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Identification of novel sequences at the 5′ terminus of the hepatitis C virus genome
Author(s) -
Trowbridge R.,
Gowans E. J.
Publication year - 1998
Publication title -
journal of viral hepatitis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.329
H-Index - 100
eISSN - 1365-2893
pISSN - 1352-0504
DOI - 10.1046/j.1365-2893.1998.00090.x
Subject(s) - biology , amplicon , genome , untranslated region , complementary dna , rna , virology , polymerase chain reaction , rapid amplification of cdna ends , hepatitis c virus , nested polymerase chain reaction , nucleotide , microbiology and biotechnology , genetics , primer (cosmetics) , polymerase , dna , virus , gene , molecular cloning , chemistry , organic chemistry
To permit accurate identification of the 5′ end of the HCV genome, we used RNA ligase‐mediated rapid amplification of cDNA ends (RLM‐RACE) where an RNA molecule of known sequence (transcribed invitro from a cDNA template) was ligated to RNA purified from a hepatitis C virus (HCV)‐infected liver sample. After ligation, the product was amplified by nested polymerase chain reaction (PCR) and amplicons that were equal to or greater in size than those predicted from the recognized 5′ terminus of the HCV genome were cloned into pBluescriptKS. Twelve clones were sequenced, including three that were identical and contained an additional eight nucleotides, namely A. Thus, the HCV 5′ untranslated region (UTR) is now recognized as comprising 349 nucleotides, although it is possible to speculate that these additional nucleotides are part of a second, as yet undetected, stem–loop at the extreme 5′ terminus of the genome.