LS forward primer. M represents a pUC19/HpaII DNA size ladder.

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