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A specialized form of ion-pair reversed-phase high-performance liquid chromatography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modulated 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 DNA chromatography and offers advantages over the use of unlabeled DNA fragments. Uniform labeling of wild-type and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels increase retention times but do not influence resolution of heteroduplexes from homoduplexes. They increase sensitivity and decrease the amount of DNA required for analysis; e.g., in the case presented here, one allele can be detected in the presence of a 500-fold excess of another allele. Furthermore, allele-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 standard, greatly reduces the complexity of fluorescence chromatograms compared with chromatograms recorded in the UV. These simplified chromatograms, in which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detection and identification of mutant alleles., (Copyright 1999 Academic Press.)

A synthetic polynucleotide sequence was developed and cloned to serve as a target in footprinting and other assays designed to characterize the sequence-selective binding of drugs and other small molecules to various forms of nucleic acids. The target sequence comprehensively represents all base quartet recognition sites in a minimal length sequence. Minimal length target sequences were found to be 144 nt long. One such target sequence was divided into two parts. One strand of each part was chemically synthesized and the complementary strands were generated using a DNA polymerase. Double-stranded sequences were then cloned into pGEM-3Zf(+/-) vectors (Promega, Inc.). The cloned target sequence can be used directly in double-stranded DNA form. Alternatively, features of the plasmid vector allow expression of the target sequences as single-stranded DNA or RNA or as RNA/DNA or RNA/RNA duplexes. These cloned target sequences designed for high information content overcome limitations to the use of natural DNA sequences for footprinting and related experiments arising from the unequal representation of base quartets and the potential for secondary structure formation in single-stranded forms.