Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning

PLoS One. 2014 Mar 11;9(3):e90896. doi: 10.1371/journal.pone.0090896. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cloning, Molecular / methods*
  • Computational Biology / methods
  • Deoxyribonucleases, Type II Site-Specific*
  • Internet
  • Nucleotide Motifs
  • Polymerase Chain Reaction*
  • Software

Substances

  • Deoxyribonucleases, Type II Site-Specific

Grants and funding

This work was supported by the Hungarian Scientific Research Fund [K-82090]; the Hungarian National Development Agency [TÁMOP-4.2.2.A-11/1/2012-0052]; and the National Excellence Program – “Elaborating and operating an inland student and researcher personal support system convergence program” [TÁMOP 4.2.4. A/2-11-1-2012-0001 to Eszter Tóth and TÁMOP 4.2.4. A/1-11-1-2012-0001 to Barbara Vodicska]. The project was subsidized by the European Union and co-financed by the European Social Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.