Genotyping of mutations in the beta-globin gene using allele specific hybridization.

Abstract

The use of DNA microarrays for genotyping is economically favorable when compared to real-time PCR and DNA sequencing. Here, we demonstrate a DNA microarray-based assay using allele-specific oligonucleotide (ASO) probes for genotyping mutations in the beta-globin gene. The assay makes use of agarose film coated glass slides as substrate, unmodified melting temperature matched ASO probes, target amplification and labeling using T7 in vitro transcription, mixing during hybridization and finally visualization using a fluorescent scanner. In this chapter we will emphasize on probe design, optimization, and validation of an allele-specific hybridization assay.