Denaturating HPLC is a reverse-phase ion pairing HPLC method used to study single nucleotide polymorphism in DNA molecules. It is an alternative to gel electrophoresis methods used to detect mutations. This method commonly resolves heteroduplexes and homoduplexes of DNA fragments ranging from 200 bp to 1000 bp. Larger fragments can be resolved in some cases. The resolution is based on differences in size and on differences in helical composition induced by temperature-modulated denaturation at 50 °C to 70 °C. The use of DHPLC for heteroduplex mutant analysis provides advantages, such as specificity, sensitivity and high throughput that can increase productivity in the screening of genetic loci.
The thermally less stable heteroduplexes denature more extensively and, therefore, are retained a shorter time on the stationary phase. The eluent contains the cationic TEAA ion (0.1 M), which interacts with the negatively charged phosphate groups on DNA molecules and also with the hydrophobic surface of the particles in the columns. The TEAA ion can be described as a bridging molecule between DNA fragments and the column. As the mobile phase is made progressively more organic, the DNA fragments are eluted in order of size.
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