A Streamlined Mutation Detection System: Multicolor Post-PCR Fluorescence Labeling and Single-Strand Conformational Polymorphism Analysis by Capillary Electrophoresis

AUTOR(ES)

Inazuka, Masakazu

FONTE

Cold Spring Harbor Laboratory Press

RESUMO

Effective use of knowledge of human genome sequences in studies of hereditary diseases or cancer heavily depends on efficient methods for detection of mutations in individual samples. We describe here a simple and efficient mutation scanning system in which PCR products are post-labeled with two different fluorescent dyes in one tube, and analyzed by an automated capillary electrophoresis system using single-strand conformation polymorphism (SSCP) conditions (PLACE–SSCP). With the appropriate use of an internal control DNA, differences in electrophoretic mobilities between a reference and samples are precisely evaluated, then the presence of mutations is statistically judged. Thirty-three of 34 known mutations in fragments of three unrelated sequence contexts up to 741 bp were detected using one electrophoresis condition at the confidence level of <0.3% false positive. All the mutations were detected by analyzing at two temperatures. The described system has the advantage of little human intervention, short analysis time, high sensitivity, and objectivity of data interpretation.

Documentos Relacionados

• Novel Algorithm Identifies Species in a Polymycobacterial Sample by Fluorescence Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism Analysis
• Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling
• PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi.
• Improved single-strand DNA sizing accuracy in capillary electrophoresis.
• Transverse temperature-gradient single-strand conformation polymorphism analysis for temperature optimization of 'Cold'-SSCP mutation detection.