For mutation detection, the PCR product from a test sample is mixed with a homozygous reference PCR product in a 1:1 (v/v) ratio.
The mixed DNA fragments are denatured at 95°C and then cooled slowly for reannealing of the DNA strands by gradually lowering the temperature at a rate of 1°C per 20 seconds from 95°C to 25°C.
A single base pair difference between the test and reference fragments will produce two heteroduplexes and two homoduplexes. The mixture of DNA samples are then analyzed by DHPLC under partially denaturing conditions.