PMID: 9186947May 1, 1997Paper

Direct blotting electrophoresis for sequencing and genotyping hepatitis C virus

Journal of Virological Methods
Sandrine CastelainG Duverlie

Abstract

Many methods have been used to differentiate the hepatitis C virus (HCV) genotypes based on, for example, type specific primers, probes and restriction fragment length polymorphism. However, determination of the nucleotide sequence remains the reference. Therefore, a simple non-radioactive cycle sequencing technique was developed for clinical tests. PCR-amplified products of the 5' non-coding region (from position -274 to -31) were sequenced using a 5' digoxygenin-labeled primer. After denaturation, the samples were loaded on a direct blotting electrophoresis system (GATC 1500). Sequencing products were blotted onto a nylon membrane during the electrophoresis. The DNA fragments were then UV-cross-linked, incubated with phosphatase-labeled anti-digoxygenin antibody and stained with a precipitating substrate. Reading the sequence of six samples were possible within 2 days. In 41 different samples, five different genotypes were found by sequence analysis from position -245 to -69, of which 17 were type 1a, 7 type 1b, 5 type 2a, 8 type 3a, 3 type 4 and 1 type 5. These results agreed with those obtained by reverse hybridization assay. Direct blotting electrophoresis offered a good non-radioactive method of performing clinical sequen...Continue Reading

References

Oct 11, 1994·Proceedings of the National Academy of Sciences of the United States of America·L StuyverG Maertens
Oct 1, 1995·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·M Martinot-PeignouxV Milotova
Nov 8, 1994·Proceedings of the National Academy of Sciences of the United States of America·H TokitaM Mayumi
May 1, 1994·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·P SimmondsD S Chen
Jan 1, 1994·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·G DusheikoP Simmonds

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Citations

Apr 3, 2007·Virology Journal·Carole FournierSandrine Castelain
Oct 7, 2004·Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology·Sandrine CastelainGilles Duverlie
Oct 9, 1999·Clinical Microbiology Reviews·M Arens

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