Detection of mutations and polymorphisms using fluorescence-based dideoxy fingerprinting (F-ddF).
We have adapted the dideoxy finger-printing (ddF) technique for detecting DNA sequence variants to fluorescence detection (F-ddF) using an Applied Biosystems Model 373A DNA Sequencer equipped with GENESCAN 672 software and an external temperature control device. The fingerprints can be precisely aligned using an internal standard run in the same lanes. This facilitates location and characterization of mobility changes resulting from sequence variants. As compared to fluorescence detected single-strand conformation polymorphism analysis (F-SSCP), F-ddF is equally efficient for detection of sequence variants, and it offers 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 fragments and analyze them in smaller subsegments. Read length and overall quality of data from F-ddF are sequence-dependent when Taq DNA polymerase 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.
NIAAA, National Institutes of Health, Bethesda, MD.
J
J
Ellison
G
G
Squires
C
C
Crutchfield
D
D
Goldman
D000818:Animals; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D005453:Fluorescence; D005914:Globins / Q000235:genetics; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D013696:Temperature
D005914:Globins