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  Comparison of PCR-based mutation detection methods and application for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates

Greber, B., Tandara, H., Lehrach, H., & Himmelbauer, H. (2005). Comparison of PCR-based mutation detection methods and application for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates. Human Mutation, 25(5), 483-490. doi:10.1002/humu.20168.

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Genre: Journal Article
Alternative Title : Hum Mutat

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Greber et al. - Human Mutation.pdf (Any fulltext), 284KB
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Greber et al. - Human Mutation.pdf
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 Creators:
Greber, Boris1, Author
Tandara, Helena1, Author
Lehrach, Hans2, Author              
Himmelbauer, Heinz2, Author              
Affiliations:
1Max Planck Society, ou_persistent13              
2Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              

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Free keywords: mouse models; mutagenesis; mutation detection; poolability; Sult1a1; Hprt1
 Abstract: 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. Hum Mutat 25:483-490, 2005. © 2005 Wiley-Liss, Inc.

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Language(s): eng - English
 Dates: 2005-04-14
 Publication Status: Published in print
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 Table of Contents: -
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 Identifiers: eDoc: 271270
DOI: 10.1002/humu.20168
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Title: Human Mutation
  Alternative Title : Hum Mutat
Source Genre: Journal
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Pages: - Volume / Issue: 25 (5) Sequence Number: - Start / End Page: 483 - 490 Identifier: ISSN: 1059-7794