Comparison of PCR-based mutation detection methods and application for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates.
Reverse genetic approaches to generate mutants of model species are useful tools to assess functions of unknown genes. Recent work has demonstrated the feasibility of such strategies in several organisms, exploiting the power of chemical mutagenesis to disrupt genes randomly throughout the genome. To increase the throughput of gene-driven mutant identification, efficient mutation screening protocols are needed. Given the availability of sequence information for large numbers of unknown genes in many species, mutation detection protocols are preferably based on PCR. Using a set of defined mutations in the Hprt1 gene of mouse embryonic stem (ES) cells, we have systematically compared several PCR-based point mutation and deletion detection methods available for their ability to identify lesions in pooled samples, which is a major criterion for an efficient large-scale mutation screening assay. Results indicate that point mutations are most effectively identified by heteroduplex cleavage using CEL I endonuclease. Small deletions can most effectively be detected employing the recently described "poison" primer PCR technique. Further, we employed the CEL I assay followed by conventional agarose gel electrophoresis analysis for screening a library of chemically mutagenized ES cell clones. This resulted in the isolation of several clones harboring mutations in the mouse Sult1a1 locus, demonstrating the high-throughput compatibility of this approach using simple and inexpensive laboratory equipment.
Greber
Boris
B
Max-Planck-Institute of Molecular Genetics, Berlin, Germany.
Tandara
Helena
H
Lehrach
Hans
H
Himmelbauer
Heinz
H
D000818:Animals; D015239:Arylsulfotransferase / Q000235:genetics*; D002999:Clone Cells; D004252:DNA Mutational Analysis / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D004622:Embryo, Mammalian / Q000166:cytology; D004720:Endonucleases; D050172:Genes, X-Linked; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013234:Stem Cells / Q000166:cytology*; D013698:Templates, Genetic
D015239:Arylsulfotransferase; C117544:SULT1A1 protein, human; C513291:Sult1a1 protein, mouse; D004720:Endonucleases; C406922:CEL I nuclease