Identification of APC gene mutations in colorectal cancer using universal microarray-based combinatorial sequencing-by-hybridization

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited form of colorectal cancer, caused mostly by mutations in the A-PC gene. Due to the wide variety of mutations found and the large size of the A-PC gene, several methods of mutation detection are used, which can be time consuming and costly. Here we demonstrate a new method of mutation detection in the APC gene using an array-based approach termed combinatorial sequencing-by-hybridization (cSBH). In cSBH, a universal probe set is attached to a support and a second one is in solution. Two-probe ligation occurs when a DNA strand from the target PCR product consecutively anneals to both unlabeled array,bound and solution phase dye,labeled probe, creating all target, complementary long-labeled probes attached to the surface. A standard array reader scores fluorescent signals at each array position. Cell lines and patient DNA with known APC gene mutations were analyzed using a cSBH, based HyChip(TM) product. Results show that this universal hexamer (6,mer) chip can successfully detect a range of mutations. Results are very robust for a continuous readout of 3.6 kb from a PCR target, with 99.97% accuracy on a single HyChip(TM) slide. cSBH is a fast, cost,efficient method for first stage mutation screening in the APC or any other gene. (C) 2004 Wiley-Liss, Inc.