An efficient method for integration of PCR fragments into adjacent or overlapping restriction sites during gene cloning
- 23 Downloads
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.
KeywordsCloning Restriction Ligation Multiple cloning sites Green fluorescent protein
Multiple cloning sites
Green fluorescent protein
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.
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.
- Geiser M, Cebe R, Drewello D, Schmitz R (2001) Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. Biotechniques 31(1):88Google Scholar
- Loukianov E, Loukianova T, Periasamy M (1997) Cloning DNA fragments between two adjacent/overlapping restriction sites using a positive stuffer. Biotechniques 22(5):912–915Google Scholar
- Reddy PK, Shekar A, Kingston JJ, Sripathy MH, Batra H (2013) Evaluation of IgY capture ELISA for sensitive detection of Alpha hemolysin of Staphylococcus aureus without staphylococcal protein A interference. J Immunol Methods 391(1–2):31–38. https://doi.org/10.1016/j.jim.2013.02.004 CrossRefGoogle Scholar