Restriction Endonuclease Abhinandan Chowdhury

What is Restriction Endonuclease? Restriction endonucleases are a family of enzymes that each cut DNA at a specific sequence within the DNA. Alternately called Restriction Enzyme. A Restriction Endonuclease can cut the DNA when it recognizes a specific sequence. These specific sequences are called Restriction Sites.

Restriction endonucleases are found in bacteria Restriction endonucleases are found in bacteria. These enzymes provide a defense against the invading viruses. When a virus infects a bacterium, the bacterium uses it’s restriction endonucleases to cut the viral DNA into pieces and protect itself.

Restriction Endonucleases act as molecular scissors.

Restriction endonucleases were first discovered as agents which ‘restrict’ the biological activity of certain exogenous DNA (i.e. bacteriophage DNA) in some host strains of bacteria but not in other strains. The molecular basis for such restriction is cleavage of DNA by a sequence specific endonuclease.

Features: Cuts in the middle of a DNA sequence. Each enzyme has a specific recognition site in the sequence where it cuts the DNA. A particular enzymes cleaves the DNA at a specific site and nowhere else. i.e. PvuI, cuts DNA only at “CGATCG” this position. PvuII, cuts at a different site, in this case “CAGCTG”.

How is host DNA protected? Host DNA is methylated at Adenine or Cytosine residues in the restriction endonuclease recognition site. So the restriction endonuclease can not cut the DNA strand.

One type of restriction endonucleases simply just cut in the middle of the recognition sequence. Make a simple double-stranded. Resulting in a blunt end.

Other restriction endonucleases cut DNA in a slightly different way Other restriction endonucleases cut DNA in a slightly different way. They do not cut both strands of DNA at exactly the same position. Cleavage is staggered, usually by two or four nucleotides. Resulting DNA fragments have short single-stranded overhangs at each end. These are called sticky or cohesive ends. Base pairing between them can stick the DNA molecule back together again.

List of Restriction Endonucleases and their cutting site

Recombinant DNA Technology Restriction Endonucleases are very useful in recombinant DNA technology. Gene of interest is cut from the foreign DNA using restriction endonuclease. Vector DNA is cut using restriction endonuclease. The gene of interest is inserted into the vector. Thus the gene of interest can be cloned.

Aims of the practical To familiarize students with the process of restriction enzyme digestion of DNA. To analyse these digests by agarose gel electrophoresis. To use data generated by this experiment to verify the relationship between the mobility of DNA fragments during electrophoresis in an agarose gel and their size. To determine the size of restriction fragments generated from λ DNA sample. To verify the restriction map for the λ to DNA.

Precautions: 1. Digests should always be set on ice. Procedure: Protocol for Restriction Digestion (20 µL) Master Mix nuclease-free water 16 µL 10X Buffer EcoRI 2 µL DNA (0.5-1 µg/µL) 1 µL EcoRI or Hind III 0.5-2 µL Mix gently and spin down for a few seconds. Incubate at 37°C for 1-16 hours Add dye & separate fragment on 1% agarose Load 5 µl of uncut λ DNA & markers Write down the sizes and number of fragments you obtain. Explain these with the help of the λ DNA diagram. Precautions: 1. Digests should always be set on ice. 2. Restriction enzyme should be added last (These do not need to be thawed as these are stored at – 200C in glycerol. 3. A separate and sterile tip should always be used for taking up R.E. 4. RE’s are available as dense solution because of the glycerol. Small amounts cannot therefore be pipetted. Master mixes with RE, appropriate buffer and water can be made for more than one reaction.

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