J. Ellison et al., DETECTION OF MUTATIONS AND POLYMORPHISMS USING FLUORESCENCE-BASED DIDEOXY FINGERPRINTING (F-DDF), BioTechniques, 17(4), 1994, pp. 742
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