Microarray-based approach for high-throughput genotyping of single-nucleotide polymorphisms with layer-by-layer dual-color fluorescence hybridization.

Abstract

Robust, inexpensive, high-throughput methodologies are needed for the analysis of molecular markers. DNA microarrays are widely used in biological and biomedical research as a high-throughput tool. At present, there are two general microarray-based methods for genotyping. One approach involves arraying thousands of short oligonucleotides on glass slides for detection of many single-nucleotide polymorphism (SNP) loci in target DNA (1). This method is particularly well suited for genotyping thousands of markers in a limited number of individuals (2)(3)(4)(5)(6). The other approach involves arraying amplified PCR products on glass slides to detect a few SNPs in a large number of samples. Recently, Flavell et al. (7) developed a tagged microarray marker approach for scoring thousands of samples for a codominant molecular marker. Biotin-terminated allele-specific PCR products are spotted unpurified on streptavidin-coated glass slides and visualized by hybridization of fluorescent detector oligonucleotides to tags attached to the allele-specific PCR primers. The tagged microarray marker approach is an efficient method for genotyping thousands of samples on a glass slide. However, reliable allele-specific PCR conditions are needed. Moreover, the expensive streptavidin-coated glass slides can be stored for only 3 days in phosphate-buffered saline. For many applications in the field of biological research, PCR products are the more convenient probe molecules (8). We have developed a microarray-based method, based on dual-color fluorescence hybridization, for genotyping of SNPs in thousands of individuals (9). The experiment successfully demonstrated that PCR products subjected to dual-color hybridization on …