Solution-based scanning for single-base alterations using a double-stranded DNA binding dye and fluorescence-melting profiles.

ABSTRACT: DNA molecules differing by as little as a single-base substitution have traditionally been distinguished by gel electrophoresis-based methodologies that exploit differences in the sequence-specific properties of double-stranded DNA (dsDNA) such as melting temperature and secondary conformational configuration. By comparison, solution-based fluorescence methods using sequence-specific probes are limited to detecting mutations restricted to very short segments of DNA ( approximately 20 bp). We describe a solution-based fluorescence method that discriminates between wild-type and mutant sequences using a dsDNA binding dye, and interrogates a region of >200 nucleotides. This method is based on melting theory and entails fluorescence monitoring of the melting temperatures of GC-clamped amplicon

SUBMITTER: Elenitoba-Johnson KS 

PROVIDER: S-EPMC1850450 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature