Linked Life Data

19835833

Source:http://linkedlifedata.com/resource/pubmed/id/19835833

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2010-1-27
pubmed:abstractText
In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
1096-0309
pubmed:author
pubmed:copyrightInfo
Copyright (c) 2009 Elsevier Inc. All rights reserved.
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
397
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
162-7
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli.
pubmed:affiliation
Institute of Biochemistry and Molecular Biology, Hubei University, Wuhan 430062, China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't