Development and applications of a BRAF oligonucleotide microarray.
We herein describe the development of a sensitive microarray hybridization method called competitive DNA hybridization (CDH) and its use for analysis of BRAF somatic mutations. These mutations have been identified in many human cancers, and fast, reliable BRAF mutation detection may one day facilitate directed therapy of BRAF-mutated tumors. Our fast, reliable mutation detection by CDH is based on the principle that competition among multiple fluorescent-labeled samples for binding to shared wild-type sequences should reduce nonspecific results and increase the positive signals of unshared mutated sequences. The positive signals can then be discriminated based on the labeling of each sample (ie, with Cy3, Cy5, or Alexa-594). For testing of this method, we developed a BRAF oligonucleotide microarray containing 65 mutation types (more than 95% of the known BRAF mutations) and validated this microarray with 20 colorectal cancer tissues/cancer cell lines with BRAF mutations and 60 BRAF-negative samples. In sum, we were able to screen up to nine cancer samples on a single BRAF microarray (three per CDH on three regions per slide), indicating that this method may dramatically decrease the experimental time, cost, and effort of mutation detection in BRAF and other genes amenable to microarray analysis.
Kim
Il-Jin
IJ
Laboratory of Cell Biology, Cancer Research Institute and Cancer Research Center, Seoul National University, Seoul, Korea 110-799.
Kang
Hio Chung
HC
Jang
Sang Geun
SG
Ahn
Sun-A
SA
Yoon
Hyun-Ju
HJ
Park
Jae-Gahb
JG
D015179:Colorectal Neoplasms / Q000235:genetics*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D017422:Sequence Analysis, DNA
D009841:Oligonucleotides; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf