Mutation detection by denaturing DNA chromatography using fluorescently labeled polymerase chain reaction products

A specialized form of ion-pair reversed-phase highperformance liquid chroma tography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modula ted heteroduplex analysis (TMHA) by chromatographic separation of partially denatured DNA heteroduplexes from homoduplexes. Here, we demonstrate that fluorescent labeling is compatible with mutation analysis by this form of D NA chromatography and offers advantages over the use of unlabeled DNA fragm ents. Uniform labeling of wildtype and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels incr ease retention times but do not influence resolution of heteroduplexes hom homoduplexes. They increase sensitivity and decrease the amount of DNA requ ired for analysis; e.g., in the case presented here, one allele can be dete cted in the presence of a 500-fold excess of another allele. Furthermore, a llele-specific wild-type probes, fluorescently labeled on one strand only, make it possible to selectively monitor specific homoduplexes and wild-type /mutant heteroduplexes. This, in combination with an internal homoduplex st andard, greatly reduces the complexity of fluorescence chromatograms compar ed with chromatograms recorded in the UV. These simplified chromatograms, i n which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detectio n and identification of mutant alleles. (C) 1999 Academic Press.