Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing.

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.