Real Science and Other Adventures

Using restriction enzymes to cut and paste

Restriction enzymes occur naturally in bacteria, but they’re also really useful tools in molecular biology. This is because they cut DNA at known sequences. This tool can be used in many ways.

Previously we looked at the symmetry of the EcoRI recognition site. This diagram shows how EcoRI cuts the DNA (I’ve put the lower strand upside down because I think it shows the symmetry better than the usual way of writing these letters up the right way):

ecori-site-and-cut

 

The DNA is cut between the G and A in both strands, leaving  AATT-overhangs that are only one DNA stand. (I’ve written the dash after the T because that’s where it joins to the double-stranded bit).

sticky-end

and

other-sticky-end

Single-stranded DNA is “sticky”. If it finds a complementary single-strand of DNA the two strands can stick together.

complementary

Any DNA cut with this enzyme has the same “sticky end”, i.e. AATT-. So any piece of DNA cut with EcoRI could potentially join to any other cut with the same enzyme. Another enzyme, a DNA ligase, can be used to seal the join.

This means you can be use restriction enzymes for cutting and joining DNA. This makes them very useful.

Bacterial DNA and mouse DNA that have been cut with EcoRI can be joined together.

mouse-to-human

A circular piece of bacterial DNA

closed-plasmid

can be opened with one cut

open-plasmid

 

and another piece of DNA (that’s been cut with the same enzyme) added

inserting-to-plasmid

 

inserted-dna

This is the process that was used to make human insulin for diabetes patients. A human insulin gene was pasted into a circular bacterial plasmid, and this recombinant DNA plasmid is be grown in massive amounts inside bacteria in the laboratory.

More:

Khan Academy has quite a detailed tutorial on restriction and ligation, including blunt ends and sticky ends.

There’s a more detailed description of how human insulin is produced in the laboratory at the DNA Learning Centre.

 

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This entry was posted on October 5, 2016 by in DNA tools and techniques, genomics, VCE Biology and tagged , , .
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