A HOMOGENEOUS, LIGASE-MEDIATED DNA DIAGNOSTIC-TEST

Single-nucleotide variations are the most widely distributed genetic m arkers in the human genome. A subset of these variations, the substitu tion mutations, are responsible for most genetic disorders. As single nucleotide polymorphism (SNP) markers are being developed for molecula r diagnosis of genetic disorders and large-scale population studies fo r genetic analysis of complex traits, a simple, sensitive, and specifi c test for single nucleotide changes is highly desirable. In this repo rt we describe the development of a homogeneous DNA detection method t hat requires no Further manipulations after the initial reaction is se t up. This assay, named dye-labeled oligonucleotide ligation (DOL), co mbines the PCR and the oligonucleotide ligation reaction ill a two-sta ge thermal cycling sequence with fluorescence resonance energy transfe r (FRET) detection monitored in real time. Because FRET occurs only wh en the donor and acceptor dyes are in close proximity, one can infer t he genotype or mutational status of a DNA sample by monitoring the spe cific ligation of dye-labeled oligonucleotide probes. We have successf ully applied the DOL assay to genotype 10 SNPs or mutations. By design ing the PCR primers and ligation probes in a consistent manner, multip le assays can be done under the same thermal cycling conditions. The s tandardized design and execution of the DOL assay means that it call b e automated for high-throughput genotyping in large-scale population s tudies.