Abstract
Molecular cloning has proven to be a powerful tool in biology, and chimeric clones are useful in a variety of fields including microbial pathogenesis and the development of vaccines. Chimeras can be created from DNA by using conventional cloning techniques, specifically restriction cleavage and DNA ligation. Such techniques, however, have limitations; most commonly, limitations result from the lack of restriction sites to provide points of entry for inserts in the desired regions or the multiplicity of restriction sites in other regions of the DNA. Because recombinant DNA molecules may be created during polymerase chain reaction (PCR) when two or more different DNA sequences are brought together (1,2), PCR-mediated recombination has been exploited to join DNA fragments of a few hundred bases (3-8). There are two drawbacks to these methods. First, they often involve multiple steps, and second, sequence errors frequently are introduced by certain thermostable polymerases during the PCR reaction (9,10).
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References
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
Meyerhans, A., Vartanian, J. P., and Wain-Hobson, S. (1990) DNA recombination during PCR. Nucl. Acids Res. 18, 1687–1691.
Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J., and Pease, L. R. (1989) Engineering hybrid gene without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68.
Horton, R. M., Cai, Z. L., Ho, S. N., and Pease, L. R. (1990) Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8, 528–535.
Yolov, A. A. and Shabarova, Z. A. (1990) Constructing DNA by polymerase recombination. Nucl. Acids Res. 18, 3483–3486.
Klug, J., Wolf, M., and Beato, M. (1991) Creating chimeric molecules by PCR directed homologous DNA recombination. Nucl. Acids Res. 19, 2793.
Gu, H., Planas, J., Gomez, R., and Wilson, D. J. (1991) Full length mouse glycoghorin gene constructed using recombinant polymerase chain reaction. Biochem. Biophy. Res. Commun. 177, 202–208.
Sandhu, G. S., Aleff, R. A., and Kline, B. C. (1992) Dual asymmetric PCR: one-step construction of synthetic genes. Biotechniques 12, 14–16.
Eckert, K. A. and Kunkel, T. A. (1991) DNA polymerase fidelity and the polymerase chain reaction. PCR Meth. Appl. 1, 17–24.
Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase-an extremely heat stable enzyme with proofreading activity. Nucl. Acids Res. 19, 4967–4973.
Coffin, J. M. (1995) HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 267, 483–489.
Chun, T. W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J. A., Taylor, H., et al. (1997) Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387, 183–188.
Myers, G. Korber, B., Foley, B., Jeang, K. T., Mellors, J. W., and Wain-Hobson, S. (1996) Human Retroviruses and AIDS. Los Alamos National Laboratory, Los Alamos, NM.
Fang, G., Weiser, B., Visosky, A., Townsend, L., and Burger, H. (1996) Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA. J. AIDS 12, 352–357.
Fang, G., Burger, H., Grimson, R., Tropper, P., Nachman, S., Mayers, D., et al. (1995) Maternal plasma human immunodeficiency virus type 1 RNA level: a determinant and projected threshold for mother-to-child transmission. Proc. Natl. Acad. Sci. USA 92, 12,100–12,104
Salminen, M. O., Koch, C., Sanders-Buell, E., Ehrenberg, P. K., Michael, N. L., Carr, J. K., et al. (1995) Recovery of virtually full-length HIV-1 provirus of diverse subtypes from primary virus cultures using the polymerase chain reaction. Virology 213, 80–86.
Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1–6.
Flaman, J. M., Frebourg, T., Moreau, V., Charbonnier, F., Martin, C., Ishioka, C., et al. (1994) A rapid PCR fidelity assay. Nucl. Acids Res. 22, 3259–3260.
Horton, R. M. (1995) SOEing together tailor-made genes. Mol. Biotech. 3, 93–99.
Fang, G., Weiser, B., Visosky, A., Moran, T., and Burger, H. (1999) PCR-mediated recom-bination: a general method applied to construct chimeric infectious molecular clones of plasma-derived HIV-1 RNA. Nat Med. 5, 239–242.
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Fang, G., Weiser, B., Visosky, A., Moran, T., Burger, H. (2002). PCR-Mediated Recombination. In: Chen, BY., Janes, H.W. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 192. Humana Press. https://doi.org/10.1385/1-59259-177-9:197
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DOI: https://doi.org/10.1385/1-59259-177-9:197
Publisher Name: Humana Press
Print ISBN: 978-0-89603-969-8
Online ISBN: 978-1-59259-177-0
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