HIGH-THROUGHPUT PARALLEL ANALYSIS OF HUNDREDS OF PATIENT SAMPLES FOR MORE THAN 100 MUTATIONS IN MULTIPLE DISEASE GENES

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis fo r rapid, accurate and cost-effective methods of mutation detection, We have developed a multiplex allele-specific diagnostic assay (MASDA) f or analysis of large numbers of samples (>500) simultaneously for a la rge number of known mutations (>100) in a single assay, MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences, Multiplex DNA samples are immobilized on a solid support and a single hybridiza tion is performed with a pool of allele-specific oligonucleotide (ASO) probes, Any probes complementary to specific mutations present in a g iven sample are in effect affinity purified from the pool by the targe t DNA. Sequence-specific band patterns (fingerprints), generated by ch emical or enzymatic sequencing of the bound ASO(s), easily identify th e specific mutation(s), Using this design, in a single diagnostic assa y, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-th alassemia mutations, two sickle cell anemia (SCA) mutations, three Tay -Sachs mutations, eight Gaucher mutations, four mutations in Canavan d isease, four mutations in Fanconi anemia, and five mutations in BRCA1, Each mutation was correctly identified. Finally, in a blinded study o f 106 of these mutations in >500 patients, all mutations were properly identified. There were no false positives or false negatives, The MAS DA assay is capable of detecting point mutations as well as small inse rtion or deletion mutations, This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.