Identification of in vivo mutations in exon 5 of the human HPRT gene in a set of pooled T-cell mutants by constant denaturant capillary electrophoresis (CDCE).
Constant denaturant capillary electrophoresis (CDCE), based on co-operative DNA melting equilibria, has the resolving power to separate single nucleotide mutants from wild type sequences. We used this technique to study mutations in a 70-bp isomelting domain of the human HPRT gene, which included the entire exon 5 and its flanking splice donor and acceptor sites. Pooled samples of 6-thioguanine selected T-cell clones from 51 healthy donors representing a total of approximately 1000 individual HPRT mutants were analysed. Slow moving peaks from the heteroduplex part of the CDCE electropherograph were collected and subjected to a second round of PCR and CDCE analysis, followed by DNA sequencing. Five independent mutations were detected. Four were splicing errors; one insertion of CC and two G-->A transitions in the splice donor site of intron 5, and one G-->C transversion in the splice acceptor site of intron 4. The fifth mutation was a missense transversion, T389>G. A reconstruction experiment, in which DNA with known mutation was mixed with wild type DNA, showed the sensitivity of mutation detection to be better than 1:100 under the conditions used in this study. These results demonstrate the high sensitivity of the CDCE-method for mutation screening.
Fält
S
S
Department of Biosciences, The Karolinska Institute, CNT/Novum, Huddinge, Sweden.
Kumar
R
R
Wennborg
A
A
Tomita-Mitchell
A
A
Thilly
W G
WG
Lambert
B
B
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary / Q000379:methods; D005091:Exons / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D009154:Mutation; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000166:cytology / Q000201:enzymology*; D013696:Temperature
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase