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J Virol Methods. 2003 Mar;108(1):67-74.

A rapid and convenient variant of fusion-PCR to construct chimeric flaviviruses.

Author information

1
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000, Leuven, Belgium.

Abstract

So far, full-length cDNAs of chimeric flaviviruses have been constructed by restriction-enzyme cleavage of the gene(s) to be exchanged or by fusion-PCR of two amplified PCR fragments. The construction of a chimeric flavivirus by a faster and more convenient variant of the standard fusion-PCR is reported. A Modoc/yellow fever chimeric virus was engineered in which the structural prM and E genes of yellow fever virus 17D were replaced by the homologous genes of Modoc virus. In two PCR steps, a fusion was made between the 3' end of the C gene of yellow fever virus and the 5' end of the prM gene of Modoc virus, and between the 3' end of the E gene of Modoc virus and the 5' end of the NS1 gene of yellow fever virus. For each of the two fusions between yellow fever and Modoc virus, a standard PCR was performed to amplify a short fragment with one overlapping end that could be used as one of the primers in the subsequent (fusion) PCR.

PMID:
12565155
DOI:
10.1016/s0166-0934(02)00259-8
[Indexed for MEDLINE]

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