The Polymerase Chain Reaction (PCR)
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:
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buffer
small amount of template DNA
an excess of primer molecules
an excess of each of the four nucleotides
Taq polymerase
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 nongenic 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.
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point mutations A B C D E A F C D E
rearrangements A B C D E A B D C E
insertions A B C D E A B INSERT C D E
delections A B C D E A B E
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.