Hi Ted, and thanks for your question.
Restriction sites are the locations within double stranded DNA where
restriction enzymes cleave (cut) the DNA. Some background information
would likely be helpful...
Here's the basic idea:
Viruses can infect bacteria. These viruses, known as phages, insert
their DNA into the bacterium. The DNA is then transcribed to make
RNA, and finally translated to make proteins that comprise a new virus
after being assembled.
As a defense mechanism, bacteria evolved to produce enzymes that could
cut certain sequences of DNA. Of course, the enzymes (or bacteria)
had to evolve such that they wouldn't cut any sequences found in the
bacteria itself, only those that are found in phages. The damaged DNA
would then be unable to be correctly transcribed and the viral life
cycle interrupted.
This was discovered by three molecular biologists, Werner Arber,
Hamilton Smith, and Daniel Nathans in the late 1960's and early
1970's. In 1978, these three scientists were awarded the Nobel Prize
for their work. You can read more here:
http://www.britannica.com/nobel/micro/722_8.html
http://nobelprize.org/medicine/laureates/1978/index.html
As an illustration, the enzyme EcoRI, isolated from E. coli bacteria,
recognizes the sequence (known as the recognition sequence)
5' GAATTC 3'
3' CTTAAG 5'
After cutting with EcoRI, the DNA is separated into two fragments.
The ends look like this:
G
The details of how each type of restriction enzyme cuts the DNA vary.
Some cut straight across the double strand leaving "blunt ends,"
others cut in a "Z" shape, leaving one or more single nucleotides
hanging off the end of each DNA fragment. With the help of a ligase
enzyme, these ends could be rejoined and the DNA would be as it was
before being cut by the restriction enzyme.
There are thousands of different restriction enzymes that have been
identified and isolated. Interestingly, some have been isolated in
so-called thermophilic bacteria that live in high temperature
environments. When using these enzymes, different conditions are used
(higher temperatures).
A bit of terminology: Restriction enzymes are also known as
restriction endonucleases, because they cut within the DNA sequence.
Enzymes that cut at the very end of a DNA sequence are known as
exonucleases.
This naturally occurring phenomenon has been used to advantage in
molecular biology for cloning DNA sequences. The term "cloning" in
this context means isolating and duplicating the sequence. Mixing DNA
with a restriction enzyme under the appropriate conditions will allow
the enzyme to cut the DNA. The appropriate (interesting) fragment of
DNA can be isolated. The "interesting DNA," of course, could be about
anything - maybe it's a gene that is used by cells to make insulin,
maybe it's a gene that makes some protein, etc.
Picking which enzymes to use, which cloning vector to use, etc.,
requires some experience and thought. You need to pick combinations
that will allow you to know what's happening at each step of the
process and check what you're doing. The best way to learn this is to
consult with a local expert and go through a simple experiment to
clone a piece of DNA.
One common way to do this is to run the mixture on an agarose gel,
which separates DNA fragments by size. The researcher will know what
size the fragment should be after cutting with the restriction enzyme
by looking at a map of the full sequence of the DNA that was
originally mixed with the restriction enzyme. Along side the DNA, one
runs a column containing a standard "ladder," which contains fragments
of DNA of known sizes, for reference. Based on the sequence map, the
researcher can see where the restriction enzyme should cut and can
count how many nucleotides should be in the fragment of interest.
The piece of the agarose gel containing the DNA of interest is cut out
of the whole gel. The DNA is purified from this chunk of gel. We can
now take a ring of DNA known as a cloning plasmid (or cloning vector)
and cut it in the same way we cut out original DNA, purify it as
before. Cutting the plasmid with the same restriction enzyme as
before will open the ring into a straight line and give us either
blunt ends or "sticky ends," depending on the specific enzyme we use.
Now we have a small tube of our interesting DNA fragment and another
small tube of cut cloning plasmid. For simplicity, let's say we have
sticky ends, where one or more single nucleotides overhangs the double
strand. Because we used the same enzyme for each tube of DNA, the
ends that are overhanging will be complimentary. I.e., if one end of
our cloning plasmid has a G (Guanine), then our interesting DNA will
have an end with a C (Cystine). G and C normally bond together within
DNA during replication, so these ends will want to join together in
the presence of a ligase enzyme (a joining enzyme) under the proper
conditions.
