Linked Life Data

10471748

Source:http://linkedlifedata.com/resource/pubmed/id/10471748

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
18
pubmed:dateCreated
1999-9-15
pubmed:abstractText
We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
1362-4962
pubmed:author
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
27
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
e18
pubmed:dateRevised
2008-11-20
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.
pubmed:affiliation
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S.