Linked Life Data

15847930

Source:http://linkedlifedata.com/resource/pubmed/id/15847930

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1-2
pubmed:dateCreated
2005-4-25
pubmed:databankReference
pubmed:abstractText
The goal of this study was to adapt a long RT-PCR technique to amplify large PCR fragments from the genome of hepatitis C virus (HCV) isolates using clinical samples. This was done by using a reverse transcriptase devoid of RNase H activity and a mixture of two antibody-bound thermostable polymerases to combine the high processivity of Taq and the high fidelity of Pwo with its 3'-->5' exonuclease activity. Other modifications included gentle handling during RNA extraction, the absence of tRNA and random primers, a two-step reverse transcription procedure to optimize cDNA synthesis, and increasing the annealing temperature for primers. With this approach, the HCV-1 genome (nucleotides 35-9282) was amplified consistently as two overlapping fragments of 5344 and 4675 bp from a pooled chimpanzee plasma sample containing approximately 10(6) genome copies of HCV RNA/ml. Using the conditions that we identified, 96% of the complete genomic sequence of a distinct HCV genotype 6 variant (km45) was determined from less than 300 microl of serum. This method should prove useful for molecular, epidemiological and clinical studies of hepatitis C where samples are limited but complete virus sequence is required, for example, identifying mutational hot spots of HCV under specific clinical conditions.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0166-0934
pubmed:author
pubmed:issnType
Print
pubmed:volume
126
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
139-48
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
A refined long RT-PCR technique to amplify complete viral RNA genome sequences from clinical samples: application to a novel hepatitis C virus variant of genotype 6.
pubmed:affiliation
Division of Gastroenterology/Hepatology, Department of Medicine, University of Kansas Medical Center, 4035 Delp, MS 1023, Kansas City, KA 66160, USA. llu@kumc.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, N.I.H., Extramural