Mutations in BRCA 1 gene are associated with breast and/or ovarian cancer development and seem to be responsible for approximately 2-5% of all breast cancer cases in the general population. The BRCA I gene, which was isolated by positional cloning, is composed of 22 coding exons distributed over approximately 100 kb. Up to now, more than 1,500 distinct variations of BRCAI have been registered in the BIC database. Breast cancer is the most common cancer in Korean women, although the incidence is lower than in the Western population. Due to rapid westernization of life style, the incidence of this disease and its associated death rates have markedly increased in Korea and BRCAI mutations are considered to be involved in a large portion of all familial breast cancer. Therefore, the need for a reliable diagnosis of the mutations is becoming more important.
We investigated immobilization efficiency of several coupling strategies which could be used in a DNA microarray. The coupling strategy of aminated DNA to glass supports modified with aldehyde is best practical method to make DNA microarrays and it does not require any coupling agent which could be a source of variability during the spotting with an automatic devices.
We developed an efficient microarray-based multiplex assay to detect Korean-specific mutations in breast cancer susceptibility gene BRCAI using direct probe/target hybridization. Allele-specific oligonucleotides were covalently immobilized on aldehyde-activated glass slide to prepare an oligonucleotide chip. From a wild type sample, two-step method was used to generate labeled multiplex PCR amplification products of genomic regions containing the mutation sites. Hybridization of the labeled PCR products to the oligonucleotide chip successfully identified all of the genotypes for the selected mutation sites.
For more reliable diagnosis, we employed a multiplex assay strategy to detect Korean-specific mutations in breast cancer susceptibility gene BRC...