Economical protocol for combined single-strand conformation polymorphism and heteroduplex analysis on a standard capillary electrophoresis apparatus.
Combined single-strand conformational polymorphism (SSCP) and heteroduplex (HD) analysis (SSCP-HD) take advantage of parallel mutation detection in single-strand and duplex fraction during the single capillary electrophoresis (CE) run. The high mutation detection rate of individual SSCP and HD in CE guarantees almost a 100% success rate of combined SSCP-HD. Described here, the protocol for SSCP-HD-CE does not require dedicated instrumentation but can be applied for any commonly available CE DNA analyzer. We focused mostly on the sample preparation step that is critical for the stability of generated fractions and reproducibility of a generated result. The application of universal primer for fluorescent labeling and omitting the PCR purification step also greatly reduce the cost of mutation detection by SSCP-HD-CE.
Kozlowski
Piotr
P
Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland.
Krzyzosiak
Wlodzimierz J
WJ
D020650:Combinatorial Chemistry Techniques / Q000191:economics / Q000295:instrumentation / Q000379:methods; D003365:Costs and Cost Analysis; D004252:DNA Mutational Analysis / Q000191:economics / Q000295:instrumentation / Q000379:methods; D019075:Electrophoresis, Capillary / Q000191:economics* / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D020180:Heteroduplex Analysis / Q000191:economics* / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D018807:Polymorphism, Single-Stranded Conformational / Q000235:genetics*