Diagnosis of BRCA1 mutations using single base extension and zip-code microarray

Abstract

We describe herein, a reliable multiplex assay strategy to detect human genetic mutations in breast cancer susceptibility gene BRCA1 on a zip-code microarray using single base extension (SBE) reaction. Multiplex PCR amplification was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in subsequent four separate multiplex SBE reactions, one with each of four different biotin-labeled dideoxy NTPs (ddNTPs) and the other three unlabeled ddNTPs. All SBE primers, terminating one base before mutation sites, were extended by a single base at each corresponding mutation site, but biotin was labeled only through the reaction with the biotin labeled ddNTP. Hybridization of the SBE products to zip-code microarray was followed by staining with streptavidin-Cy3, leading to successful genotyping of several selected BRCA1 mutation sites with a wild-type and heterozygote mutant samples from breast cancer patients. This work represents one of the few successful verification of the DNA microarray-based reliable diagnosis of human genetic mutations