title: Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.
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.
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Eszter
authors.initials: E
authors.lastname: Tóth
authors.affiliation: Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Huszár
authors.affiliation: Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Petra
authors.initials: P
authors.lastname: Bencsura
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Péter István
authors.initials: PI
authors.lastname: Kulcsár
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Barbara
authors.initials: B
authors.lastname: Vodicska
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Antal
authors.initials: A
authors.lastname: Nyeste
authors.affiliation: Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Zsombor
authors.initials: Z
authors.lastname: Welker
authors.affiliation: Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Szilvia
authors.initials: S
authors.lastname: Tóth
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary; Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Ervin
authors.initials: E
authors.lastname: Welker
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D015252:Deoxyribonucleases, Type II Site-Specific; D020407:Internet; D059372:Nucleotide Motifs; D016133:Polymerase Chain Reaction; D012984:Software
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific