Methods and compositions for mutation analysis of polynucleotides by liquid chromatography

Abstract

Methods, compositions, and kits for separating heteroduplex and homoduplex DNA molecules in a test mixture by temperature-compression denaturing high performance liquid chromatography (tcDHPLC). The method includes use of nitrogen-containing additives in the mobile phase that allow detection of diverse heteroduplex molecules to be performed at the same pre-selected temperature. An example of a preferred additive is betaine. Standard mixtures of DNA fragments, such as mutation standards containing known heteroduplex and homoduplex molecules, can be used to select the concentration of additive and temperature. Compositions and kits including the mobile phase, mutation standards, PCR primers, separation media, and DNA polymerase are also provided.