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 APC gene. Due to the wide variety of mutations found and the large size of the APC 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 trade mark 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 trade mark slide. cSBH is a fast, cost-efficient method for first stage mutation screening in the APC or any other gene.
Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania, USA.
Shannon
S
Cowie
Snezana
S
Drmanac
Donald
D
Swanson
Kathleen
K
Delgrosso
Steve
S
Huang
Desirée
D
du Sart
Radoje
R
Drmanac
Saul
S
Surrey
Paolo
P
Fortina
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics*; D005091:Exons / Q000235:genetics; D017491:Genes, APC; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D004273:DNA, Neoplasm