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Nucleic Acids Research, 1990, Vol. 18, No. 4 913-918
© 1990

MOLECULAR BIOLOGY

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

Czeslaw Wychowski1,2,*, Suzanne U. Emerson1, Jonathan Silver2 and Stephen M. Feinstone3

1Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases NIH, Bethesda, MD 20892 2Laboratory of Molecular Biology, National Institute of Allergy and Infectious Diseases NIH, Bethesda MD 20892 3Laboratory of Hepatitis Research, Division of Virology CBER/FDA, Bethesda, MD 20892, USA 4Unité de Virologie Moléculaire, UA CNRS 545, Institut Pasteur Pans, France

*To whom correspondence should be addressed

Received October 16, 1989. Revised January 12, 1990. Accepted January 12, 1990.

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 polyrnerase 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 in 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 oligonucieotides 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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