Linked Life Data

7835714

Source:http://linkedlifedata.com/resource/pubmed/id/7835714

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1995-3-1
pubmed:databankReference
pubmed:abstractText
A simple procedure for in vitro site-directed mutagenesis (SDM) was developed using two oligodeoxyribonucleotide (oligo) primers, one serving as selection primer and the other for mutagenesis in the target DNA (mutagenic primer). The mutant clones can be selected by reversion mutations of dual amber (am) to dual Gln codons in the cat gene, resulting in a chloramphenicol-resistant phenotype in sup(O) host cells. Unlike previously reported procedures, the new method requires neither special chemical reagents, nor single-stranded DNA purification, nor any additional biochemical treatments such as multiple PCRs, restriction digestion and so on, but utilises one of the oligo-directed dual am (ODA) plasmids, i.e., pKF16c, pKF17c, pKF18c or pKF19c. The two amber (am) mutations located two codons apart from each other in cat (catam2) can be simultaneously reverted with a 20-nucleotide primer (CQ, pAACCAGACCGTTCAGCTGGA) during primer extension. In a model experiment using the lacZ' gene, an am mutation or a single-bp deletion (sbd) mutation was frequently co-introduced in lacZ' using a mixture of am- or sbd-encoding mutagenic primer for lacZ', and the CQ primer for catam2. Applying this procedure to the human AT cDNA (encoding antithrombin), a missense mutation (Arg406-->Met) in one of the human AT variants, AT-Kyoto, involved in congenital thrombosis disease, was introduced efficiently into the wild-type cDNA (> 85%).
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0378-1119
pubmed:author
pubmed:issnType
Print
pubmed:day
23
pubmed:volume
152
pubmed:geneSymbol
lacZ
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
271-5
pubmed:dateRevised
2006-5-1
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis.
pubmed:affiliation
Department of Biochemistry and Molecular Genetics, Kyoto Prefectural University of Medicine, Japan.
pubmed:publicationType
Journal Article