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InFusion cloning for the generation of biologically relevant HCV chimeric molecular clones

King, Barnabas; Urbanowicz, Richard; Tarr, Alexander W.; Ball, Jonathan K.; McClure, C. Patrick

Authors

Barnabas King

Richard Urbanowicz

Alexander W. Tarr

Jonathan K. Ball

C. Patrick McClure



Contributors

Mansun Law
Editor

Abstract

This chapter describes how to generate chimeric molecular cassettes that are ready to receive PCR-amplified E1/E2 genes using new DNA cloning technology. The method is divided into three sections: (1) generation of a ΔCore-NS2 cassette based upon the full-length JFH-1 molecular clone; (2) insertion of a “structural gene” fragment encoding the Core, p7, and NS2 genes of a given genotype reference sequence, to generate a ΔE1/E2 cassette; and (3) insertion of patient-isolated E1/E2 genes that are genotype-matched to the structural genes. The final assembled chimeric genomes can then be analyzed in the HCV cell culture system. These cassettes allow characterization of the extensive in vivo viral diversity without the need to isolate and clone whole virus genomes. This method can be readily applied to the study of other HCV genes and other viruses.

Publication Date Jan 1, 2019
Journal Methods in Molecular Biology; Hepatitis C Virus Protocols
Electronic ISSN 1940-6029
Publisher Springer
Pages 93-104
Series Title Methods in Molecular Biology
Series Number 1911
Book Title Hepatitis C Virus Protocols
Chapter Number 6
ISBN 9781493989751
APA6 Citation King, B., Urbanowicz, R., Tarr, A. W., Ball, J. K., & McClure, C. P. (2019). InFusion cloning for the generation of biologically relevant HCV chimeric molecular clones. In M. Law (Ed.), Hepatitis C Virus Protocols, 93-104. Springer. doi:10.1007/978-1-4939-8976-8_6
DOI https://doi.org/10.1007/978-1-4939-8976-8_6
Publisher URL https://link.springer.com/protocol/10.1007%2F978-1-4939-8976-8_6
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