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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
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Abstract

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.

 


Keywords

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

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