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

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....

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Autores principales:	Eszter Tóth, Krisztina Huszár, Petra Bencsura, Péter István Kulcsár, Barbara Vodicska, Antal Nyeste, Zsombor Welker, Szilvia Tóth, Ervin Welker
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/be21df070ba745f58af2e1bafcce9e3e
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spelling	oai:doaj.org-article:be21df070ba745f58af2e1bafcce9e3e2021-11-18T08:28:47ZRestriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.1932-620310.1371/journal.pone.0090896https://doaj.org/article/be21df070ba745f58af2e1bafcce9e3e2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24618593/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Eszter TóthKrisztina HuszárPetra BencsuraPéter István KulcsárBarbara VodicskaAntal NyesteZsombor WelkerSzilvia TóthErvin WelkerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 3, p e90896 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Eszter Tóth Krisztina Huszár Petra Bencsura Péter István Kulcsár Barbara Vodicska Antal Nyeste Zsombor Welker Szilvia Tóth Ervin Welker Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.
description	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.
format	article
author	Eszter Tóth Krisztina Huszár Petra Bencsura Péter István Kulcsár Barbara Vodicska Antal Nyeste Zsombor Welker Szilvia Tóth Ervin Welker
author_facet	Eszter Tóth Krisztina Huszár Petra Bencsura Péter István Kulcsár Barbara Vodicska Antal Nyeste Zsombor Welker Szilvia Tóth Ervin Welker
author_sort	Eszter Tóth
title	Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.
title_short	Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.
title_full	Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.
title_fullStr	Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.
title_full_unstemmed	Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.
title_sort	restriction enzyme body doubles and pcr cloning: on the general use of type iis restriction enzymes for cloning.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/be21df070ba745f58af2e1bafcce9e3e
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Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

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