Improvement of TA Cloning Method to Facilitate Direct Directional Cloning of PCR Products

Abstract:

Directional cloning is a prerequisite for the construction of expression vectors in molecular biology laboratories. Although TA cloning is widely used to clone unmodified PCR (polymerase chain reaction) products, a major disadvantage of this technique is that cloning is not directional. Here we reported a novel PCR products cloning vector with one deoxythymidine overhang and one deoxycytidine overhang at two 3'-ends respectively. With the choice of nucleotides of 5'-ends of PCR primers, PCR products can be cloned to this vector both directly and directionally. The feasibility and efficacy of this cloning method were confirmed by using a pET-17b derivative vector and a green fluorescent protein gene (EGFP) and a red fluorescent protein reporter (Ds-Red) gene. This cloning strategy may be useful in the high-throughput construction of expression vectors and could be viewed as an interesting improvement of existing TA cloning method.

• Periodical: Applied Mechanics and Materials (Volume 565)
• Pages: 3-8
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.565.3
• Online since: June 2014
• Authors: Ji Gang Li*, Guo Ying Han, Xiu Min Li, Jiao Jiao Sun, Ke Jing Song, Ting Zhang
• Keywords: Direct Directional Cloning, Expression Plasmid Vector Construction, Polymerase Chain Reaction, Terminal Transferase, Thermostable DNA Polymerase

References

[1] D.A. Mead, N.K. Pey, C. Herrnstadt, R.A. Marcil, L.M. Smith, A universal method for the direct cloning of PCR amplified nucleic acid. Biotechnology (N Y), 9 (7): 657-663, (1991).

DOI: 10.1038/nbt0791-657

[2] J.M. Clark, Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Research, 16 (20): 9677-9686, (1988).

DOI: 10.1093/nar/16.20.9677

[3] G. Hu, DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biology, 12 (8): 763-770, (1993).

DOI: 10.1089/dna.1993.12.763

[4] V.L. Magnuson, D.S. Ally, S.J. Nylund, Z.E. Karanjawala, J.B. Rayman, J.I. Knapp, A.L. Lowe, S. Ghosh, F.S. Collins, Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. Biotechniques, 21 (4): 700-709, (1996).

DOI: 10.2144/96214rr03

[5] M.J. Brownstein, J.D. Carpten, J.R. Smith, Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques, 20 (6): 1004-1006, 1008-1010, (1996).

DOI: 10.2144/96206st01

[6] D. Esposito, L.A. Garvey, C.S. Chakiath, Gateway cloning for protein expression. Methods in molecular biology, 498: 31-54, (2009).

DOI: 10.1007/978-1-59745-196-3_3

[7] D. Horn, Directional enrichment of directly cloned PCR products. Biotechniques, 39 (1): 40, 42, 44, 46, (2005).

DOI: 10.2144/05391bm03

[8] Q. Wu, X. Zhong, C. Zhai, J. Yang, X. Chen, L. Chen, W. Wang, L. Ma, A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products. Biotechnology letters, 32 (3): 439-443, (2010).

DOI: 10.1007/s10529-009-0166-3

[9] R.H. Peng, A.S. Xiong, J.G. Liu, F. Xu, C. Bin, H. Zhu, Q.H. Yao, Adenosine added on the primer 5' end improved TA cloning efficiency of polymerase chain reaction products. Analytical Biochemistry, 363 (1): 163-165, (2007).

DOI: 10.1016/j.ab.2007.01.014