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    • Fulltext

       

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      Permanent link:
      https://www.ias.ac.in/article/fulltext/jbsc/038/05/0857-0866

    • Keywords

       

      Directional cloning; exonuclease; Klenow fragment; sticky end

    • Abstract

       

      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.

    • Author Affiliations

       

      Guojie Zhao1 Jun Li1 Tianyu Hu1 Hua Wei2 Yifu Guan1

      1. Department of Biochemistry and Molecular Biology, Preclinical Medicine College, China Medical University, Shenyang, Liaoning Province, P.R. China
      2. Animal Science and Veterinary Medicine College, Shenyang Agricultural University, Shenyang, China

    • Dates

       
      Published
      December 2013
      Manuscript received
      7 May 2013
      Accepted
      8 August 2013

    • Supplementary Material

       

  • Journal of Biosciences | News

      Forthcoming Special issue.


    • 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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