A STREAMLINED MUTATION DETECTION SYSTEM - MULTICOLOR POST-PCR FLUORESCENCE LABELING AND SINGLE-STRAND CONFORMATIONAL POLYMORPHISM ANALYSIS BY CAPILLARY ELECTROPHORESIS

Effective use of knowledge of human genome sequences in studies of her editary diseases or cancer heavily depends on efficient methods for de tection 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 c onformation polymorphism (SSCP) conditions (PLACE-SSCP). With the appr opriate use of an internal control DNA, differences in electrophoretic mobilities between a reference and samples are precisely evaluated, t hen 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 detec ted by analyzing at two temperatures. The described system has the adv antage of little human intervention, short analysis time, high sensiti vity, and objectivity of data interpretation.