Enhanced protocol for determining the 3′ terminus of hepatitis C virus

The determination of viral 3′ ends is a routine practice in molecular biology. However, this has been a challenging task for hepatitis C virus (HCV), an enveloped single-stranded, positive-sense RNA virus classified into the Flaviviridae family. The extreme end of HCV 3′ untranslated region (3′UTR), the so-called 3′ X tail, was not identified at the time of HCV discovery. Complete HCV 3′UTR sequences occupy a very small percentage of the exponentially growing HCV sequence databases. Although commercial kits and experimental protocols are available, these methods are both tedious and not reproducible. A stepwise optimization procedure was developed as a simple and robust protocol for determining the complete HCV 3′UTR from clinical samples. The availability of abundant authentic sequence information for the complete HCV 3′UTR will allow full investigation of its biological role in the life cycle of HCV.