The major application of DHPLC is to screen for unknown mutations and putative SNPs. To achieve this, the partially denaturing HPLC mode is used and the column temperature is maintained above 50°C, but below 70°C. The column temperatures vary with different DNA fragments to be analyzed and depend on the melting domains in the fragments. Before the DHPLC analysis, the DNA fragments are amplified by PCR.
The PCR product from a test sample is mixed with a homozygous reference PCR product in equal volume. The mixed DNA fragments are denatured and allowed to reanneal by gradually lowering the temperature. With this process of heteroduplex formation, two dsDNA molecules that differ by a single base pair (e.g., A-T vs G-C) will give two heteroduplexes and two homoduplexes. Stability of the DNA duplexes determines the order of elution from the column: the more stable the duplexes, the longer the elution time. Heteroduplexes with mismatches are less stable than and are thus eluted before homoduplexes.
The partially denaturing HPLC allows separation of homoduplexes and heteroduplexes produced as a result of even a single base difference between two otherwise identical dsDNA molecules at an optimized column temperature. With reference to a homozygous wild type control, any difference in the elution profile is indicative of the presence of a sequence variation. In fact, a 4-peak pattern is not frequently seen. The test DNA samples are then sequenced to confirm the presence and characterize the nature of the mutations.
Nevertheless, false positive results can sometimes be obtained and an altered elution pattern is demonstrated for DNA fragments without sequence variations. The ideal size of PCR products is 150–450 bp for detection of unknown sequence variations although mutations have been detected in fragments as large as 1500 bp. Long DNA fragments tend to have more than 1 melting domain and hence require several column temperatures for complete screening of the fragment.