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 acrucial need in the areas of medical genetics and genome analysis forrapid, accurate and cost-effective methods of mutation detection. We havedeveloped a multiplex allele-specific diagnostic assay (MASDA) for analysisof large numbers of samples (> 500) simultaneously for a large number ofknown mutations (> 100) in a single assay. MASDA utilizesoligonucleotide hybridization to interrogate DNA sequences. Multiplex DNAsamples are immobilized on a solid support and a single hybridization isperformed with a pool of allele-specific oligonucleotide (ASO) probes. Anyprobes complementary to specific mutations present in a given sample are ineffect affinity purified from the pool by the target DNA. Sequence-specificband patterns (fingerprints), generated by chemical or enzymatic sequencingof the bound ASO(s), easily identify the specific mutation(s). Using thisdesign, in a single diagnostic assay, we tested samples for 66 cysticfibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cellanemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations,four mutations in Canavan disease, four mutations in Fanconi anemia, andfive mutations in BRCA1. Each mutation was correctly identified. Finally,in a blinded study of 106 of these mutations in > 500 patients, allmutations were properly identified. There were no false positives or falsenegatives. The MASDA assay is capable of detecting point mutations as wellas small insertion or deletion mutations. This technology is amenable toautomation and is suitable for immediate utilization for high-throughputgenetic diagnostics in clinical and research laboratories.