RESUMEN
As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large 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 hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion 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.
Asunto(s)
Análisis Mutacional de ADN/métodos , Enfermedades Genéticas Congénitas/diagnóstico , Pruebas Genéticas/métodos , Mutación , Alelos , Cartilla de ADN , Sondas de ADN , Electroforesis en Gel de Poliacrilamida , Enfermedades Genéticas Congénitas/genética , Pruebas Genéticas/instrumentación , Humanos , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
We examined DNA damage induced by the enediyne-containing antitumor antibiotic C-1027 in intracellular nuclear and mitochondrial DNA targets using the episome-containing cell line 935.1. Strand-scission activity of the C-1027 holoantibiotic was measured by the topological forms conversion assay in episomal and mitochondrial DNA, as well as in cell-free plasmid DNA. Genomic DNA damage was quantitated by filter elution analysis. Comparisons were made to the well-characterized enediyne neocarzinostatin. From these studies, mixed single- and double-strand breaks were observed not only in cell-free, plasmid DNA but also in intracellular episomal, mitochondrial, and genomic DNA at low nanomolar concentrations. C-1027 cleaved DNA 285-fold more efficiently in cells than in a cell-free environment, and displayed preference for intracellular DNA species in the following rank order: episome > mitochondrial DNA >> genomic. NCS also damaged the non-histone-associated mitochondrial DNA, but not the episome. Cleavage of the 935.1 cell episome by C-1027 occurred at specific sites including the BPV origin of replication and E6/E7 open reading frame regions, as well as the MMTV LTR promoter region.