Array-based mutation detection of BRCA1 using direct probe/target hybridization.
We describe here an efficient microarray-based multiplex assay to detect Korean-specific mutations in breast cancer susceptibility gene BRCA1 using direct probe/target hybridization. Allele-specific oligonucleotides were covalently immobilized on an aldehyde-activated glass slide to prepare an oligonucleotide chip. From a wild-type sample, a two-step method was used to generate labeled multiplex polymerase chain reaction (PCR) amplification products of genomic regions containing the mutation sites. Amino allyl-dUTP, an amine-modified nucleotide, was incorporated during multiplex PCR amplifications and a monofunctional form of cyanine 3 dye was subsequently attached to the reactive amine group of the PCR products. Hybridization of the labeled PCR products to the oligonucleotide chip successfully identified all of the genotypes for the selected mutation sites. This work demonstrates that oligonucleotides chip-based analysis is a good candidate for efficient clinical testing for BRCA1 mutations when combined with the indirect strategy to prepare labeled target samples.
Yim
Seong-Chun
SC
Department of Chemical and Biomolecular Engineering, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Park
Hyun Gyu
HG
Chang
Ho Nam
HN
Cho
Dae-Yeon
DY
D019313:BRCA1 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics; D004252:DNA Mutational Analysis; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D007723:Korea; D009154:Mutation / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D019313:BRCA1 Protein; D015345:Oligonucleotide Probes