Figure 2. HRMA detects the presence of mutant alleles in mixed populations of wildtype and mutant genomes.

(A) Schematic illustration of the principle of HRMA. A small region of the genome that includes a DNA polymorphism is amplified by PCR from a mixed population of wildtype (WT) and mutant template genomes. Heat denaturation of the amplicons followed by rapid re-annealing of DNA strands produces two kinds of homoduplex and two kinds of heteroduplex products. Heteroduplex products are likely to be particularly unstable and exhibit a Tm that differs from the homoduplex products. (B) HRMA of lef1 PCR amplicons generated from template genomes that were either WT (lef1^+/+) or heterozygous for an 18 bp deletion (lef1^Δ18/+). PCR amplifications were performed in the presence of LC Green Plus dye (Idaho Technology), which fluoresces upon binding double strand DNA, allowing the detection of intact duplex molecules. The DNAs amplified from the lef1^+/+ genome (grey curves) comprise a homogeneous population of duplexes with a single Tm. In contrast, re-annealed amplicons derived from the lef1^Δ18/+ genome (red curve) are composed of multiple duplex populations, which display distinct Tms (*). (C) Sensitivity of HRMA for detecting the presence of the lef1^Δ18 mutation in mixtures of lef1^+/+ WT and lef1^Δ18/+ heterozygous genomes. Heterozygous DNA was mixed with increasing amounts of WT DNA; fractions indicate the relative abundance of mutant haploid genomes in the mixtures. A constant total amount of genomic template DNA and primers was used to generate each set of amplicons. LightScanner Call-IT Software (Idaho Technology) was used to identify melt curves that differed significantly from WT. HRMA detected the lef1 18 bp deletion allele when it was present as only 1/70^th of the total haploid genomes.