Fast and reliable screening of mutations in human tumors: Use of multiple fluorescence-based long linker arm nucleotides assay (mf-LLA)

Human tumor-samples were screened for point mutations by adapting a mobilit y-shift assay to automated DNA sizing. This screen identifies the type of p oint mutation and relative amount of mutated DNA sequences present in a sam ple. Test samples having known hypoxanthine-guanine phosphoribosyl transfer ase: (hprt)/exon-3 sequence mutations were characterized by: (i) PCR amplif ication, (ii) fluorescent dye-primer extension with 36-atom linker derived deoxycytosine or deoxyuridine triphosphate and the remaining three natural nucleotides and (iii) sizing of the resulting fluorescently labeled modifie d strands, using an automated DNA sequencer Routinely, a range of sizes is observed among the sequence variants of a single DNA target sequence. This is because nucleotide analogs are incorporated into DNA Strands in a sequen ce-dependent manner resulting in composition-dependent electrophoretic mobi lity. Thus, point mutations are identified as shifts in, mobility, between the fluorescently labeled modified strands of the control and test samples. The twenty different hprt/exon-3 single-base substitution mutations tested were easily identified even at fourfold dilution with control DNA.