Linked Life Data

3224824

Source:http://linkedlifedata.com/resource/pubmed/id/3224824

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1989-4-5
pubmed:databankReference
pubmed:abstractText
A method to introduce multiple mutations and to reconstruct genes, using a single oligodeoxyribonucleotide and DNA polymerase with high processivity, such as modified T7 DNA polymerase [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 84 (1987a) 4767-4771], is described. A eukaryotic cDNA, coding for porcine growth hormone (pGH), was reconstructed in this study to delete 75 bp and to introduce a G----A transition. The deletion removes 75 bp and brings an ATG just upstream from the codon for the first amino acid in the mature protein. Moreover, the G----A substitution creates a new PvuII restriction site to facilitate further manipulation of the gene. Maximum mutation frequency with this multisite-directed mutagenesis is reached within 15 min with an efficiency approaching 50%, when using the modified T7 DNA polymerase. No multisite-directed mutants were obtained when T4 DNA polymerase or Klenow (large) fragment of DNA polymerase I were used. The described method is also applicable to simple single site-directed mutations as well as to more complex gene reconstruction strategies.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0378-1119
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
69
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
81-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1988
pubmed:articleTitle
A multisite-directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene.
pubmed:affiliation
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't