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  • DNA Digestion

    Molecules of DNA can be hundreds of thousands of basepairs long, so to efficiently work with them, it is oftentimes necessary to cut the DNA into smaller, more manageable lengths. This cutting of DNA is called digestion, as it is carried out using enzymes. Known as restriction endonucleases, these enzymes are highly specialized and digest DNA at a specific sequence of nucleotides. This stretch of DNA is known as a recognition sequence. When the enzyme encounters this sequence, it hydrolyzes the phosphodiester backbone of the DNA. The exact point within the recognition sequence at which the phosophodiester backbone is cleaved is known as the restriction site. Each enzyme recognizes a specific recognition sequence and this inherent specificity allows for exquisitely fine-tuning of DNA products. While each enzyme recognizes a specific sequence, it is possible for multiple enzymes to recognize the same site. Different enzymes that recognize the same site are called isoschizomers. Currently, over 4000 restriction endonucleases have been identified. Of these, greater than 600 are commercially available for laboratory use. Restriction endonucleases can be further delineated by their cut position, cofactor requirements, and modification preference: Types I, II, III, and IV. Type II enzymes are the most commonly-used for molecular biology, as they recognize the canonical 4-8 nucleotide sequence, cut at a reliable position within that site, and requires only magnesium as a cofactor. Unlike other restriction enzymes, Type IV enzymes preferentially cleave methylated DNA and have become useful as tools for epigenetics.

    1. Standard Protocol for Restriction Enzyme Digests

      Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.

    2. Why is My Restriction Enzyme Not Cutting DNA?

      Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.

    3. Restriction Enzyme Digest Problem: Too Many DNA Bands

      Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.

    4. What is Restriction Enzyme Star Activity?

      Learn what Star Activity is, why it is detrimental to accurate restriction enzyme digestion, and how NEB's HF enzymes are engineered to avoid it.

    5. Setting-up Restriction Enzyme Digests with RE-Mix Master Mixes

      RE-Mix® Restriction Enzyme Master Mixes offer simplified reaction setup. Learn more about digesting DNA with RE-Mix.

    6. RE-Mix Double Digest Protocol

      Double digests are now easier than ever! Learn how to set up your next double digest with RE-Mix®.

    7. Reduce Star Activity with High-Fidelity Restriction Enzymes

      NEB has engineered HF® enzymes to eliminate star activity. Learn how, and what this means for your digests.

    8. NEB Restriction Enzyme Double Digest Protocol

      Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using.

    9. Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

      Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.

    10. Restriction Enzyme Digest Protocol: Cutting Close to DNA End

      When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.

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    DNA Digestion includes these areas of focus:

    Restriction Enzyme Digestion
    DNA Nicking

    Protocols for DNA Digestion