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). Mutat Res 2000 Jul 20;452(1):57-66
Date
07/15/2000Pubmed ID
10894891DOI
10.1016/s0027-5107(00)00046-4Scopus ID
2-s2.0-0343962276 (requires institutional sign-in at Scopus site)Abstract
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.
Author List
Fält S, Kumar R, Wennborg A, Tomita-Mitchell A, Thilly WG, Lambert BAuthor
Aoy Tomita Mitchell PhD Professor in the Surgery department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Base SequenceDNA
DNA Mutational Analysis
Electrophoresis, Capillary
Exons
Humans
Hypoxanthine Phosphoribosyltransferase
Male
Mutation
Nucleic Acid Denaturation
Point Mutation
Sensitivity and Specificity
T-Lymphocytes
Temperature
