• Tianyu Hu

      Articles written in Journal of Biosciences

    • 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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      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.

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