Golden Gate Cloning

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The Golden Gate Cloning (English Golden Gate Assembly ) is a biochemical method of cloning . It was developed by Sylvestre Marillonet to allow high throughput cloning. With the help of type IIs restriction enzymes and T4 DNA ligase , the simultaneous assembly of several DNA fragments in vitro in the correct orientation is possible. The type IIs restriction enzymes used in this method, such as Bsa I , Bsm BI and Bbs I , cut outside of their recognition sequence and can therefore generate non- palindromic , 4 base-pair long overhangs that no longer have any cleavage sites after ligation . Since the restriction cleavage sites are not part of the resulting construct, restriction digestion and ligation can be carried out simultaneously. The method is u. a. used for the production of TALEN for genome editing .

Golden Mutagenesis

The Golden Gate cloning principle can also be used for mutagenesis and is called Golden Mutagenesis. The technique is easy to implement as there is a web tool for primer design .

Individual evidence

  1. ↑ C. Engler, R. Kandzia, S. Marillonet: A One Pot, One Step, Precision Cloning Method with High Throughput Capability . In: PLOS One Volume 3 (11), 2008, e3647, PMID 18985154 .
  2. ^ E. Weber, C. Engler, R. Gruetzner, S. Werner, S. Marillonnet: A modular cloning system for standardized assembly of multigene constructs. In: PloS one. Volume 6, number 2, 2011, p. E16765, doi : 10.1371 / journal.pone.0016765 , PMID 21364738 , PMC 3041749 (free full text).
  3. ↑ C. Engler, R. Kandzia, S. Marillonnet: A one pot, one step, precision cloning method with high throughput capability. In: PloS one. Volume 3, number 11, 2008, p. E3647, doi : 10.1371 / journal.pone.0003647 , PMID 18985154 , PMC 2574415 (free full text).
  4. ^ NE Sanjana, L. Cong, Y. Zhou, MM Cunniff, G. Feng, F. Zhang: A Transcription Activator-Like Effector Toolbox for Genome Engineering . In: Nature Protocols Volume 7 (1), (2012), pp. 171-192. PMID 22222791 .
  5. ↑ Leibniz Institute for Plant Biochemistry : Golden Mutagenesis
  6. ↑ Pascal Püllmann, Chris Ulpinnis, Sylvestre Marillonnet, Ramona Gruetzner, Steffen Neumann: Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design . In: Scientific Reports . tape 9 , no. 1 , July 29, 2019, ISSN  2045-2322 , p. 1–11 , doi : 10.1038 / s41598-019-47376-1 ( nature.com [accessed March 5, 2020]).