 |
|
|
Polymerase Chain Reaction
Polymerase Chain Reaction, better
known as PCR, was developed by a man named Kerry Mullis who received
the Nobel Prize for Chemistry in 1993 because his technique has
made studying DNA so much easier. In short, PCR is a technique
which allows a scientist to make a whole lot of copies of a portion
of a DNA strand starting from just a few DNA molecules. It "amplifies"
the DNA exponentially in just a few hours. PCR became possible
with the discovery of an enzyme found in bacteria which live in
the hydrothermal vents at the bottom of the oceans. The temperature
of the water in these hydrothermal vents is near boiling. Therefore,
their DNA polymerase (the enzyme which copies their DNA) is stable
and functional at very high temperatures. This enzyme is called
Taq polymerase. The DNA polymerases found in other organisms,
such as humans, which do not live in boiling water, are destroyed
at temperatures not far above the organism's normal range. DNA
polymerases are the enzymes which build a "new" strand
of DNA along the "old" strand in DNA replication.
There are basically three steps in
PCR, which are repeated over and over again:
(1) Separation: In this step double-stranded DNA molecules are heated up to 94°C which separates or "denatures" them so that they become single-stranded. These single strands of DNA then become the templates for the new DNA strands to be made upon.
(2) Bind primers: In order for the Taq polymerase to start building a new strand of DNA, it must have something to hook onto. A "primer" is used to start the process. A primer is a short (10 to 20 base-pairs long) piece of DNA which will anneal, or stick, to the DNA template strand where it finds a sequence complementary to its own sequence. Scientists use specific primers which will find sequences that will "flank" the portion of the DNA they are interested in amplifying. In order for the primer to anneal to the template strand, the temperature must be lowered to a temperature which will allow it to stick long enough to start making a new strand. 55°C for 30-60 seconds seems to be optimum.
(3) Extension: During this step of the cycle, the Taq polymerase will extend the primer by bringing in complementary nucleotides as it moves along the template strand. The Taq polymerase works best at temperatures between 72-75°C so the temperature is raised so that the enzyme can work efficiently. All four nucleotides are added to the reaction mixture so that the Taq builds a new strand complementary to the template strand.
When these three steps are repeated
over and over again, the newly made strands are separated from
the template strands by heat. The new strands then go on to serve
as template strands for yet more new strands to be made in each
new cycle. In this way, the number of double-stranded fragments
of DNA grows exponentially. In order to keep the reaction going
for many cycles (usually 40-50) the scientist must plan his/her
reaction carefully.
Into each reaction tube, the scientist
places the following:
Using PCR to Make a "DNA
Fingerprint"
With PCR, a researcher ordinarily chooses two primers, each with a sequence that is specific to the beginning and end of a gene that they wish to study. In this case, the researcher is choosing one, specific segment of DNA that is amplified in the PCR reaction. However, if the scientist used a primer with a sequence that was randomly generated, he/she would be able to amplify regions along the template DNA wherever the primer happened to find its complementary sequence. If this primer was short, say 10 base pairs long, there would be many places along the template DNA molecule where the primer would find its complementary sequence. The locations where the primer would bind would be "random".
The genomes of higher organisms contain
two general types of DNA: genes and non-genic DNA (sometimes called
"junk" DNA because it doesn't code for any protein).
The genes of one individual to another within a species usually
have the same sequence because if a mutation occurs in a gene
it is usually harmful or fatal. When a mutation in a gene occurs
the organism usually doesn't produce offspring so that this change
in the gene "dies" with the organism. In this way, genes
are "conserved" from one individual to another. However,
this is not true of non-genic DNA. Because this DNA does not code
for any proteins, if a mutation occurs here, it doesn't hurt the
organism. It may still live and reproduce at the same rate as
before. Thus, changes in nongenic DNA are not eliminated and are
passed from one generation to another. The net result of this
is that non-genic DNA is highly variable from one individual to
another.
Changes in chromosomes occur quite rarely, but when they do it can happen in a variety of ways.
The changes in chromosomes are called
polymorphisms. If a change affects a primer site,
then the pattern of bands in the DNA profile changes.
Individual A
Individual B
When a scientist uses a short, random
primer some of the amplified regions are probably from genes and
some are probably from non-genic DNA. As a result, some of the
fragments generated by PCR will be the same size and some will
be different from one individual to another. This is because the
fragments generated from genes will probably be similar and the
fragments generated from non-genic DNA will very probably be different.
Unless two individuals are genetically almost identical, each
should generate a different set of fragments of DNA. Using PCR
with short, random primers can provide a "DNA fingerprint"
for each individual.
| Science Education Connection
Department of Biochemistry The University of Arizona May 1, 1997 warder@u.arizona.edu
http://biology.arizona.edu/sciconn/lessons/alongi/ |