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. 1990 Feb 25;18(4):913–918. doi: 10.1093/nar/18.4.913

Construction of recombinant DNA molecules by the use of a single stranded DNA generated by the polymerase chain reaction: its application to chimeric hepatitis A virus/poliovirus subgenomic cDNA.

C Wychowski 1, S U Emerson 1, J Silver 1, S M Feinstone 1
PMCID: PMC330345  PMID: 2156236

Abstract

In order to study the importance of VP4 in picornavirus replication and translation, we replaced the hepatitis A virus (HAV) VP4 with the poliovirus (PV1) VP4. Using a modification of oligonucleotide site directed mutagenesis and the polymerase chain reaction (PCR), we created a subgenomic cDNA chimera of hepatitis A virus in which the precise sequences coding for HAV VP4 capsid protein were replaced by the sequences coding for the poliovirus VP4 capsid protein. The method involved the use of PCR primers corresponding to the 3' and 5' ends of the poliovirus VP4 sequence and that had HAV VP4 3' and 5' flanking sequences on their 5'ends. Single stranded DNA of 240 and 242 nt containing the 204 nt coding for the complete poliovirus VP4 were produced by using a limiting amount of one of the primers in a PCR reaction. These single stranded PCR products were used like mutagenic oligonucleotides on a single stranded phagemid containing the first 2070 bases of the HAV genome. Using this technique, we precisely replaced the HAV VP4 gene by the poliovirus VP4 gene as determined by DNA sequencing. The cDNA was transcribed into RNA and translated in vitro. The resulting protein could be precipitated by antibody to poliovirus VP4 but not to HAV VP4.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson D. A. Cytopathology, plaque assay, and heat inactivation of hepatitis A virus strain HM175. J Med Virol. 1987 May;22(1):35–44. doi: 10.1002/jmv.1890220106. [DOI] [PubMed] [Google Scholar]
  2. Baroudy B. M., Ticehurst J. R., Miele T. A., Maizel J. V., Jr, Purcell R. H., Feinstone S. M. Sequence analysis of hepatitis A virus cDNA coding for capsid proteins and RNA polymerase. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2143–2147. doi: 10.1073/pnas.82.7.2143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Cohen J. I., Ticehurst J. R., Feinstone S. M., Rosenblum B., Purcell R. H. Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture. J Virol. 1987 Oct;61(10):3035–3039. doi: 10.1128/jvi.61.10.3035-3039.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen J. I., Ticehurst J. R., Purcell R. H., Buckler-White A., Baroudy B. M. Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses. J Virol. 1987 Jan;61(1):50–59. doi: 10.1128/jvi.61.1.50-59.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coulepis A. G., Locarnini S. A., Westaway E. G., Tannock G. A., Gust I. D. Biophysical and biochemical characterization of hepatitis A virus. Intervirology. 1982;18(3):107–127. doi: 10.1159/000149314. [DOI] [PubMed] [Google Scholar]
  7. Emini E. A., Dorner A. J., Dorner L. F., Jameson B. A., Wimmer E. Identification of a poliovirus neutralization epitope through use of neutralizing antiserum raised against a purified viral structural protein. Virology. 1983 Jan 15;124(1):144–151. doi: 10.1016/0042-6822(83)90297-0. [DOI] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gust I. D., Coulepis A. G., Feinstone S. M., Locarnini S. A., Moritsugu Y., Najera R., Siegl G. Taxonomic classification of hepatitis A virus. Intervirology. 1983;20(1):1–7. doi: 10.1159/000149367. [DOI] [PubMed] [Google Scholar]
  11. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Higuchi R. G., Ochman H. Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Res. 1989 Jul 25;17(14):5865–5865. doi: 10.1093/nar/17.14.5865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Kammann M., Laufs J., Schell J., Gronenborn B. Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Res. 1989 Jul 11;17(13):5404–5404. doi: 10.1093/nar/17.13.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mead D. A., Skorupa E. S., Kemper B. Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a 'stretched' preproparathyroid hormone. Nucleic Acids Res. 1985 Feb 25;13(4):1103–1118. doi: 10.1093/nar/13.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  17. Mitchell L. G., Merril C. R. Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction. Anal Biochem. 1989 May 1;178(2):239–242. doi: 10.1016/0003-2697(89)90631-3. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scharf S. J., Horn G. T., Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. doi: 10.1126/science.3461561. [DOI] [PubMed] [Google Scholar]
  20. Schold M., Colombero A., Reyes A. A., Wallace R. B. Oligonucleotide-directed mutagenesis using plasmid DNA templates and two primers. DNA. 1984 Dec;3(6):469–477. doi: 10.1089/dna.1.1984.3.469. [DOI] [PubMed] [Google Scholar]
  21. Siegl G., Frösner G. G., Gauss-Müller V., Tratschin J. D., Deinhardt F. The physicochemical properties of infectious hepatitis A virions. J Gen Virol. 1981 Dec;57(Pt 2):331–341. doi: 10.1099/0022-1317-57-2-331. [DOI] [PubMed] [Google Scholar]
  22. Siegl G., Weitz M., Kronauer G. Stability of hepatitis A virus. Intervirology. 1984;22(4):218–226. doi: 10.1159/000149554. [DOI] [PubMed] [Google Scholar]
  23. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vallette F., Mege E., Reiss A., Adesnik M. Construction of mutant and chimeric genes using the polymerase chain reaction. Nucleic Acids Res. 1989 Jan 25;17(2):723–733. doi: 10.1093/nar/17.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  26. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  27. Williams P. M., Williamson K. J., Emerson S. U., Schubert M. Deletion mapping analyses indicate that epitopes for monoclonal antibodies to the NS phosphoprotein of VSV are linear and clustered. Virology. 1988 May;164(1):176–181. doi: 10.1016/0042-6822(88)90634-4. [DOI] [PubMed] [Google Scholar]
  28. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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