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. 1993 Apr 25;21(8):1889–1893. doi: 10.1093/nar/21.8.1889

Construction of recombinant DNA by exonuclease recession.

Y S Yang 1, W J Watson 1, P W Tucker 1, J D Capra 1
PMCID: PMC309429  PMID: 8388100

Abstract

We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions. This recombinant DNA can be transformed directly into bacteria without a further ligase-dependent reaction. Using this approach, we have constructed recombinant DNA molecules rapidly, efficiently and directionally. This method can effectively replace conventional protocols for PCR cloning, PCR SOEing, DNA subcloning and site-directed mutagenesis.

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Selected References

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

  1. Aslanidis C., de Jong P. J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 1990 Oct 25;18(20):6069–6074. doi: 10.1093/nar/18.20.6069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  4. Hemsley A., Arnheim N., Toney M. D., Cortopassi G., Galas D. J. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 1989 Aug 25;17(16):6545–6551. doi: 10.1093/nar/17.16.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  7. Innis M. A., Myambo K. B., Gelfand D. H., Brow M. A. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. doi: 10.1073/pnas.85.24.9436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jones D. H., Howard B. H. A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles. Biotechniques. 1990 Feb;8(2):178–183. [PubMed] [Google Scholar]
  9. Jones D. H., Sakamoto K., Vorce R. L., Howard B. H. DNA mutagenesis and recombination. Nature. 1990 Apr 19;344(6268):793–794. doi: 10.1038/344793a0. [DOI] [PubMed] [Google Scholar]
  10. Kaluz S., Kölble K., Reid K. B. Directional cloning of PCR products using exonuclease III. Nucleic Acids Res. 1992 Aug 25;20(16):4369–4370. doi: 10.1093/nar/20.16.4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Shuldiner A. R., Scott L. A., Roth J. PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products. Nucleic Acids Res. 1990 Apr 11;18(7):1920–1920. doi: 10.1093/nar/18.7.1920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Stoker A. W. Cloning of PCR products after defined cohesive termini are created with T4 DNA polymerase. Nucleic Acids Res. 1990 Jul 25;18(14):4290–4290. doi: 10.1093/nar/18.14.4290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]

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