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A simple, general procedure for purifying restriction endonucleases

T A Bickle, V Pirrotta, R Imber

    Nucleic Acids Research
    |August 1, 1977
    PubMed
    Summary
    This summary is machine-generated.

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    A straightforward method purifies restriction enzymes using polyethyleneimine precipitation and heparin-agarose affinity chromatography. This technique yields enzymes suitable for DNA sequencing, often requiring minimal additional purification steps.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Enzymology

    Background:

    • Restriction endonucleases are crucial tools in molecular biology for DNA manipulation.
    • Efficient purification of these enzymes is essential for reliable downstream applications like DNA sequencing.
    • Current purification methods can be complex and time-consuming.

    Purpose of the Study:

    • To develop a simple and general method for purifying restriction endonucleases.
    • To assess the efficiency of the proposed method for various restriction enzymes.
    • To determine if the purified enzymes are suitable for demanding applications such as DNA sequencing.

    Main Methods:

    • Nucleic acid precipitation from crude cell extracts using polyethyleneimine (PEI).
    • Affinity chromatography utilizing heparin covalently linked to agarose beads.

    Related Experiment Videos

  • Optional supplementary ion-exchange chromatography for enhanced purity.

    • Main Results:

    • The described method successfully purified most of the sixteen tested restriction endonucleases.
    • Enzymes purified by this method achieved a purity level adequate for DNA sequencing.
    • In most cases, only the PEI precipitation and heparin-agarose chromatography steps were necessary.

    Conclusions:

    • A simple, general, and effective purification strategy for restriction endonucleases has been established.
    • The method is broadly applicable to a range of restriction enzymes.
    • The purified enzymes meet the stringent requirements for DNA sequencing applications, highlighting the method's utility.