Linked Life Data

20194456

Source:http://linkedlifedata.com/resource/pubmed/id/20194456

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2010-4-23
pubmed:abstractText
The G-less cassette is a 365-nucleotide (nt) segment of DNA lacking guanine (G) residues on the nontemplate strand. In principle, a full-length transcript can be generated in an in vitro reaction lacking GTP, an omission that leads to suppression of most random, nonspecific transcription throughout the plasmid. This method, like runoff transcription, generates radiolabeled RNA products, directly bypassing the necessity and extra time required to perform primer extension or other indirect mRNA product measurements. Unlike runoff transcription, which requires a cleaved end, the G-less assay can be performed on a circular, supercoiled plasmid, which in many systems is a more efficient template. In practice, most crude systems, such as HeLa nuclear extracts, contain low amounts of contaminating GTP that lead to small amounts of background transcription and occasionally can cause random upstream transcription to read through the G-less cassette. To minimize these artifacts, the reaction generally contains 3'-O-Me-GTP, a chain-terminating analog of GTP that causes transcription to cease when it is incorporated into a growing transcript, much like the dideoxy analogs used in DNA sequencing. The reaction products are cleaved with T1 RNase, which cleaves RNAs at G residues, further reducing background transcription; the G-less mRNA remains intact, whereas small random RNAs are digested.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
1559-6095
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
2010
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
pdb.prot5387
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
G-less cassette in vitro transcription using HeLa cell nuclear extracts.
pubmed:publicationType
Journal Article