Single nucleotide polymorphisms (SNP) genotype analysis looks for small changes in sequences instead of simply identifying the amplicon. One of the best ways to detect SNPs is to compare melting curves. The use of real-time PCR to create melting curves can detect differences as small as one base pair.
Melting curve analysis is an assessment of the disassociation characteristics of double stranded DNA when exposed to heat. The strength of the hydrogen bond of each piece of DNA is dependent upon several different factors: the length of the DNA segment, the amount of guanine and cytosine pairs, and the degree of complement.
In real-time PCR the fluorescence that is given off by the probes will decrease once the strand of DNA disassociates. After the DNA or RNA is amplified, the temperature of the sample is slowly increased while the fluorescence is recorded. The melting points should show up as peaks when plotting temperature against fluorescence. These peaks allow one to differentiate between homozygous wild type, heterozygous, and homozygous mutant alleles.
One clinical use of this method is to detect both HIV-1 and HIV-2 in samples.