Abstract
This chapter describes the preparation of template DNA from poxvirus-infected cells, plaques, or crude virus stocks for polymerase chain reaction (PCR) amplification. The advantages of this technique are that it is rapid, inexpensive, and, most importantly, reliable, requiring only centrifugation, detergent, and protease treatment. The template preparation is suitable for PCR amplification for screening viruses, cloning, transfection, and DNA sequencing.
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References
Joklik, W. K. and Becker, Y. (1964) The replication and coating of vaccinia DNA. J. Mol. Biol. 10, 452–474.
Erlich, H. A., ed. (1989) PCR Technology: Principles and Applications for DNA Amplification. M Stockton Press, New York.
Sung, T.-S., Roper, R. L., Shang, Y., Rudge, S. A., Temel, R., Hammond, S. M., et al. (1997) Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity. EMBO J. 16, 4519–4530.
Roper, R. L. and Moss, B. (1999) Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein. J. Virol. 73, 1108–1117.
Roper, R. L., Wolffe, E. J., Weisberg, A., and Moss, B. (1998) The envelope protein encoded by the A33R gene is required for formation of actin-containing microvilli and efficient cell to cell spread of vaccinia virus. J. Virol. 72, 4192–4204.
Wolffe, E. J., Katz, E., Weisberg, A., and Moss, B. (1997) The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus. J Virol. 71, 3904–3915.
Shisler, J. L., Isaacs, S. N., and Moss, B. (1999) Vaccinia virus serpin-1 deletion mutant exhibits a host range defect characterized by low levels of intermediate and late mRNAs. Virology 262, 298–311.
Roper, R. L., Payne, L. G., and Moss, B. (1996) Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene. J. Virol. 70, 3753–3762.
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© 2004 Humana Press Inc., Totowa, NJ
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Roper, R.L. (2004). Rapid Preparation of Vaccinia Virus DNA Template for Analysis and Cloning by PCR. In: Isaacs, S.N. (eds) Vaccinia Virus and Poxvirology. Methods in Molecular Biology, vol 269. Humana Press. https://doi.org/10.1385/1-59259-789-0:113
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DOI: https://doi.org/10.1385/1-59259-789-0:113
Publisher Name: Humana Press
Print ISBN: 978-1-58829-229-2
Online ISBN: 978-1-59259-789-5
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