Linked Life Data

6096101

Source:http://linkedlifedata.com/resource/pubmed/id/6096101

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
1985-2-21
pubmed:abstractText
This paper presents a simple and efficient method for oligonucleotide-directed mutagenesis using vectors derived from single-stranded phage. This modification of our previously published procedure (Zoller and Smith, 1982) features the use of two primers, one of which is a standard M13 sequencing primer and the other is the mutagenic oligonucleotide. Both primers are simultaneously annealed to single-stranded template DNA, extended by DNA polymerase I (large fragment), and ligated together to form a mutant wild-type gapped heteroduplex. Escherichia coli is transformed directly with this DNA; the isolation of covalently closed circular DNA as in our previous report is not necessary. Mutants are identified by plaque lift hybridization using the mutagenic oligonucleotide as a probe. As an example of the method, a heptadecanucleotide was used to create a T----G transversion in the MATa gene of Saccharomyces cerevisiae cloned into the vector M13mp5. The efficiency of mutagenesis was approximately 50%. Production of the desired mutation was verified by DNA sequencing. The same procedure has been used without modification to create insertions of restriction sites as well as specific deletions of 500 bases.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0198-0238
pubmed:author
pubmed:issnType
Print
pubmed:volume
3
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
479-88
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1984
pubmed:articleTitle
Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't