Original Research
Copyright ©The Author(s) 1997. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Mar 15, 1997; 3(1): 12-15
Published online Mar 15, 1997. doi: 10.3748/wjg.v3.i1.12
Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing
Lai Wei, Yu Wang, Hong-Song Chen, Qi-Min Tao
Lai Wei, Yu Wang, Hong-Song Chen, Qi-Min Tao, Institute of Hepatology, People’s Hospital of Beijing Medical University, Beijing 100044, China
Lai Wei, PhD, MD, male, born on Novermber 14, 1961, from Nanjing in Jiangsu Province, graduated from the Department of Medicine of Xuzhou Medical College, Associate Professor, engaged in infectious diseases
Author contributions: All authors contributed equally to the work.
Supported by The China Medical Board of America (No.93-582)
Correspondence to: Dr. Qi-Min Tao, Professor Institute of Hepatology, People’s Hospital of Beijing Medical University, Beijing 100044, China
Telephone: +86-10-68314422-5722
Received: September 9, 1996
Revised: January 1, 1997
Accepted: March 1, 1997
Published online: March 15, 1997

AIM: To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome and establish a new sequencing method in China.

METHODS: Polymerase chain reaction (PCR) was combined with a DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE) and polyethylene glycol (PEG) respectively. Then, in the presence of a 5’ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC-42 and HC-49) were sequenced.

RESULTS: PCR directed sequencing worked best using PCR amplified DNA purified by electrophoresis as a sequencing template. When sequencing a large number of templates, the purification step can be bypassed by using a lower concentration of dNTPs (40 μmol of each dNTP) and primers (10 pmol of each primer) in the first stage of PCR. The aliquot of the first stage of PCR mixture was then directly used for amplification of chain terminated products but the sequencing ladders generated were of low intensity. Polyethylene glycol (PEG) could not remove nonspecific products of PCR, which affected the sequencing result to a certain extent and generated a background in sequencing ladders. Compared with the reported HCVJ and HC-C2, a new three nucleotide deletion was found in HC-42.

CONCLUSION: PCR directed sequencing is a rapid, simple and effective method, especially for sequencing large samples. A three nucleotide deletion was first reported.

Keywords: Hepatitis C virus DNA, Viral DNA, Complementary polymerase chain reaction, Sequence analysis, DNA mutation