3 Biotech

, 8:197 | Cite as

An efficient method for integration of PCR fragments into adjacent or overlapping restriction sites during gene cloning

  • Shivakiran Makam
  • Krupanidhi Srirama
  • Vijaya Ramu Dirisala
  • Prakash Narayana Reddy
Original Article


In the present work, a simple and straightforward method was developed to clone any PCR-amplified products into restriction sites that are very close, adjacent or overlapping in the expression vector. The novelty of the methodology involves a crucial primer-designing step by adding appropriate overhangs to the 5′ ends of primers based on the multiple cloning sites (MCS) (polylinker) region of expression vector. After PCR amplification, actual cloning is performed not in adjacent RE sites, but in sites that are little distant in the MCS. However, the sites lost during this cloning step are maintained intact since they are provided by the cloned PCR product (through the primer overhangs). Gene for green fluorescent protein (GFP) was cloned and expressed employing this strategy to demonstrate its simplicity. This method is highly useful for vector modification without losing the restriction sites present in the MCS.


Cloning Restriction Ligation Multiple cloning sites Green fluorescent protein 



Restriction enzyme


Multiple cloning sites


Green fluorescent protein


Pyrococcus furiosus



The authors thank the administration and management of Vignan’s Foundation for Science, Technology and Research for providing the necessary facilities for undertaking this work. Prakash Narayana Reddy is a DST-INSPIRE Faculty [DST/INSPIRE/04/2017/000565] sponsored by INSPIRE division, Department of Science and Technology, Govt. of India.

Author contributions

SM participated in conceiving and performing the experiments. KS helped in technical discussion, writing the manuscript and proofreading. VRD helped in discussions, correcting the English language and proofreading the manuscript. PNR participated in conceiving, performing the experiments and writing the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interests.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Shivakiran Makam
    • 1
  • Krupanidhi Srirama
    • 1
  • Vijaya Ramu Dirisala
    • 1
  • Prakash Narayana Reddy
    • 1
  1. 1.Department of BiotechnologyVignan’s Foundation for Science, Technology and Research (VFSTR)Guntur DistrictIndia

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