Applied Biochemistry and Biotechnology

, Volume 189, Issue 4, pp 1318–1326 | Cite as

An Efficient Approach for Two Distal Point Site-Directed Mutagenesis from Randomly Ligated PCR Products

  • Jafar Khezri
  • Bagher Yakhchali
  • Ali Asghar Karkhane
  • Mohammad Hossein SangtarashEmail author


Site-directed mutagenesis is one of the most important tools in molecular biology. The majority of the mutagenesis methods have been developed to mutate one region of target DNA in each cycle of mutagenesis, while in some cases there is a need to mutate several distal points. We used a new method to simultaneously mutate two distal points in the target DNA. Different regions of the target DNA were amplified in three separate PCR reactions. The PCR products were back-to-back and together they made the complete length of the template DNA. Mutations were introduced to PCR products by middle mutagenic primers. PCR products were mixed and ligated with random blunt ligation, and then the desired mutated DNA fragments were selected in two steps by flanking restriction enzyme digestion and size selection. Selected fragments were amplified in another PCR reaction using flanking primers and finally cloned into the plasmid vector. This mutagenesis process is simple, there is no need to use modified primers and long or difficult PCR reactions.


Site-directed mutagenesis Random ligation PCR 


Authors’ Contribution

B. Yakhchali, A. A. Karkhane, and M. H. Sangtarash conceived and designed the research. J. Khezri performed experiments. All the authors read and approved the final manuscript.

Funding Information

This study was supported by the National Institute for Genetic Engineering and Biotechnology and the University of Sistan and Baluchestan.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiologyUniversity of Sistan and BaluchestanZahedanIran
  2. 2.Department of Industrial and Environmental BiotechnologyNational Institute for Genetic Engineering and Biotechnology (NIGEB)TehranIran

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