1997
DOI: 10.1099/0022-1317-78-11-2751
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Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.

Abstract: The synthesis of long cDNA molecules encoding the complete genome of RNA viruses has recently been demonstrated ; this major improvement has numerous practical applications such as construction of infectious cDNA clones or study of sequence variability at the level of a single RNA molecule. Using hepatitis C virus (HCV) as a model, we established an RT-PCR technique for amplification of cDNA fragments with a length of about 5 kb. The RT reaction was carried out with a Moloney murine leukaemia virus reverse tra… Show more

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Cited by 26 publications
(19 citation statements)
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“…Total RNA was isolated from 100 l of serum by proteinase K digestion and phenol-chloroform extraction as previously described (31) and dissolved in 20 l of sterile water. To generate a long fragment of cDNA, RT was performed on 10 l of RNA with Expand reverse transcriptase (Roche Diagnostics GmbH) and primer 5460, 8625, or 3ЈUTR (Table 1 and Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Total RNA was isolated from 100 l of serum by proteinase K digestion and phenol-chloroform extraction as previously described (31) and dissolved in 20 l of sterile water. To generate a long fragment of cDNA, RT was performed on 10 l of RNA with Expand reverse transcriptase (Roche Diagnostics GmbH) and primer 5460, 8625, or 3ЈUTR (Table 1 and Fig.…”
Section: Methodsmentioning
confidence: 99%
“…HCV plasma samples were treated with or without 0.1% Triton X-100 in phosphate-buffered saline at 37°C for 1 h; subsequently, 5 U of RNase A (QIAGEN) was added and incubation continued for 1 h. In order to degrade RNase A before RNA extraction, proteinase K was added to each reaction mixture and incubated at 37°C for 15 min prior to adding lysis buffer. RNA was extracted as previously described (23) and was analyzed by RT-PCR.…”
Section: Methodsmentioning
confidence: 99%
“…Total RNA was isolated from 100 l serum by proteinase K digestion and phenol-chloroform extraction as previously described (23) and dissolved in 20 l sterile water. To generate a long fragment of cDNA, reverse transcription (RT) was performed on 10 l RNA with Expand reverse transcriptase (Roche Diagnostics GmbH) and primer 5460 or 8625 or the 3ЈUTR, according to the manufacturer's instructions, in a total volume of 30 l at 42°C for 2 h as described previously (21).…”
Section: Methodsmentioning
confidence: 99%
“…However, research on HCV replication and pathogenesis, as well as the development of therapeutic strategies, has been severely hindered because of the lack of a reliable cell culture system and an adequate animal model for HCV infection and propagation (3)(4)(5). Nevertheless, HCVencoded proteins have been identified by in vitro and ex vivo systems expressing cloned viral cDNA (6)(7)(8)(9). Recent research efforts have thus focused on the properties and functions of individual HCV gene products in the interest of unraveling the mechanisms of viral pathogenicity.…”
mentioning
confidence: 99%