Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease

Kazuki Shinomiya, Tomoaki Mori, Yasuhiro Aoyama, Takashi Sera

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1. h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation. Because sandwiched ZFNs can recognize and cleave a variety of long (≥26-bp) target DNAs, they may not only expand the utility of ZFNs for construction of recombinant plasmids, but also serve as useful meganucleases for synthesis of artificial genomes.

Original languageEnglish
Pages (from-to)733-736
Number of pages4
JournalBiochemical and Biophysical Research Communications
Volume414
Issue number4
DOIs
Publication statusPublished - Nov 4 2011
Externally publishedYes

Keywords

  • DNA cloning
  • Meganuclease
  • Sandwiched zinc finger nuclease
  • Single-chain FokI dimer
  • Zinc finger protein

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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