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 segments (approximately 1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5' or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microtiter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5' labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known 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 two-fold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day.
Rowley
G
G
Division of Medical & Molecular Genetics, United Medical & Dental Schools of Guy's & St Thomas's Hospitals, London, United Kingdom.
Saad
S
S
Giannelli
F
F
Green
P M
PM
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005164:Factor IX / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D002836:Hemophilia B / Q000235:genetics*; D006801:Humans; D006898:Hydroxylamines / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics* / Q000378:metabolism; D009993:Osmium Tetroxide / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism; D013997:Time Factors
D006898:Hydroxylamines; D009692:Nucleic Acid Heteroduplexes; D010880:Piperidines; D005164:Factor IX; D004247:DNA; D009993:Osmium Tetroxide