Hemophilia B Mutational Analysis

Since the cloning of the factor IX gene in 1982 (1 ), there have been several strategies employed for the identification of mutations in the mutationally heterogeneous hemophilia B population. Initially, such strategies inevitably employed Southern blotting to screen for gross deletions (2 ) or restriction site alterations (3 ), and cloning of the patients genomic DNA (4 ). However, with the advent of polymerase chain reaction (PCR) using a thermostable DNA polymerase (5 ), cloning has become superfluous, and factor IX mutations can be identified simply by direct DNA sequencing of PCR-amplified sections of the factor IX gene (6 ). From 1988 onwards, a new method of screening PCR products for mutations was developed in our laboratory (7 ) based on the chemical cleavage of mismatch method which was first used on cloned DNA (8 ). This procedure, capable of detecting 100% of mutations, is useful for screening a large number of patients who are all expected to have different mutations, prior to sequencing the PCR product. However, it is probably quicker simply to sequence the products straightaway if only a handful of patients are to be examined (6 ).