Biotechnologists use what nature already does; there is no reason to reinvent the wheel.
Bacteria defend themselves from viruses by chopping up their DNA.
This process is called restriction because the bacteria are able to restrict the growth and replication of viruses.
The enzymes responsible for doing this are known as restriction enzymes or restriction endonucleases.
The bacteria protect themselves from their own enzymes by adding methyl groups to their DNA (CH3).
Figure 16.1, Purves's Life: The Science of Biology, 7th Edition
Restriction Enzyme Biology
Each restriction enzyme recognizes and cuts at a specific DNA sequence.
Enzymes are named for the bacteria in which they are discovered.
The first restriction enzyme was named EcoR1.
It was isolated from E. coli and hence the name.
Over 100 different restriction enzymes have now been identified.
Many restriction enzymes only cut at sequences that form palindromes.
In biochemistry, a palindrome is a sequence that reads that same 5' → 3' on one strand as it does 5' → 3' (the opposite direction) on the other strand.
Many restriction enzymes also make staggered cuts so they create sticky ends that can bind to other complementary sticky ends.
Part of figure 20.2, page 366, Campbell's Biology, 5th Edition
Two “Miracle” Enzymes and What They Can Do
Two “miracle” enzymes for biotechnology are:
Restriction enzymes
DNA ligase
What they can do:
The same restriction enzyme can be used to cut up DNA from two different organisms.
The sticky ends from the different pieces can then form hydrogen bonds.
DNA ligase can then join the different DNA molecules into a single molecule with covalent bonds.
The result:
DNA from one organism can be transferred to another organism.
This is called recombinant DNA, because two pieces of DNA have been combined creating a new piece of DNA that contains both.
An animation of this process can be found by following this link and choosing Techniques → Cutting and Pasting → “Cutting and Pasting” and “Recombining DNA.”
Create Different Length DNA Fragments and Separate the Fragments
Different pieces of DNA will have different restriction sites.
Even two alleles of the same gene can have different restriction sites!
If the same restriction enzyme is used to cut the two different pieces of DNA, different size DNA fragments will be created.
Example:
Allele 1 has two restriction sites, and therefore if a restriction enzyme is added to allele 1 three different size fragments of DNA will be produced (w, x, and y).
Allele 2 has only one restriction site, and therefore if a restriction enzyme is added to allele 2 two different size fragments of DNA will be produced (y and z).
Separating Different Length DNA Fragments: Gel Electrophoresis
Gel electrophoresis is a technique that separates large molecules based on how quickly they move through an agarose gel under the influence of an electric field.
DNA samples that have been cut up by restriction enzymes are loaded into wells in the gels.
The phosphate backbone of the DNA double helix gives the DNA molecule an overall negative charge.
Therefore, when the power is turned on the DNA moves toward the positive electrode.
The gel provides a matrix through which the DNA fragments move.
The smaller fragments move faster than the larger fragments because they can weave their way through the gel matrix more easily.
An animation of this process can be found by following this link and choosing Techniques → Sorting & Sequencing → “Gel Electrophoresis.”
Figure 16.2, Purves's Life: The Science of Biology, 7th Edition
Technical Considerations
DNA is invisible, so a special blue loading dye that is visible is added.
It is smaller than the smallest DNA fragment and will always win the race through the gel.
This allows researchers to know when to turn off the power supply so the DNA does not run off the gel!
Once the gel has been run it needs to be stained.
The gel is put in a liquid called ethidium bromide.
After the gel has been stained by ethidium bromide, it still cannot be seen!
The stained DNA is, however, now visible under UV light; the ethidium bromide causes the DNA to fluoresce pink.
