Linked Life Data

19333780

Source:http://linkedlifedata.com/resource/pubmed/id/19333780

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2010-2-3
pubmed:abstractText
In the post-genomic era, providing a detailed description of protein functions poses a formidable challenge. To gain functional insights, we have to construct many kinds of expression vectors. DNA recombination based on polymerase chain reaction (PCR) and digestion followed by ligation is the preferred method for vector construction. However, this existing pattern is intrinsically limited by the selection of restriction endonuclease, prompting researchers to use commercial suppliers rather than amplifying genes themselves. Moreover, this method would introduce additional bases into the PCR products, which may be undesired for the construction of epitope expressing plasmid. A PCR-based gene synthesis method, referred to as competitive priming PCR (CP-PCR), is described here to efficiently assemble the plasmid expressing fusion protein of fibrinogen alpha chain and binding domain of galactose transcription factor. A pair of competitive sense primers were designed for the same target sequence. With the presence of antisense primer, PCR amplification of target sequence was performed in the same one system. The PCR product was underwent single digestion by using PstI, followed by ligation with the vector pCMV-BD linearized with EcoRIand PstI. The reconstructed plasmid was validated by sequencing and the fusion protein was affirmed by western blot. CP-PCR combines the superior convenience and precision of PCR. Moreover, it is perfectly capable of generating nearly all kinds of cohesive terminuses, which are ready to recombination in the presence of single digestion or even in the absence of digestion. We demonstrate, by using CP-PCR, the feasibility of directed cloning interested sequence only in the requirement of single digestion or even in the absence of digestion. Competitive priming PCR is demonstrated with convenience and precision equivalent to the traditional method. More than that, "seamless" DNA recombination may be achieved by this novel strategy.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
1573-4978
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
37
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1421-5
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Competitive priming PCR: a versatile method to generate cohesive terminus.
pubmed:affiliation
Division of Gastroenterology, Union Hospital of Fujian Medical University, Fujian Province, China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't