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 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.
Inazuka
M
M
Division of Genome Analysis, Institute of Genetic Information, Kyushu University, Fukuoka 812-82, Japan.
Wenz
H M
HM
Sakabe
M
M
Tahira
T
T
Hayashi
K
K
D001331:Automation; D002138:Calibration; D002874:Chromosome Mapping / Q000379:methods*; D027081:DNA Gyrase; D004252:DNA Mutational Analysis / Q000379:methods; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D005091:Exons; D015894:Genome, Human; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II