Simultaneous detection of multiple point mutations using allele-specific oligonucleotides

Abstract

This unit describes high-throughput mutation analysis using hybridization with pooled allele-specific oligonucleotide (ASO) probes. The approach can be used to screen one gene for many allelic mutations or to screen several loci for several allelic mutations each. Because tetramethyl ammonium chloride (TMAC) is added to the hybridization solution, the melting temperature of each oligonucleotide is independent of G-C content and oligonucleotides of the same length can be hybridized simultaneously. The pooled probes will give a positive hybridization signal from any PCR-amplified DNA sample containing a sequence complementary to any of the ASOs in the pool of oligonucleotide sequences. If many PCR-amplified samples are spotted onto a single membrane, multiple individuals can then be screened simultaneously for many mutant sequences. This multiple ASO hybridization technique is appropriate only for circumstances when hybridization with any one of the pooled probes is expected to be uncommon.