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Restriction Enzymes

Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a DNA molecule at a particular place. They are essential tools for recombinant DNA technology. The enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to six nucleotides. Once it finds this recognition sequence, it stops and cuts the strands. This is known as enzyme digestion. On double stranded DNA the recognition sequence is on both strands, but runs in opposite directions. This allows the enzyme to cut both strands. Sometimes the cut is blunt, sometimes the cut is uneven with dangling nucleotides on one of the two strands. This uneven cut is known as sticky ends.

A blunt end may look like this:

blunt ends

A sticky end like this:

sticky ends

Most plasmids used for recombinant technology have recognition sequences for a number of restriction enzymes. This allows a scientist to choose from a number of places to cut the plasmid with a restriction enzyme. Ligation enzymes can then be used to sort of paste in new genomic sequences. These mutated, or recombined, plasmids can then be grown up in bacterial cells and used for a number of purposes, including the addition of genes to mammalian genomes.

You always want to read carefully the information sheet that comes with your enzymes as well as the catalogue information. The better you know your enzyme, the more likely you will be to have a successful digestion.  Most enzymes come in glycerol solution as a storage buffer, but enzymes don't work well in the presence of high glycerol concentration. You want to be sure to dilute the glycerol content down to less than 5% to ensure proper enzymatic activity.

Problems with enzyme activity can occur under the following conditions:

  • High glycerol concentration
  • Enzyme-to-DNA ratio is too high
  • pH is too high
  • Organic solvents, particularly ethanol, interfere with your DNA

Some other helpful tips for working with enzymes include:

  • Wear gloves. This protects you as well as protecting your sample from contamination from you.
  • Keep the enzymes cold.
  • Don't reuse tips. Contamination will ruin your experiment.
  • Know your enzyme. Know what makes it work and what causes problems with it. Know what buffers to use.

 

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