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Rapid, High Throughput Mutation Discovery Using Fluorescent SSCP on the ABI PRISM 3100 and 3100Avant Genetic Analyzers

The availability of multiple dyes permits the use of internal size standards for normalization of mobility rates. The internal size standards align data in various capillaries, thus eliminating capillary- to-capillary or run-to-run variability. This ensures the accuracy and precision that are critical for successful SSCP analysis.

Analysis at varying temperatures

The 3100 and 3100 Avant analyzers provide automated electrophoresis of a single sample at stable, uniform temperatures ranging from 18C to 65C. Running samples at varying temperatures maximizes the probability of detecting novel mutations. This flexibility allows researchers to determine the optimal temperature for differentiating mutant and wild-type strands.

Fluorescent labeling of forward and reverse strands

The ABI PRISM 3100 and 3100 Avant multi-fluorescence systems offer unique differential labeling of forward and reverse strands. Four-color fluorescent labeling eliminates the safety concerns inherent in isotopic-dye systems while increasing the probability of detecting novel mutations and reducing error. For example, under a given condition, more than one stable conformation of each DNA strand may exist. Labeling the two strands with different colors simplifies data comparison and ensures that the same strands are being compared from sample-to-sample. In addition, residual double-stranded products display both colors and are easily distinguished from single-stranded DNA molecules. Overlapping single strands may also show this pattern.

Internal size standards for detecting mobility differences

The availability of multiple dyes permits the use of internal size standards for normalization of mobility rates. The internal size standards align data in various capillaries, thus eliminating capillary- to-capillary or run-to-run variability. T
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