Linked Life Data

3199441

Source:http://linkedlifedata.com/resource/pubmed/id/3199441

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1989-1-24
pubmed:abstractText
We have modified current methods to create a very efficient technique for cloning cDNAs in a defined orientation, into plasmid vectors bearing phage SP6 and T7 polymerase promoters. First strand synthesis is primed at the poly(A) tail with a 26-mer synthetic oligonucleotide linker/primer, the RNA is hydrolyzed and the cDNA is tailed with 10 to 15 dG residues. The cDNA is then annealed to two prepared vector fragments specific for the two ends of the cDNA (one bearing a dC10-15 tail and the other bearing a 14-nucleotide cohesive end complementary to the linker/primer). After ligation the second strand is synthesized with the large fragment of DNA polymerase I. Libraries of up to 8 x 10(6) independent transformants have been obtained from 1 microgram of Drosophila poly(A)+ RNA. The design of the method and careful optimization of first strand synthesis have permitted cloning of several large (4.3 to 6.5 kb), low abundance cDNAs. Transcription of essentially full-length clones with phage SP6 RNA polymerase produces RNAs that are efficiently translated in vitro to give complete, unfused products, thus permitting rapid characterization of the clones via the encoded polypeptides. Antisense RNAs can also be produced by transcription with phage T7 RNA polymerase.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0022-2836
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
203
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
425-37
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1988
pubmed:articleTitle
Functional cDNA libraries from Drosophila embryos.
pubmed:affiliation
Department of Cellular and Developmental Biology, Harvard University Biological Laboratories, Cambridge, MA 02138.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't