Fig. 2.

Identification of the g.13,732C>T SNP (encodes p.S195L) by (a) electrophoretic or (b–f) real-time detection of PCR products from 3-primer (hF2– 50, hF2–79 and hF2–52r) allele-specific amplification. (a) Lane 1: MspI cut pBR322. Lanes 2–7 are control reactions: 2, minus template; 3, synthetic heterozygote composed of reference plasmid + g.13,732C>T plasmid DNA; 4, reference plasmid DNA; 5, g.13,732C>T plasmid DNA; 6, genomic DNA from a reference individual; 7, genomic DNA with added g.13,732C>T plasmid DNA. Lanes 8–15 are products from genomic DNA from individual Hispanic-American samples. (b) Melting curve showing control reactions with simulated homozygotes and a non-mutant genomic sample: orange tracing of the 77°C peak is from the 65-bp g.13,732C>T plasmid DNA product; blue and pink 81°C peaks are from the 78-bp products from reference plasmid and genomic DNA, respectively. (c) Melting curve showing PCR products from control reactions with synthetic heterozygotes; green, mixed plasmid DNA; teal, non-mutant genomic DNA with g.13,732C>T plasmid DNA. (d) Superimposed melting curves of PCR products from the genomic DNA of eight Hispanic-American individuals. These melting curves are shown separated into their components including two heterozygotes (e), and six homozygotes encoding non-mutant p.S195 (f).