Detection of single base substitutions in total genomic DNA

Abstract

Certain single base substitutions causing genetic diseases^1–4 or resulting in polymorphisms linked to mutant alleles^5–7, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known^8,9. In the case of β-thalassaemia, where 22 different single base mutations have been identified¹⁰, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S₁ nuclease treatment of heteroduplexes formed between wild-type and mutant DNA¹¹. Although certain single base mismatches are cleaved by S₁ nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea¹², but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system¹³. We show that four different human β-thalassaemia alleles with known single base mutations can be detected with as little as 5 µg of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.