Abstract
Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence1. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR)2, followed by ligase detection reaction (LDR)3. Mutations were identified by screening reaction products with a universal DNA microarray4, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT)5 as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR2 products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.
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Acknowledgements
We thank Michael Osborne and Alvaro Monteiro for helpful discussion and members of the Barany lab for their suggestions and technical assistance. Support for this work was provided by the National Cancer Institute (P01-CA65930 and RO1-CA81467) and a grant from PE Biosystems.
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Favis, R., Day, J., Gerry, N. et al. Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2. Nat Biotechnol 18, 561–564 (2000). https://doi.org/10.1038/75452
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DOI: https://doi.org/10.1038/75452
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