Linked Life Data

9918117

Source:http://linkedlifedata.com/resource/pubmed/id/9918117

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
1999-4-5
pubmed:abstractText
Recently, a novel strategy for nucleotide exchange of target DNA using chimeric RNA/DNA oligonucleotides (CO) was reported. The CO can easily be transfected into cells, remain stable within the cells, and migrate to the nucleus. We have in this study used 42 similar constructs for targeting six different human and canine loci. A variety of cationic lipids, electroporation, and microinjection were used for transfection of the CO into lymphoblastoids, Huh7, HT 1080, and Jurkat cell lines, and canine primary fibroblasts and hepatocytes. However, no nucleotide exchange was detected in any of the targeted loci. Using PCR followed by restriction enzyme analysis, nucleotide exchange in approximately 2%-10% of the PCR products was observed during the first 3 days after transfection with CO-vWF-28S2 designed for repairing a mutation in the von Willebrand gene. Surprisingly, the observed exchange reverted after culturing the cells for a longer period of time (14 days). Furthermore, a positive indication of gene conversion (5%) was also obtained using an allele-specific PCR method for analysis of the PAI-1 gene. However, cloning of the PCR products revealed no nucleotide exchange. In our view, the most likely explanation is that the initial false positive result originates from a PCR artifact created by the CO itself. Our results imply that an independent method, that is, Southern blotting, must be used to verify an observed nucleotide exchange.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1087-2906
pubmed:author
pubmed:issnType
Print
pubmed:volume
8
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
531-6
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Failure to achieve gene conversion with chimeric circular oligonucleotides: potentially misleading PCR artifacts observed.
pubmed:affiliation
Department of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't