Figure 2

TSP genotyping by high resolution melt (HRM) analysis of a T/C SNP. A. Schematic showing the position of TSP primers for amplification of a size-defined target region harboring a SNP. B. HRM amplification products were generated with 0.1 μM LS primers (forward 5'-GAAGGGCGGAGCTTTGTGGA and reverse 5'-GGTGGAGCCCTGATGGCAGT) and 0.5 μM NLS primers designed to position the T/C SNP in the centre of the 142 bp amplicon (forward 5'-GGC CTCATCCTTCTGGTA and reverse 5'-GGC GCTTTTACATTTGTTAT). Underlined sequence indicates the non-complementary 5'-tail region. Following amplification, the reaction was completed with a melt gradient from 65°C to 95°C to discriminate the alleles. The presence of the T allele is represented by the blue line, the presence of the C is represented by the red line and heterozygous samples are represented by the green line. C. Following amplification and analysis, a subset of the reaction products were separated on a 2% agarose gel to demonstrate the specific amplification and accumulation of only the 142 bp product of the NLS primer pair. M represents a pUC19/HpaII DNA size ladder.