Here's a simple example:
End of cloning plasmid:
...ATTTCG
...TAAAG
Interesting DNA fragment:
...GCCC
...CGG
The extra G and the extra C want to come together, but generally won't
without the help of a ligase enzyme, which finds complimentary
nucleotides and bonds them together.
In this way, we can insert our interesting DNA fragment into the
cloning plasmid. We would now have a ring of DNA containing our
original cloning plasmid sequence with the sequence of our interesting
DNA inserted into the cloning site.
The University of Michigan has a very nice step-by-step lab page on
this cloning technique using standard lab reagents, which might be a
good initial project to try.
http://www-personal.umd.umich.edu/~mparsons/474/cloning_week1.pdf
===================
Why is this useful?
Now that our interesting DNA is inside the cloning plasmid, it is
under control of the sequences in the plasmid DNA. For example,
cloning plasmids are usually designed to contain DNA sequences that
allow replication of the DNA within bacteria, for example. The
interesting DNA on it's own has no such sequences and would not be
replicated in bacteria.
This means that our new plasmid can be inserted into bacteria, grown
up in a vat of broth and shazam - in the morning we have a big soup of
bacteria, all containing a ring of DNA (plasmid) with our interesting
DNA within the cloning sequence. If we now extract the DNA from the
bacteria, we will have orders of magnitude more DNA than we started
with. This is how a small amount of interesting DNA can be
"amplified" or multiplied to give a much larger quantity for use in
other experiments, etc.
_____________________________
This site from Access Excellence give a very nice overview of
restriction enzymes and restriction sites from the discovery of DNA
through examples of cutting DNA by these enzymes.
http://www.accessexcellence.org/AE/AEC/CC/restriction.html
You can find an online restriction mapper here, which allows you to
paste in your DNA sequence, select the enzyme(s) you wish to digest
with, and then see the results.
http://www.restrictionmapper.org/
One of the largest suppliers of restriction enzymes is New England
Biolabs. They have superb online resources and a very useful catalog,
which I would recommend requesting. Resources include specific
protocols and conditions for using their enzymes. Here's their site:
http://www.neb.com/nebecomm/products/category1.asp?
You can request a free catalog here:
http://www.neb.com/nebecomm/neb_mail_form.asp?
New England Biolabs also has a page of more technical information
about restriction enzymes here, including how to set up a typical
reaction:
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp
You can find sequence maps of many cloning plasmids and other DNA
sequences at NEB's sequence page:
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dna_sequences_maps.asp?
The lower case "p" before the name of a DNA sequence by convention
means that the DNA is in the form of a plasmid (a ring of DNA) as
opposed to a linear strand of DNA. These maps typically show where
various enzymes cut so that you can predict how big fragments would be
and where one's interesting DNA will be inserted. Having such a map
is a requirement for planning a cloning project. You can make a map
such as these using the complete sequence of the cloning plasmid,
which NEB also includes on it's website.
Another good overview can be found at Wikipedia:
http://en.wikipedia.org/wiki/Restriction_enzyme
Another brief look at the use of restriction enzymes in cloning can be
found here, complete with scanned pictures of agarose
(electrophoresis) gels, etc.:
http://www.mtholyoke.edu/courses/sdecatur/chem210/lecturfigs/2900.pdf
Restriction enzymes are also used to identify or differentiate DNA
specimens using restriction fragment length polymorphism analysis
(RFLP). If this is of interest to you, you can read more here:
http://www.cfsan.fda.gov/~frf/rflp.html
http://www.bio.davidson.edu/courses/genomics/method/RFLP.html
Restriction enzymes can also be used for physical sequencing of long
DNA sequences. This is a difficult and somewhat complicated process,
not likely to be of much interest to you at this stage. If you are
interested, you can get an overview here:
http://binf.gmu.edu/jamison/binf732f04/lecture_5.pdf
_____________________________
I hope this information was useful. Please feel free to request any clarification.
-welte-ga |
