Handling DNA

Restriction endonuclease digestion of DNA

Many applications require conversion of genomic DNA into conveniently sized fragments by restriction endonuclease digestion. This yields DNA fragments of a convenient size for downstream manipulations. Restriction endonucleases are bacterial enzymes that bind and cleave DNA at specific target sequences. Type II restriction enzymes are the most widely used in molecular biology applications. They bind DNA at a specific recognition site, consisting of a short palindromic sequence, and cleave within this site, e.g., AGCT (for AluI), GAATTC (for EcoRI), and so on. Isoschizomers are different enzymes that share the same specificity, and in some cases, the same cleavage pattern. 

Tip: Isoschizomers may have slightly different properties that can be very useful. For example, the enzymes MboI and Sau3A have the same sequence specificities, but MboI does not cleave methylated DNA, while Sau3A does. Sau3A can therefore be used instead of MboI where necessary.

The following factors need to be considered when choosing suitable restriction enzymes:

  • Fragment size 
  • Methylation sensitivity 
  • Blunt-ended/sticky-ended fragments 
  • Compatibility of reaction conditions (where more than one enzyme is used)

Restriction enzymes with shorter recognition sequences cut more frequently than those with longer recognition sequences. For example, a 4 base pair (bp) cutter will cleave, on average, every 44 (256) bases, while a 6 bp cutter cleaves every 46 (4096) bases.

Tip: Use 6 bp cutters for mapping genomic DNA or YACs, BACs, or P1s, as these give fragments in a suitable size range for cloning.

Many organisms have enzymes called methylases that methylate DNA at specific sequences. Not all restriction enzymes can cleave their recognition site when it is methylated. Therefore the choice of restriction enzyme is affected by its sensitivity to methylation. In addition, methylation patterns differ in different species, also affecting the choice of restriction enzyme.

  • The CpG dinucleotide occurs about 5 times less frequently in mammalian DNA than would be expected by chance, and most restriction enzymes with a CpG dinucleotide in their recognition site do not cleave if the cytosine is methylated. Therefore many enzymes with CpG in their recognition site, such as EagI, NotI, and SalI, cleave mammalian DNA only rarely. 
  • Drosophila, Caenorhabditis, and some other species do not possess methylated DNA, and have a higher proportion of CpG dinucleotides than mammalian species. Rare-cutter enzymes therefore cleave more frequently in these species. 
  • Plant DNA is highly methylated, so for successful mapping in plants, choose enzymes that either do not contain a CpG dinucleotide in their recognition site (e.g., DraI or SspI) or that can cleave methylated CpG dinucleotides (e.g., BamHI, KpnI, or TaqI). 

Tip: Methylation patterns differ between bacteria and eukaryotes, so restriction patterns of cloned and uncloned DNA may differ. 

Tip:
Methylation patterns also differ between different eukaryotes (see bullets above), affecting the choice of restriction enzyme for construction genomic DNA libraries.

Some restriction enzymes cut in the middle of their recognition site, creating blunt-ended DNA fragments. However, the majority of enzymes make cuts staggered on each strand, resulting in a few base pairs of single-stranded DNA at each end of the fragment, known as “sticky” ends. Some enzymes create 5' overhangs and others create 3' overhangs. The type of digestion affects the ease of downstream cloning:

  • Sticky-ended fragments can be easily ligated to other sticky-ended fragments with compatible single-stranded overhangs, resulting in efficient cloning. 
  • Blunt-ended fragments usually ligate much less efficiently, making cloning more difficult. However, any blunt-ended fragment can be ligated to any other, so blunt-cutting enzymes are used when compatible sticky-ended fragments cannot be generated – for example, if the polylinker site of a vector does not contain an enzyme site compatible with the fragment being cloned.
If a DNA fragment is to be cut with more than one enzyme, both enzymes can be added to the reaction simultaneously provided that they are both active in the same buffer and at the same temperature. If the enzymes do not have compatible reaction conditions, it is necessary to carry out one digestion, purify the reaction products, and then perform the second digestion.
  • Water 
  • DNA 
  • Buffer 
  • Enzyme 

The amount of DNA digested depends on the downstream application and the genome size of the organism being analyzed. We recommend using a minimum of 10 µg DNA per reaction for Southern blotting of mammalian and plant genomic DNA. For mapping of cloned DNA, 0.2–1 µg DNA per reaction is adequate. 

Tip: DNA should be free from contaminants such as phenol, chloroform, ethanol, detergents, or salt, as these may interfere with restriction endonuclease activity.

One unit of restriction endonuclease completely digests 1 µg of substrate DNA in 1 hour. However, supercoiled plasmid DNA generally requires more than 1 unit/µg to be digested completely. Most researchers add a 10-fold excess of enzyme to their reactions in order to ensure complete cleavage.

Tip: Ensure that the restriction enzyme does not exceed more than 10% of the total reaction volume; otherwise the glycerol in which the enzyme is supplied may inhibit digestion.

Most digests are carried out in a volume between 10 and 50 µl. (Reaction volumes smaller than 10 µl are susceptible to pipetting errors, and are not recommended.)
  1. Pipet reaction components into a tube and mix well by pipetting. 

    Tip:
    Thorough mixing is extremely important. 

    Tip: The enzyme should be kept on ice and added last. 

    Tip: When setting up large numbers of digests, make a reaction master mix consisting of water, buffer, and enzyme, and aliquot this into tubes containing the DNA to be digested.

  2. Centrifuge the tube briefly to collect the liquid at the bottom. 
  3. Incubate the digest in a water bath or heating block, usually for 1–4 h at 37°C. However, some restriction enzymes require higher (e.g., 50–65°C) while others require lower (e.g., 25°C) incubation temperatures.
  4. For some downstream applications it is necessary to heat-inactivate the enzyme after digestion. Heating the reaction to 65°C for 20 min after digestion inactivates the majority of enzymes that have optimal incubation temperature of 37°C. Note that some restriction enzymes are not fully inactivated by heat treatment.
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