Linked Life Data

15832303

Source:http://linkedlifedata.com/resource/pubmed/id/15832303

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2005-4-26
pubmed:databankReference
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
1098-1004
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
25
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
483-90
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Comparison of PCR-based mutation detection methods and application for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates.
pubmed:affiliation
Max-Planck-Institute of Molecular Genetics, Berlin, Germany.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't