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    • Volume 38 Issue 5 December 2013 pp 857-866 Articles

      Realizing directional cloning using sticky ends produced by 3ʹ-5ʹ exonuclease of Klenow fragment

      Guojie Zhao Jun Li Tianyu Hu Hua Wei Yifu Guan

      More Details Abstract Fulltext PDF

      The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5′-3′ polymerase activity can extend the 5′ overhanging sticky end to the blunt end, and 3′-5′ exonuclease activity can cleave the 3′ overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3′-5′ exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5′ overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.

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    • To trigger further research on plant mitochondria, the Journal of Biosciences is bringing out a special issue titled "Plant Mitochondria: Properties and Interactions with Other Organelles".


      Plant mitochondria are quite distinct and have unique features, such as a cyanide-insensitive alternate pathway. They also interact with chloroplasts to optimize photosynthetic carbon assimilation.


      Submissions are welcome until 30 July 2023. The contributions can be original articles, short communications, reviews, or mini-reviews on any topic related to plant mitochondria.


      Authors can submit their articles online at https://www.editorialmanager.com/jbsc/default2.aspx

      Posted on April 12, 2023
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