Detection of point mutations by capillary electrophoresis with temporal temperature gradients.
By combining the advantages of capillary electrophoresis and temperature gradient gel electrophoresis, a method was developed to detect point mutations in polymerase chain reaction (PCR) fragments. Increasing and decreasing temporal temperature gradients were established by means of a computer-controlled Peltier module. Native and denaturing conditions were achieved by cooling to 25 degrees C and heating to 70 degrees C, respectively, a thermostating liquid surrounding the capillary. To separate nucleic acid fragments, a sieving media, containing 4% linear polyacrylamide, 1 x Tris borate EDTA buffer (TBE) and 6 M urea, was found appropriate. Renewal of the sieving matrix before each run significantly improved the reproducibility of fragment separation. The ability of this capillary electrophoresis system to detect point mutations is demonstrated with the human prion-protein gene.
Schell
J
J
Institut für Physikalische Biologie and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität, Düsseldorf, Germany.
Wulfert
M
M
Riesner
D
D
D000818:Animals; D004247:DNA / Q000032:analysis / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D017354:Point Mutation; D013696:Temperature
D004247:DNA