MUTATION DETECTION BY FLUORESCENT CHEMICAL CLEAVAGE - APPLICATION TO HEMOPHILIA-B

Chemical mismatch detection when combined with PCR or RT-PCR amplifica tion represents one of the most efficient mutation screening methods. This procedure has been used for the analysis of both large population s of mutants and large and complex genes because it detects and locate s 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 ge l scanning system, the efficiency of the method has been increased fou rfold 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 e xamined in a single gel lane and how the increase in efficiency thus o btained can be used either totally to maximize the DNA screened per la ne or partly to locate the mutation unequivocally so that only one seq uencing reaction is needed to characterize it fully.