Identification of eplication-ompetent Dimeric Human Hepatitis B Viral Using PCR Screening Technique

Nam, Seok Hyun; Cho, Sung Won

Abstract

BACKGROUND/AIMS
Transfection of the hepatitis B virus (HBV) genome requires the cloning of tandem HBV sequences inserted into a plasmid vector, which is usually screened for by the restriction enzyme digestion of plasmid minipreparation from at least a dozen of bacterial colonies. The aim of this study was to develop a simple alternative screening method for bacterial colonies harbouring tandem HBV sequences by a PCR.
METHODS
A set of polymerase chain reaction (PCR) primer was designed to detect the bacterial colonies harbouring "head to tail" dimeric HBV DNA. PCR which amplifies the head to tail junction site of two tandem HBV molecules was performed.
RESULTS
PCR products with appropriate size (1.2kb) were obtained. The accurate detection by PCR screening technique was confirmed by enzyme digestion.
CONCLUSIONS
These results suggest that PCR screening technique is a simple and rapid method for the identification of bacterial colonies containing tandem HBV sequences.