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Denaturing HPLC (DHPLC)

s of the homozygous sample at temperatures from 52 to 67 °C are made. The retention time of the resulting single peak should increase as the temperature is increased. At the denaturing temperature, the retention time will decrease with the increase in temperature.

Optimisation of the DHPLC temperature may also be determined mathematically, using Stanford University’s Melt Calculator http://insertion.stanford.edu/melt.html.

Check for successful PCR amplification by running the homozygous sample under non denaturing conditions , ie less than 50 C and then at the optimal DHPLC temperature. If the PCR conditions have been optimised then a single peak will be seen.

Run the heterozygous sample at the optimum DHPLC temperature. More than one peak should be seen.

If the heterozygous sample does not give a significantly different peak pattern than that seen for the homozygous sample, increase or decrease the column temperature until a different pattern is seen.


Typical Chromatograms:


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