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Generating Nested Deletions with Exonuclease III

  • George Murphy
Protocol
  • 166 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Exonuclease III (Exo III) will digest double-stranded DNA in a 3′ to 5′ direction if the DNA is blunt ended or possesses a 5′ overhang. It will not digest if there is a 3′ overhang of three or more bases, or if the 3′ end has had thiophosphate-containing bases incorporated into it. In order to generate a set of insert deletions using Exo III, it is necessary to cut the polylinker twice with different restriction enzymes so that the cut end nearest the primer site possesses a 3′ overhang, or has thiophosphate residues at the 3′ end, and the end of the polylinker attached to the insert possesses a 5′ overhang or blunt end. If this can be achieved, digestion with Exo III will result in progressive deletion of the 3′ end of the insert, leaving a single-stranded 5′ overhang that can be removed by treatment with mung bean nuclease (1) or exonuclease VII (Exo VII). The blunt ends thus formed are ligated, a suitable host is transformed, and colonies are picked at random and screened for insert size (2), following which a suitable range of inserts is then sequenced.

Keywords

Sodium Acetate Luria Bertani Insert Size Digestion Rate Mung Bean Nuclease 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Henikoff, S. (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351–359.PubMedCrossRefGoogle Scholar
  2. 2.
    Wen-Qin, X. and Potts, M. (1989) Quick screening of plasmid deletion clones carrying inserts of desired sizes for DNA sequencing. Gene Anal. Tech. 6, 17–20.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • George Murphy
    • 1
  1. 1.John Innis Centre for Plant Science ResearchNorwichUK

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