Efficacy of fluorescence-based PCR-SSCP for detection of point mutations.
We assessed the efficiency of fluorescence-based PCR single-strand conformation polymorphism analysis (PCR-SSCP) using an automated DNA sequencer and analysis software. We evaluated 48 point mutations in a 191-bp stretch of mouse beta-globin. The mutations included 10 transversions and 38 transitions; and both types of mutation were compared at six different locations in the PCR fragment. Mobilities of the red dye-labeled internal standard fragments were non-proportional to size but highly reproducible and were used to normalize or correct minor differences in apparent electrophoretic mobility between lanes. Forty-six of forty-eight mutants (96%) were distinguished from wild type when run in separate lanes using one set of conditions. Co-electrophoresis of wild type and mutant in the same lane resolved 100% of 45 mutants from wild type. Under conditions of this study, the magnitude of mobility shifts resulting from the globin mutations were largely determined by position of the mutation, rather than by the nature of the substitution (transition vs. transversion). However, examination of paired mutations at the same position revealed that some substitutions cause greater mobility shifts than others.
Ellison
J
J
Laboratory of Neurogenetics, NIAAA, NIH, Rockville, MD 20852.
Dean
M
M
Goldman
D
D
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000737:chemistry*; D015246:Deoxyribonuclease EcoRI; D005456:Fluorescent Dyes; D005914:Globins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D005914:Globins; D004247:DNA; D015246:Deoxyribonuclease EcoRI