• Logo
  • SBMUJournals

An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

Masoumeh Hajirezaei, Mojtaba Darbouy, Manoochehr Rasouli, Bahram Kazemi




Background: The homologous recombination (HR) is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.

Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1(+) plasmid.

Results: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing.

Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method.



Homologous recombination, Gene cloning, Heat , pcDNA3.1, IgG1


Chames P, Regenmortel MV, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. BJP. 2009;157:220–33.

Frentzena A, Yua YA, Chena N, Zhanga Q, Weibelb S, Raabb V, et al. Anti-VEGF single-chain antibody GLAF-1 encoded by the oncolytic vaccinia virus significantly enhances antitumor therapy. PNAS. 2009;106(31):12915-20.

Fellouse FA, Sidhu SS. In Phage Display in Biotechnology and Drug Discovery. 1st ed. Taylor and Francis Group, Boca Raton, Florida. 2005;709–40.

Nissim A, Chernajovsky Y. Historical development of monoclonal antibody therapeutic. Therapeutic antibodies. Handbook of experimental pharmacology. 2008;181:3-17.

Binyamin L, Borghaei H, Weiner LM. Cancer therapy engineered monoclonal antibodies. Cancer therapeutics. 2006;147-57.

Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood. 1998;91:1644–52.

Rang HP. Pharmacology, Edinburgh: Churchill Livingstone, for the examples infliximab, basiliximab, abciximab, daclizumab, palivusamab, palivusamab, gemtuzumab, alemtuzumab, etanercept and rituximab, and mechanism and mode. 2003;Page 24.

Takahashi T, Yamaguchi S, Chida K, Shibuya M. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO. 2001;20(11):2768-78.

Goldenberg MM. Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. Clin. Ther. 1999;21:309–18.

Trikha M, Yan L, Nakada MT. Monoclonal antibodies as therapeutics in oncology. Curr Opin Biotechnol. 2002;13(6):609-14

Herbst RS, Langer CJ. Epidermal growth factor receptors as a target for cancer treatment: the emerging role of IMC-C225 in the treatment of lung and head and neck cancers. Semin Oncol. 2002;29:27-36.

Prewett M, Rockwell P, Rockwell RF, Giorgio NA, Mendelsohn J, Scher HI, et al. The biologic effects of C225, a chimeric monoclonal antibody to the EGFR, on human prostate carcinoma.J Immunother Emphasis Tumor Immunol. 1996;19:419–27.

Court DL, Sawitzke JA, Thomason LC. Genetic engineering using homologous recombination. Journal of Annual Reviews Genetic. 2002;36:361–88.

Yu D, Ellis H.M, Lee EC, Jenkins NA, Copeland NG, Court DL. An efficient recombination system for chromosome gineering in Escherichia coli. Proc Natl Acad Sci U S A. 2000;97(11):5978-83.

Li MZ, Elledge SJ. Harnessing homologous recombination invitro to generation recombinant DNA via SLIC. Nature Method. 2007;4(3):251-6.

Hajirezaei M, Darbouyi M, Kazemi B. Cloning and expression of the functional human anti-vascular endothelial growth factor (VEGF) using the pcDNA3.1 vector and the human chronic myelogenous leukemia cell line K562. The Protein Journal. 2014;33(1):100-9.

Bateson P. William Bateson: a biologist ahead of his time. Genetics. 2002;81(2):49–58.

Lobo I, Shaw K. Thomas Hunt Morgan, genetic recombination, and gene mapping. Nature Education. 2008;1(1):205.

Creighton HB, McClintock BA. Correlation of Cytological and Genetical Crossing-Over in Zea Mays. Proc Natl Acad Sci U S A. 1931;17(8):492–7.

Heyer WD. Biochemistry of eukaryotic homologous Recombination. Molecular Genetics of Recombination. 2007;17:95-133.

Liu P, Jenkins NA, opeland NG. A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations. Genome Research. 2003;13:476-84.

Myers RS and Stahl FW. Chi and the RecBCD enzyme of Escherichia coli. Annu Rev Genet. 1994;28:49–70.

Sharan SK, Thomason LC, Kuznetsov SG, Court DL. Recombineering: a homologous recombination-based method of genetic engineering. Nat Protocol. 2009;4(2):206-23.

Lee SG, Song JJ, Lee JM, Ha JS. Method for cloning and expressing target gene by homologous recombination. US patent. 2007;0148775 Al.

Oliner JD, Kinzler KW, Vogelstein B. In vivo cloning of PCR products in E. coli. NAR. 1993;21(22):5192-7.

Filippo JS, Sung P, Klein H. Mechanism of Eukaryotic Homologous Recombination. Annual Reviews Biochemistry. 2008;77:229–57.

Gao G, McMahon C, Chen J, Rong YS. A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila. PNAS. 2008;105(37):13999–4004.

Lopez E, Blázquez J. Effect of Sub inhibitory Concentrations of Antibiotics on Intra chromosomal Homologous Recombination in Escherichia coli. AAC. 2009;53(8):3411-5.

Lee DJ, Bingle LEH, Heurlier K, Pallen MJ, Penn CW, Busby SJW et al. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiology. 2009;9:252.

Hanahan D. Studies on transformation of Escherichia coli with plasmids. Molecular Biology. 1983;98:503-17.

Thomason L, Court DL, Bubunenko M, Costantino N, Wilson H, Datta S, et al. Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination. Current Protocols in Molecular Biology. 2007;1:1-16.

DOI: https://doi.org/10.22037/nbm.v3i4.7971