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....
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24618593/pdf/?tool=EBI |
| _version_ | 1830162734532001792 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-17T15:25:36Z |
| format | Article |
| id | doaj.art-be21df070ba745f58af2e1bafcce9e3e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-17T15:25:36Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-be21df070ba745f58af2e1bafcce9e3e2022-12-21T21:43:18ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0193e9089610.1371/journal.pone.0090896Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.Eszter TóthKrisztina HuszárPetra BencsuraPéter István KulcsárBarbara VodicskaAntal NyesteZsombor WelkerSzilvia TóthErvin WelkerThe 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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24618593/pdf/?tool=EBI |
| spellingShingle | 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. PLoS ONE |
| title | 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_short | 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 |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24618593/pdf/?tool=EBI |
| work_keys_str_mv | AT esztertoth restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT krisztinahuszar restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT petrabencsura restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT peteristvankulcsar restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT barbaravodicska restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT antalnyeste restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT zsomborwelker restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT szilviatoth restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning AT ervinwelker restrictionenzymebodydoublesandpcrcloningonthegeneraluseoftypeiisrestrictionenzymesforcloning |