[Amplification of hepatitis C virus 5' untranslated region gene by RACE and its secondary structure analysis].
OBJECTIVE
To obtain very end full-length cDNA of hepatitis C virus (HCV) 5' untranslated region (5' UTR), and analyse its primary and secondary structure.


METHODS
By reverse transcription-nested polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP), a patient infected with genotype 2a HCV was found. Total RNA isolated from the serum as template, the cDNA of 5' noncoding region was amplified using rapid amplification of cDNA ends methods (RACE), the fragments were recombined by A-T clone strategy, the recombinants were confirmed by RFLP and PCR then sequenced. Secondary structures were analysed by RNA draw.


RESULTS
Very end full-length cDNA of 2a genotype HCV 5' UTR was obtained by RACE. In five clones obtained, three contained full-length 5' UTR cDNA, and A21G, G170A, T222C, T247C, C339T substitutions were found compared with HC-J6. he homologies with HCV-1,HC-J6,HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93.0%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions did not alter the secondary structure. Two out of five clones were deleted to have 53 and 144 bases at 5' terminus of HCV 5' UTR, respectively.


CONCLUSIONS
RACE is rapid and effective, works well to obtain very end of virus genome. With that, Authors obtained full-length cDNA of genotype 2a of HCV 5' UTR. There are genes deleted at 5' terminus circulated in hepatitis C patients.
Qin
Zhaoxi
Z
Institute of Hepatology, People's Hospital, Beijing University, Beijing 100044, China.
Cong
Xu
X
Jiang
Dong
D
Ha
Minghao
M
Chen
Hongsong
H
Wei
Lai
L
D020121:5' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D016174:Hepacivirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D004279:DNA, Viral