Abstract
The polymerase chain reaction (PCR) is a versatile, widely used method for the production of a very large number of copies of a specific DNA molecule (1,2). For some applications, it is advantageous to subclone the PCR product into a plasmid vector for subsequent replication in bacteria (3–6). Subcloning the PCR product into a plasmid vector has several advantages: The amplified fragment can be sequenced with greater reliability, only one allele is sequenced per clone, and the vector containing the PCR product may be used for other molecular biological experiments, e.g., in vitro transcription, transfection, and further amplification in bacteria.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985) Enzymatic amplification of beta-globin genomic sequence and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
Scharf, S. J., Horn, G. T., and Erlich, H. A. (1986) Direct cloning and sequence analyses of enzymatically amplified genomic sequences. Science 233, 1076–1078.
Lee, C. C., Wu, X., Gibbs, R. A., Cook, R. G., Muzny, D. M., and Caskey, C. T. (1988) Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science 239, 1288–1290.
Higuchi, R. (1989) Using PCR to engineer DNA, in PCR Technology (Erlich, H. A., ed.), Stockton, New York, pp. 61–70.
Scharf, S. J. (1990) Cloning with PCR, in PCR Protocols (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic, San Diego, pp. 84–91.
Kaufman, D. L. and Evans, G. A. (1990) Restriction endonuclease cleavage at the termini of PCR products. BioTechniques 9, 304–306.
Shuldiner, A. R., Scott, L. A., and Roth, J. (1990) PCR induced subcloning polymerase chain reaction (PCR) products. Nucleic Acid Res. 18, 1920.
Shuldiner, A. R., Tanner, K., Scott, L. A., and Roth, J. (1991) Ligase-free subcloning: a versatile method of subcloning polymerase chain reaction (PCR) products in a single day. Anal. Biochem. 194, 9–15.
Higuchi, R., Krummel, B., and Saiki, R. K. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acid Res. 16, 7351–7367.
Horton, R. M, Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68.
Del Sal, G., Manfioletti, G., and Schneider, C. (1989) A one-tube DNA mini-preparation suitable for sequencing. Nucleic Acids Res. 16, 9878.
Maas, R. (1983) An improved cloning hybridization method with significantly increased sensitivity for detection of single genes. Plasmid 10, 296–298.
Gussow, D. and Clackson, T. (1989) Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acid Res. 17, 4000.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Shuldiner, A.R., Tanner, K. (2000). Rapid (Ligase-Free) Subcloning of Polymerase Chain Reaction Products. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:295
Download citation
DOI: https://doi.org/10.1385/1-59259-038-1:295
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-459-4
Online ISBN: 978-1-59259-038-4
eBook Packages: Springer Book Archive