Chapter 17. PCR the polymerase
chain reaction and its many
uses
Prepared by Woojoo Choi
Polymerase chain reaction
1) Polymerase chain reaction (PCR): artificial amplification of a DNA
sequence by repeated cycles of replication and strand separation
Fundamentals of PCR
1) We begin with a segment of DNA that we want to amplify in order to
generate many copies.
2) What do we need?
– We need a few molecules of DNA (template) which include the DNA
segment we want to amplify.
Fundamentals of PCR
– We need two PCR primers
• Primers: short segment of DNA that binds to the longer template
strand and allows DNA synthesis to get started
Fundamentals of PCR
– An enzyme to manufacture the DNA copies
• The PCR procedure involves a couple of high temperature steps
so we use a heat resistant DNA polymerase.
• Taq polymerase: heat resistant DNA polymerase
• A supply of nucleotides is needed for the polymerase to use
when making the new DNA.
Fundamentals of PCR
– Finally we need a PCR machine to keep changing the temperature.
• The PCR process requires cycling around through several different
temperatures.
• Thermocycler: machine used to rapidly shift samples between
several temperatures in a pre-set order. Used for PCR
Cycling through the PCR
1) To separate the strands, we start by heating our template DNA to 90℃
or so for a minute or two.
Cycling through the PCR
2) We drop the temperature to around 50℃ to 60℃, allowing the primers
to anneal to the complementary sequences on the template strands.
Cycling through the PCR
3) We maintain the temperature
at 70℃ for a minute or two
to allow the polymerase to
elongate new DNA strands
starting at the primers.
Cycling through the PCR
4) Now repeat the cycle of
events. The second cycle goes
as shown in Figure 17.9.
Cycling through the PCR
5) As we continue to cycle through
the PCR, the single strand
overhang are ignored and are
rapidly outnumbered by
segments of DNA containing
only the target sequence.
6) The cycle is repeated over and
over again.
7) Once past the first two or three
cycles, the vast majority of the
product is double stranded
target sequence with flush ends.
Use of PCR in medical diagnosis
1) PCR can be used in a variety of diagnostic tests.
2) If the DNA sample tested is indeed from another sample of the original
organism, we will get a nice band of DNA of the predicted length (such
as unknown sample No. 2).
3) If the test DNA is not from the same organism, no band will be
generated (such as unknown sample No. 1).
4) We can test DNA by using PCR primers specific for sequences found only
in the genes of virus or bacteria (AIDS, Mycobacterium).
Use of PCR in medical diagnosis
Use of PCR in medical diagnosis
5) It is possible to identify an organism from an extremely small trace of
DNA-containing material.
Degenerate primers
1) Degenerate primers: primer
with several alternative bases
at certain positions
2) They are used when there is
some information but no
complete sequence to go on.
3) We make degenerate DNA
primers that have a mixture
of all possible bases in every
third position.
4) A degenerate primer is
actually a mixture of closely
related primers.
5) Many segments of DNA have
been PCR’ed successfully by
using sequence data from
close relatives.
Inverse PCR
1) To generate DNA by PCR, we need two regions of known sequence, for
binding primers on either side of the unknown target sequence
2) However, the present situation is exactly the opposite of that.
Inverse PCR
3) Inverse PCR is a more sneaky way than degenerate primers.
– First, we convert our target molecule of DNA into a circle
• If we go around a circle, we eventually get back to where we
started.
• To make circle, choose a restriction enzyme that recognizes a sixbase sequence.
Inverse PCR
– Then, use two primers corresponding to the known region and facing
outwards around the circle
4) Overall, the PCR reaction gives multiple copies of a chunk of DNA, we
want to explore, containing some DNA to the right and left of our
original known region.
Random Amplified Polymorphic DNA (RAPDs)
1) Random amplified polymorphic DNA (RAPD)
– method for testing generic relatedness using PCR to amplify
arbitrarily chosen sequence
– The purpose is to test how closely related two organisms are.
– The principle is statistically based.
– For example, to find any particular five-base sequence, probability is
one of every 45 stretches of five bases.
Random Amplified Polymorphic DNA (RAPDs)
2) For RAPDs, we do not want to be unique, just rare.
