Skip to main content
Log in

A novel three primers PCR (TP-PCR) method to obtain recombinant DNA molecule independent of restriction enzyme

  • Notes
  • Published:
Chinese Science Bulletin

Abstract

In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapidness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Nelson, M. R., Creutz, C. E., Comparison of the expression of native and mutant bovine annexin IV in Escherichia coli using four different expression systems, Protein Exp. Purif., 1995, 6: 132.

    Article  CAS  Google Scholar 

  2. Boylan, M. O. Pelletier, J., Dhepagnon, S., et al., Construction of a fused LuxAB gene by site-directed mutagenesis. J. Biolumin. Chemilumin, 1989, 4: 310.

    Article  PubMed  CAS  Google Scholar 

  3. Mullis, K., Faloona, F., Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction, Methods in Enzymol., 1987, 155: 335.

    Article  CAS  Google Scholar 

  4. Ryan, C. A., Proteinase inhibitor gene families: Strategies for transformation to improve plant defenses against herbivores, Bioassays, 1989, 10: 20.

    Article  CAS  Google Scholar 

  5. Hilder, V. A., A novel mechanism of insect resistance engineered into tobacco, Nature, 1987, 300: 160.

    Article  Google Scholar 

  6. Wandelt, C. I., Khan, M. R. I., Craig, S., et al., Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants, Plant J., 1992, 2(2): 181.

    PubMed  CAS  Google Scholar 

  7. Hammond, R. W., Foard, D. E., Larkins, B. A., Molecular cloning and analysis of a gene coding for the Bowman-Birk protease inhibitor in soybean, J. Biol. Chem., 1984, 259: 9883.

    PubMed  CAS  Google Scholar 

  8. Pelham, H. R. B., The retention signal for soluble proteins of the endoplasmic reticulum, Trends in Biochem. Sci., 1990, 15: 483.

    Article  Google Scholar 

  9. Song, S. I., Kim, C. H., Baek, S. J., et al., Nucleotide sequences of cDNAs encoding the precursors for soybean (Glycine max) trypsin inhibitors (Kunitz type), Plant Physiol., 1993, 101: 1401.

    Article  PubMed  CAS  Google Scholar 

  10. Sambrook, J., Maniatis, T., Fritsch, E. F., Molecular Cloning: A Laboratory Annual, 2nd ed., New York: Cold Spring Harbor Lab., C.S.H., 1989.

    Google Scholar 

  11. Horton, R. M., Hunt, H. D., Ho, S. N., et al., Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension, Gene, 1989, 77: 61.

    Article  PubMed  CAS  Google Scholar 

  12. Takimoto, I., Christensen, A. H., Quail, P. H., et al., Non-systemic expression of a stress-responsive maize polyubiquitin gene (Ubi-1) in transgenic rice plants, Plant Mol. Biol., 1994, 26(3): 1007.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Zhen Zhu.

Additional information

These authors contributed equally to this work.

About this article

Cite this article

Deng, C., Song, G., Xu, J. et al. A novel three primers PCR (TP-PCR) method to obtain recombinant DNA molecule independent of restriction enzyme. Chin.Sci.Bull. 47, 2067–2070 (2002). https://doi.org/10.1360/02tb9448

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1360/02tb9448

Keywords

Navigation