Linked Life Data

1595905

Source:http://linkedlifedata.com/resource/pubmed/id/1595905

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1992-6-30
pubmed:abstractText
We describe an efficient site-specific mutagenesis procedure that is effective with virtually any plasmid, requiring only that the target plasmid carry a unique, nonessential restriction site. The procedure employs two mutagenic oligonucleotide primers. One primer contains the desired mutation and the second contains a mutation in any unique, nonessential restriction site. The two primers are annealed to circular single-stranded DNA (produced by heating circular double-stranded DNA) and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mut S) Escherichia coli strain, which increases the probability that the two mutations will cosegregate during the first round of DNA replication. Transformants are selected en masse in liquid medium containing an appropriate antibiotic and plasmid DNA is prepared, treated with the enzyme that recognizes the unique, nonessential restriction site, and retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently. Plasmids with mutations in the unique restriction site are resistant to digestion, remain circular, and transform bacteria efficiently. By linking a selectable mutation in a unique restriction site to a nonselectable mutation, the latter can be recovered at frequencies of about 80%. Since most plasmids share common vector sequences, few primers, targeted to shared restriction sites, are needed for mutagenizing virtually any plasmid. The procedure employs simple procedures, common materials, and it can be performed in as little as 2 days.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:volume
200
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
81-8
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1992
pubmed:articleTitle
Site-directed mutagenesis of virtually any plasmid by eliminating a unique site.
pubmed:affiliation
Department of Cancer Biology, Harvard University School of Public Health, Boston, Massachusetts 02115.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.