Cloning and Assembly of Complex Libraries of Full-Length HCV cDNA Clones

Many studies of the molecular biology of hepatitis C virus (HCV) begin by obtaining representative cDNA clones of the viral genome. Most cloning strategies have been devised to deal with the low levels of HCV RNA present in starting material used for RNA isolation and cDNA synthesis. Typical sources include patient sera, liver samples, sera and tissues from infected chimpanzees, or in some cases, viral RNA obtained after replication in cell culture. Thus far, patient samples are the most common source of HCV RNA for cDNA cloning. Titers of HCV RNA in patient sera are low, typically ranging from 10³ to 10⁸ mol/mL. Except for a few instances, where cDNA libraries were obtained directly by isolating RNA from large volumes of high-titer sera (1 ,2 ), PCR is used for amplifying HCV cDNA prior to cloning. Since this procedure requires smaller amounts of HCV RNA, it is more generally useful for construction of complex cDNA libraries from a wide variety of HCV-containing samples.