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Description of the DHPLC Melt Program

DHPLC is a method generally successful at separating DNA duplexes that differ in the identity of one or more base pairs. The method is believed to be most efficient near the site melting temperature of the duplex at the site of the mutation (i.e., the temperature at which the site has a 50% chance of being Watson-Crick bonded rather than open).

If a DNA duplex is modeled as a string of open and closed states, it is mathematically equivalent to an Ising-like model that is analytically solvable (see Poland, Biopolymers, 13:1859 (1974)). It is a simple mathematical extension of this model to include individual stacking energies for different pairs of bases. In addition, entropy effects of open loops enclosed between closed base pairs can be incorporated in this model (see Wartell, Nuc. Acids Res., 4:2779 (1977)), and the percentage of duplexes that are completely dissociated can be calculated from appropriate partition functions (see Poland and Scheraga, "Theory of Helix Coil Transition in Biopolymers," Acad. Press, New York (1970)).

Published values of the parameters for this model produce inaccurate melting temperatures when applied to the HPLC system (the temperatures are about 10 degrees too high). Our new software uses parameters obtained by the simplest of simulated annealing algorithms (evenly distributed random steps with generalized Metropolis acceptance rates and 1/t dependence of annealing temperature) used to fit melting temperature predictions to experimental data for 10 mutations.

This program, developed by Nancy Fisher Hansen and Peter Oefner, appears to give good results. In more than half the cases, it predicts a temperature within 1 degree Celsius of the experimentally determined temperature (this experiment involves running the apparatus at several temperatures, a time-consuming procedure). The program runs in under a minute using only the sequence as input. The program is still being improved, and suggestions, as well as sequences for which it is inaccurate (off by two or more degrees) can be forwarded to Peter Oefner.

 

DHPLC Melt Program | OTL Information (Office of Technology Licensing)

 

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last updated 07/07/2003 | © Stanford Genome Technology Center