Analysis of enzymatically amplified β-globin and HLA-DQα DNA with allele-specific oligonucleotide probes

Abstract

Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA^1–3. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions⁴. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure⁵ to enzymatically amplify a specific segment of the β-globin or HLA-DQα gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a >10⁵-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple ‘dot blot’ for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.