– We make PCR primers with our arbitrarily chosen sequence and run a
PCR reaction using the total DNA of our organism as a template.
– Every now and then our primers will find a correct match, purely by
chance, on the template.
Random Amplified Polymorphic DNA (RAPDs)
– For PCR to happen, we need two such sites facing each other on
opposite strands of the DNA.
– We also need the sites to be more than a few thousand bases apart
for the reaction to work well.
– The likelihood of two correct matches in this arrangement is quite
low.
Random Amplified Polymorphic DNA (RAPDs)
3) We repeat this several times with primers of different sequence.
4) The result is a diagnostic pattern of bands that will vary in different
organisms, depending on how closely they are related.
Adding artificial restriction sites
1) To clone something we need convenient cut sites for restriction enzymes.
2) We are unlikely to find such sites just at the ends of our PCR fragment.
3) In order to overcome problems, we add artificial restriction enzyme cut
sites at the far end of the primers.
4) This allows us to cut the PCR fragment with the restriction enzyme, and
then clone it into a convenient plasmid.
PCR in genetic engineering
1) Changing one or two bases of a DNA sequence
– If we want to alter the T in the middle to an A, we simply make a
PCR primer with the base alteration.
– Using this mutant primer in PCR, the DNA product will incorporate
the change we made in the primer.
Primer with base alteration:
AAG CCG GTG GCG CCA
AAG CCG GAG GCG CCA
PCR in genetic engineering
2) Rearranging large stretches of DNA
– Making a hybrid gene
– The crucial point is that we use an overlap primer that matches part
of both gene segments.
Reverse transcriptase PCR
1) If we clone the DNA from higher organism and put into a bacterial cell,
the gene will not be expressed properly because the RNA will not be
processed and the introns will not be cut out.
2) It would be nice to get a copy of the gene without the introns (mRNA).
3) If we obtain the mRNA, we have an intron-free sequence.
Reverse transcriptase PCR
4) To convert RNA to DNA, we use
reverse transcriptase.
– Reverse transcriptase: enzyme
that starts with RNA and
makes a DNA copy of the
genetic information
5) RT-PCR: combination of reverse
transcriptase with PCR which
allows DNA copies to be
manufactured in bulk from mRNA
– Complementary DNA (cDNA):
the DNA sequence
complementary to an RNA
sequence,
Reverse transcriptase PCR
6) RT-PCR has other uses, transcriptome.
7) There will be many more copies of the mRNA in the cell than of the
original gene.
Reverse transcriptase PCR
8) Carrying out RT-PCR on an
organism under different
growth conditions, we can see
when the gene under scrutiny
was switched on.
9) If this gene was expressed we
will get a PCR band, whereas if
the gene was switched off, no
band will be generated.
10)We can tell which
environmental factors bring
about expression of our
favorite gene.
Differential display PCR
1) It is used to specifically amplify mRNA from eukaryotic cells.
2) This technique is a combination of RAPDs with RT-PCR and uses oligo-dT
primers.
3) After making cDNA, we run a PCR reaction with two primers
① An oligo-dT primer that binds to the 3’ end of all DNA copies of
mRNA
② As we do not know the sequences at the other end of the mRNAs,
our second primer is actually a mixture of random primers similar to
those used in RAPDs.
4) The result is that we end up with lots of DNA corresponding to each of
mRNA molecules in the original mixture.
Real time PCR
1) Real time PCR: PCR where the
synthesis of new DNA is
monitored directly by using a
fluorescent probe
2) Fluorescence indicates the
amount of PCR product
produced.
3) To be sure that the correct
target DNA was amplified, more
sophisticated fluorescent probes
can be made that are specific
for a particular sequence of
DNA probe attached.
4) Only when the DNA probe
binds to the correct target DNA
does the fluorescence increase.
Jurassic park PCR
1) Some scientists have looked for DNA in fossils.
2) DNA enough to yield valuable information have been extracted from
museum specimens and fossils.
3) The problem lies in the preservation of the fossil DNA.
4) The older the fossil, the more decomposed the DNA will be.
5) Normal rates of decay should break up DNA into fragments less than
1,000 bp long in 5,000 years or so.