ULTRARAPID MUTATION DETECTION BY MULTIPLEX, SOLID-PHASE CHEMICAL CLEAVAGE

The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segme nts (similar to 1.8 kb), and its efficiency has been well tested in th e analysis of both average (e.g., coagulation factor IX) and large, co mplex genes (e.g., coagulation factor VIII and dystrophin). In the lat ter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two sp ecific chemical reactants (hydroxylamine and osmium tetroxide) and pip eridine cleavage of the above procedure to develop a very fast mutatio n screening method. This is based on: (1) 5' or internal fluorescent l abeling to allow concurrent screening of three to four DNA fragments a nd (2) solid-phase chemistry to use a microtiter format and reduce the time required for the procedure, from amplification of sequence to ge l loading inclusive, to one person-working-day. We test the two variat ions of the method, one entailing 5' labeling of probe DNA and the oth er uniform labeling of both probe and target DNA, by detecting 114 kno wn hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure m ay offer very efficient though less than 100% screening for sequence c hanges with only hydroxylamine. The full method with two chemical reac tions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in th ree ABI 373 gels in 1 day. (C) 1995 Academic Press, Inc.