Artificial zinc finger nucleases for DNA cloning

Vardit Zeevi, Andriy Tovkach, Tzvi Tzfira

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

5 Scopus citations

Abstract

DNA cloning is fundamental for modern cell research and biotechnology. Various restriction enzymes have been isolated, characterized, and purified to facilitate the digestion and ligation of DNA molecules of different origins. Nevertheless, the very small numbers of enzymes capable of digesting novel and long DNA sequences and the tedious and nearly impossible task of re-engineering existing enzymes with novel specificities greatly limit the use of restriction enzymes for the construction of complex and long DNA molecules. Zinc finger nucleases (ZFNs) - hybrid restriction enzymes that can be tailor made for the digestion of both native and artificial DNA sequences - offer a unique opportunity for expanding the repertoire of restriction enzymes useful for various DNA cloning tasks. Here we present protocols for the assembly, expression, and purification of cloning-grade ZFNs and their use for DNA cloning. We focus our discussion on the assembly of a dual-cassette plant transformation vector, as an example of a task that is nearly impossible to perform using the current collection of naturally occurring and recombinant 6-8 bp long restriction enzymes.

Original languageEnglish
Title of host publicationEngineered Zinc Finger Proteins
Subtitle of host publicationMethods and Protocols
EditorsJoel Mackay, David Segal
Pages209-225
Number of pages17
DOIs
StatePublished - 1 Dec 2010
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume649
ISSN (Print)1064-3745

Keywords

  • Cloning
  • binary vector
  • multigene
  • plant transformation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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