One of the simplest and most powerful tools for the detection of point mutations and other DNA sequence variants is single strand conformation polymorphism analysis (SSCP) of DNA amplified by PCR. However, detection efficiencies using standard SSCP have ranged from 35% to 100% with highest detection rates most often requiring several different SSCP conditions. We assessed the efficiency of fluorescence-based SSCP (F-SSCP) using the 373A automated DNA sequencer with GENESCAN 672 software from ABI. In addition to extremely sensitive laser detection of four fluorescent dyes, this system permits a red dye-label internal standard to be run in the same lane as DNA being examined. We examined 48 point mutations in a 191 bp stretch of mouse beta-globin using F-SSCP. The mutations include 10 transversions and 38 transitions; both types of mutation were compared at 6 different locations in the PCR fragment. Mobilities of the internal standard fragments were nonproportional to size but highly reproducible and were used to normalize or correct for minor differences in apparent electrophoretic mobility between lanes. Forty-six of 48 mutants (96%) were distinguished from wild type when run in separate lanes using one set of conditions. Coelectrophoresis of wild type and mutant in the same lane resolved 100% of 45 mutants from the wild type using a single set of conditions. The magnitude of mobility shifts resulting from the globin mutations was largely determined by position of the mutation, rather than by nature of the substitution (transition vs. transversion); however, examination of paired mutations revealed that some substitutions cause greater mobility shifts than others. This work has demonstrated that F- SSCP increases the sensitivity of the technique enabling detection of mutations that result in very small SSCP mobility shifts. In addition, F- SSCP is capable of higher sample throughput at lower cost than direct DNA sequencing. We have begun using F-SSCP to screen the tryptophan hydroxylase gene, and other candidate genes for alcoholism vulnerability will be screened similarly. Variants will be identified by sequencing.
