A strategy for obtaining near full-length HCV cDNA clones (assemblicons) by assembly PCR.
Hepatitis C virus (HCV) genotype is a predictor of response, and guides the duration of antiviral therapy. However, with the exception of HCV genotype 1a, 1b and 2a, a limited number of clones from other genotypes exist. Here we report the optimization of long RT-PCR to generate three overlapping amplicons that span the near full length HCV genome from a panel of HCV genotypes (1a, 1b, 2a, 2b, 3a, 4a, 5a). Assembly-PCR (As-PCR) was used to construct near full-length cDNA clones (assemblicons) for each genotype. The optimization of the long RT-PCR on genotype 1a and 1b indicated that QIAamp Viral RNA kit (Qiagen, UK), Expand RT and Expand Long Template PCR system (Roche, UK), were the most efficient in producing the requisite three overlapping amplicons and assemblicons for each genotype. The genotype of each assemblicon was confirmed. Assemblicon generation was only possible when the overlapping amplicons were biotinylated. As-PCR obviated the need for time consuming ligations and cloning. The use of three overlapping amplicons in the construction of HCV assemblicons minimised the chimeric nature of the resultant clone. As-PCR may prove a methodological avenue through which a larger panel of consensus HCV clones could be made available for HCV in vitro investigation.
Sheehy
P
P
Hepatitis C Unit, Department of Medicine, National University of Ireland Cork (NUIC), Clinical Science Building, Cork University Hospital, Wilton, Cork City, Ireland. P.Sheehy@ucc.ie
Scallan
M
M
Kenny-Walsh
E
E
Shanahan
F
F
Fanning
L J
LJ
D018076:DNA, Complementary / Q000032:analysis; D004279:DNA, Viral; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic