Restriction enzyme body doubles and PCR cloning: On the general use of Type IIS restriction enzymes for cloning

Eszter Tóth, Krisztina Huszár, Petra Bencsura, Péter István Kulcsár, Barbara Vodicska, Antal Nyeste, Zsombor Welker, Szilvia Tóth, Ervin Welker

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Abstract

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease - a Body Double of the Type IIP enzyme - is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body-double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Original languageEnglish
Article numbere90896
JournalPloS one
Volume9
Issue number3
DOIs
Publication statusPublished - Mar 11 2014

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Tóth, E., Huszár, K., Bencsura, P., Kulcsár, P. I., Vodicska, B., Nyeste, A., Welker, Z., Tóth, S., & Welker, E. (2014). Restriction enzyme body doubles and PCR cloning: On the general use of Type IIS restriction enzymes for cloning. PloS one, 9(3), [e90896]. https://doi.org/10.1371/journal.pone.0090896