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Biology SL/HL: Restriction enzymes in gene engineering

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HL Biology: Why might you want to cut a strand of DNA with a restriction enzyme? And what unique feature may be present at the ends of the cut strands?

A restriction enzyme, also known as an endonuclease, is used to cut strands of DNA specifically for gene engineering. The reason for this is that restriction enzymes are known to always cut DNA at particular sequences in a very specific manner. These are known as target sequences.

The unique feature which may be present at the end of cut strands are 'sticky ends'. These are overhangs on the end of DNA chains where you have unpaired nucleotides and which are most commonly created by restriction endonucleases. They are frequently palindromic (meaning they can be read the same forwards as backwards).
An example (from Wikipedia!) would be:

5'-ATCTGACT + GATGCGTATGCT-3'
3'-TAGACTGACTACG CATACGA-5'


You can see that the 'overhang' on the bases matches the shorter ends so that if you joined the whole thing up, it would form a complete double stranded chain of DNA. For instance the GATGC of the blue strand 3' on the top line matches with CTACG (in terms of pairing A-T and C-G) which is the overhang on 3' on the second line.

The importance of this is that you get very SPECIFIC overhangs with SPECIFIC types of endonuclease. In gene engineering you want to know exactly where you are cutting different sections of DNA and also to be able to specifically join them together in order to introduce new genes. Restriction enzymes are therefore extremely useful because they provide a source of specificity in the field of gene engineering.

The syllabus states Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase. You need to be able to do this!

Plasmids are circles of DNA which are present in micro-organisms such as E. Coli, which is a bacterium. Restriction enzymes can be used to cleave and remove sections of the plasmid at particular target sequences. Cleaving the desired DNA from another organism (for instance, DNA that codes for the hormone insulin) using the same restriction enzyme means that you are going to have complementary sticky ends on both this desired DNA and also in the plasmid. You can then add the desired DNA to the plasmid and use an enzyme such as ligase to bind it together - which works, thanks to using these sticky ends. Now you have a plasmid that codes for your desired DNA, in this case insulin! This recombinant plasmid can now be inserted back into an E. Coli which should produce human insulin, which can then be removed, purified and used to help treat human diabetics.

Revision tip!
If you're struggling to understand this, I recommend drawing it out as really it's very simple once you've really grasped the concept. Imagine it all like a stencil - stencils have very specific shapes. You use one stencil to cut piece of paper number 1 and then the same stencil to cut piece of paper number 2. Because you used the same stencil on both of them, they ought to match up when you put them on top of each other. Restriction enzymes essentially just act a bit like stencils, providing a cut which is:
1. Reliably always in the same place and of the same shape
2. Will match up with other things cut with the same stencil (due to sticky ends!)


Any questions please post them here or in the Biology forum.

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