Abstract
The use of polymerase chain reaction (PCR)-based mutagenesis to create chimeric genes is presently not cost-effective because of the size and number of primers as well as the number of PCRs required. We have developed two strategies based on inverse PCR that exploit limited homologies between two DNA molecules to create in-frame chimeric plant viral genes. This report also contains a compilation of information useful for determining possible restriction sites at a given common dipeptide coding motif between any genes of interest.
References
Allison R, Johnston RE, Dougherty WG: The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein. Virology 154: 9–20 (1986).
Atreya CD, Atreya PL, Pirone TP: Selection of deletion mutants by polymerase chain reaction. Biochem Biophys Res Comm 173: 1344–1346 (1990).
Bloch W: A biochemical perspective of the polymerase chain reaction. Biochemistry 30: 2735–2747 (1991).
Clark JM: Novel non-templated nucleotide addition reactions by procaryotic and eucaryotic DNA polymerases. Nucl Acids Res 16: 9677–9686 (1988).
Clarkson T, Winter G: ‘Sticky-feet’ mutagenesis and its application to swapping antibody domains. Nucl Acids Res 17: 10163–10170 (1989).
Domier LL, Franklin KM, Shahabuddin M, Hellman GM, Overmeyer JH, Hiremath ST, Siaw MFE, Lomonossoff GP, Shaw JG, Rhoads RE: The nucleotide sequence of tobacco vein mottling virus RNA. Nucl Acids Res 14: 5417–5430 (1986).
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR: Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68 (1989).
Ochman H, Aijoka JW, Garza D, Hartl DL: Inverse polymerase chain reaction. In: Erlich HA (ed) PCR Technology-Principles and Applications for DNA Amplification, pp. 105–111. Stockton Press, New York (1989).
Saiki R, Scharf S, Faloona F, Mullis KB, Erlich H, Arnheim N: Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354 (1985).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor University Press, Cold Spring Harbor, NY (1989).
Villarreal XC, Long LG: A general method of polymerase chain reaction enabled protein domain mutagenesis: construction of a human protein S-Osteonectin gene. Anal Biochem 197: 362–367 (1991).
Wychowski C, Emerson SU, Silver J, Feinstone SA: 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/polio virus subgenomic cDNA. Nucl Acids Res 18: 913–918 (1990).
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Atreya, C.D., Atreya, P.L. & Pirone, T.P. Construction of in-frame chimeric plant viral genes by simplified PCR strategies. Plant Mol Biol 19, 517–522 (1992). https://doi.org/10.1007/BF00023403
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DOI: https://doi.org/10.1007/BF00023403