restriction enzymes and mapping

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Restriction enzymes identify DNA sequences

Restriction enzymes cut dsDNA

Naming restriction enzymes

Restriction enzymes in mapping

ReBase - restriction enzyme database

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Restriction enzymes identify short DNA sequences
A restriction map shows the positions at which specific short base sequences (i.e. restriction enzyme recognition sites) occur in a DNA molecule. Restriction maps are made using a specific type of endonuclease (an enzyme which cuts DNA within the molecule NOT at the ends!).

All cells contain a range of nucleases, most have fairly broad preferences as to where they will attack nucleic acids. Bacterial cells produce a special type of endonuclease, called restriction endonucleases (REs), which cut double-stranded DNA only at specific, short base sequences.

REs protect bacterial cells from attack by bacteriophages. Phage DNA entering the cell is disabled by RE attack. The cell's own DNA is protected by addition of a methyl group to one of the bases at the site where the endonuclease would cut it. Methylation is carried out by an enzyme called a methylase which matches with the RE present in the cell.

There are three types of RE (Types I, II and III). They are grouped according to their mechanism of action.

Type II REs, more commonly known as restriction enzymes, are the most researched and are used widely in DNA manipulation and analysis. They recognise DNA sequences from 4-16 bp long, depending on the enzyme, and cut between specific bases within this sequence. (The sequence is called the recognition site). The recognised sequence and site of cut is shown for two commonly used restriction enzymes, EcoR1 and Not 1 are shown below.

Two examples of restriction enzyme target sites. The arrows show the positions at which the two DNA strands are cut, the red line shows that for these enzymes, the cut leaves a short section of single-stranded DNA at the end of each cut fragment.

Roll the cursor over the diagram to see the effect of cutting the DNA.

Naming restriction enzymes.

Restriction enzymes are named for the bacterial species and strain from which they were isolated. EcoR 1 was isolated from E. coli strain R and was the 1st restriction enzyme to be isolated from this bacterium.

Not 1 was the 1st restriction enzyme isolated from Nocardia otitidis-cavarium

Details of many more restriction enzymes can be found at the www ReBase site and in molecular biology suppliers catalogues and websites.

Using restriction enzymes in mapping

When DNA from the same source is digested with a particular restriction enzyme it will always give a set of the same sized fragments. For example if lambda bacteriophage DNA is cut with EcoR1 we know that it will give six fragments of the sizes: 21.23, 7.42, 5.8, 5.65, 4.87, 3.53 kbp. This is because, mutations apart, the phage sequence will always be the same, and so EcoR1 cutting sites will always be present in the same places. The fragments can be separated and their sizes determined by agarose gel electrophoresis.

We can use the positions of restriction enzyme sites as convenient markers along DNA sequences. The map obtained can be used for DNA identification and to plan DNA manipulations.

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last updated 01/01