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

Abstract

Single-strand conformation polymorphism (SSCP) analysis on the ABI PRISM 3100 and the 3100 Avant Genetic Analyzers provides fast, easy detection of small variations in DNA sequences, including singlebase substitutions, insertions, or deletions.

SSCP Analysis

SSCP analysis is based on the principle that under non-denaturing conditions, single-stranded DNA molecules assume unique conformations that vary depending on their nucleotide sequence. The change of only one base may cause a conformational change in the DNA molecule that is sufficient to be detected on a non-denaturing polymer, e.g., electrophoretic mobility differences compared with wild-type (wt) sequences.

The SSCP technique includes three processes:

PCR amplification using primers that flank the DNA region of interest;

Denaturation of the resulting double-stranded PCR product, followed by rapid chilling to prevent re-annealing of the strands;

Electrophoretic separation of the single-stranded DNA on a nondenaturing sieving medium, such as a flowable polymer.

The ABI PRISM 3100 Genetic Analyzer, a 16-capillary instrument, performs fluorescent SSCP rapidly and efficiently, separating fragments of up to 350 bases in less than 35 minutes with a minimum throughput of 654 samples per 24 hours. Fluorescent SSCP analysis on the 3100 system can be an ideal first-pass discrimination test for screening a large number of samples. Subsequent sequence analysis on this instrument can determine the exact base change of the mutation. This can be much more cost-effective than sequencing every sample, especially for populations in which the mutation frequency is low.

For studies involving fewer samples and lower throughput, the ABI PRISM 3100 Avant Genetic Analyzer
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