Mutation detection by fluorescent chemical cleavage: application to hemophilia B.
Chemical mismatch detection when combined with PCR or RT-PCR amplification represents one of the most efficient mutation screening methods. This procedure has been used for the analysis of both large populations of mutants and large and complex genes because it detects and locates virtually all sequence variations in segments of DNA of up to 1.7 kb cleaving at, and/or adjacent to, mismatches in heteroduplexes formed by target and probe DNA. By using fluorescent tags and an automatic gel scanning system, the efficiency of the method has been increased fourfold with gains also in the safety and quality control of probes and in the flexibility of the procedure. Using 12 hemophilia B patients, 8 with known mutations and 4 chosen at random, we show how the chemical mismatch cleavage of up to four DNA segments can be multiplexed and examined in a single gel lane and how the increase in efficiency thus obtained can be used either totally to maximize the DNA screened per lane or partly to locate the mutation unequivocally so that only one sequencing reaction is needed to characterize it fully.
Division of Medical and Molecular Genetics, United Medical School of Guy's Hospital, London, UK.
I I
II
Haris
P M
PM
Green
D R
DR
Bentley
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F
Giannelli
D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D005453:Fluorescence; D002836:Hemophilia B / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction