DETECTION OF MUTATIONS AND POLYMORPHISMS USING FLUORESCENCE-BASED DIDEOXY FINGERPRINTING (F-DDF)

We have adapted the dideoxy fingerprinting (ddF) technique for detecti ng DNA sequence variants to fluorescence detection (F-ddF) using an Ap plied Biosystems Model 373A DNA Sequencer equipped with GENESCAN(TM) 6 72 software and an external temperature control device. The fingerprin ts can be precisely aligned using an internal standard run in the same lanes. This facilitates location and characterization of mobility cha nges resulting from sequence variants. As compared to fluorescence det ected single-strand conformation polymorphism analysis (F-SSCP), F-ddF is equally efficient for detection of sequence variants, and it offer s additional advantages. These include information regarding location of the sequence variation, greater reliability for distinguishing one sequence variant from another and the capacity to generate large PCR f ragments and analyze them in smaller subsegments. Read length and over all quality of data from F-ddF are sequence-dependent when Taq DNA pol ymerase is used, but reducing terminator concentration can extend read length. The strengths and weakness of F-ddF and F-SSCP are different. Thus F-ddF may work better in a given situation than F-SSCP and vice versa. A strategy for using F-ddF to circumvent limitations of F-SSCP is described