Oligonucleotide microarray based detection of repetitive sequence changes

Prior studies of oligonucleotide microarray-based mutational analysis have demonstrated excellent sensitivity and specificity except in circumstances where a frameshift mutation occurs in the context of a short repeated seque nce. To further evaluate this circumstance, a series of nucleic acid sample s having heterozygous mutations within repetitive BRCA1 sequence tracts was prepared and evaluated. These mutations included single nucleotide inserti ons and deletions in homopolymer runs, insertions and deletions of trinucle otide repeats, and duplications. Two-color comparative hybridization experi ments were used wherein wild type reference and test targets are co-hybridi zed to microarrays designed to screen the entire BRCA1 coding sequence for all possible sequence changes. Mutations in simulated heterozygote samples were detected by observing relative losses of test target hybridization sig nal to select perfect match oligonucleotide probes. While heterozygous muta tions could be readily distinguished above background noise in 9/19 cases, it was not possible to detect alterations in a poly dA/dT tract, small trip ler repeat expansions, and a 10 bp direct repeat. Unexpectedly, samples con taining (GAT)(3) tripler repeat expansions showed significantly higher affi nity toward specific perfect match probes relative to their wild type count erparts. Therefore, markedly increased as well as decreased test sample hyb ridization to perfect march probes should be used to raise a suspicion of r epetitive sequence changes. Hum Mutat 16:354-363, 2000. (C) 2000 Wiley-Liss , Inc.