Introduction to the Polymerase Chain Reaction
Molecular Biology Lab #9
Background:
Polymerase chain reaction or PCR is a method that has revolutionized molecular biology.
The method was first conceived in the early 1970s but it never became popular due to
two basic problems. First, there was no thermostabile Taq polymerase and second, most
genes had not been sequenced at this time. Thus, it was not possible to perform
automated PCR and there were no known sequences with which to design primers. This
changed by the late 1980s when the thermostabile Taq polymerase was discovered in
bacteria growing in hot springs (Thermus aquaticus). This polymerase was not denatured
by repeated cycles of heating to 94C and it was ideal for automated PCR. A large
number of genes had also been sequenced by the late 1980s.
The standard PCR reaction has several components. The first is the thermostabile DNA
polymerase, usually Taq polymerase. The second component is a pair of synthetic
oligonucleotides to prime DNA synthesis (primers). The proper design of these primers is
perhaps the most critical factor for the success of the PCR reaction. They must bind
specifically to the DNA target, but they should not bind specifically to other sequences,
they should have no secondary structure, and they should not bind to the other primer.
Primers work best if they are about 20 base pairs long and contain equal amounts of the 4
bases. PCR reactions usually are set up to contain an excess of primers. The third
component of the PCR reaction is the deoxynucleotide triphosphates dATP, dCTP,
dGTP, and dCTP, which are also added in excess amounts (200 M). The fourth
component is a divalent cation. All Taq polymerases require divalent cations for activity
and PCR reactions are typically supplemented with magnesium. The amount of Mg+2 is
often 1.5 mM but this concentration can vary widely in different PCR reactions
depending on different factors. The fifth component of the reaction is a buffer to maintain
the pH at 7. Usually Tris buffers are used. The sixth component is a monovalent cation
such as potassium. The final component of the PCR reaction is the template DNA that
contains the target sequence for the reaction. Although PCR should technically work
even if there were only one copy of the target DNA molecule, it actually works much
better when several thousand copies of template DNA are present in the reaction (usually
about 1 g of high molecular weight DNA).
The basic PCR reaction is simply a repetitive process that consists of three main steps.
These include denaturation of the template DNA by heating, annealing of the template
and primers by cooling slightly, and extension of the primers by a thermostabile Taq
DNA polymerase at slightly higher temperature. The first step, denaturation, is usually
carried out at 94-95C which is the highest temperature that the enzyme can survive for
multiple cycles. A higher temperature may be required if the DNA has a higher ratio of
GC. The annealing step allows the primers to hybridize to the target sequence. This
temperature is very critical, about 3C cooler than the melting temperature of the DNA
primer hybrids. If the annealing temperature is too low, you have nonspecific
hybridization and formation of nonspecific bands. If the temperature of annealing is set
too high, you have poor formation of hybrids and little or no product. If the melting
temperature of the primers is unknown, you can perform touchdown PCR (see below).
The third step is extension of the primers at 72-78C, the optimal temperature for the
polymerase to work. The rate of polymerization is about 2000 nucleotides/min, however,
the reaction is usually set up to allow 1 min of polymerization for every 1000 nucleotides
of product. From the third cycle onward, the product delimited by the area between the
two primers is amplified exponentially whereas the larger products from the first two
reactions accumulate arithmetically. The total number of cycles that are required depends
on two factors: how much template was present at the beginning and the efficiency of the
PCR reaction. Once the reaction enters the exponential phase, it continues until one of the
reactants becomes limiting. This usually occurs after about 30 cycles.
Early PCR reactions in the 1980s were performed manually by having the scientist
rapidly switch reaction tubes between three different water baths. Usually each cycle
requires 30-60 seconds depending on product size. This process was tedious and time
consuming. Now, PCR reactions are performed using a programmable thermocycler. This
instrument automatically cycles the temperature of the block containing the reaction
tubes. You simply enter your parameters into the thermocycler and the machine does the
rest. You can relax and enjoy life for an hour or two while the reaction cycles 25-30
times.
However, there can be problems even when using a thermocycler. For example, there are
a number of substances that inhibit PCR reactions. The major culprits are proteinase K
(left over from the DNA extraction) which digests the polymerase. Another problem is
phenol which denatures the polymerase molecule. EDTA is also a problem because it
chelates Mg++ that is needed for the reaction. Finally, the presence of detergents can
inhibit PCR. As is obvious from these sources, most problems with PCR reactions are
due to poor template DNA. If the PCR reaction fails, it is usually possible to make it
work by cleaning up the DNA (ethanol precipitation or dialyzing the sample).
Another major problem with PCR is contamination. This is a problem because the PCR
method has such high sensitivity. If any of your PCR reagents (tips, pipetters, bench top,
tubes, etc) are contaminated with a trace of another template DNA you can easily obtain
the wrong results. One way to guard against this problem is to always use a negative
control consisting of a reaction containing absolutely no target DNA. If you obtain a band
in your PCR with this control, you know that you have problems. At this point it is
probably wise just to throw all of your reagents away and start fresh.
There are several interesting variations on the basic PCR reaction. In multiplex PCR you
set up the reaction using more than just one set of primers. The goal is to amplify several
sequences on a target DNA simultaneously. This saves time and expense. An example of
multiplex PCR would be DNA finger printing with primers for several different regions
of target DNA containing microsatellites. Hot start PCR is another variation where the
polymerase is withheld until the reaction has been heated to a temperature that inhibits
nonspecific priming (sometimes the primers bind DNA nonspecifically at lower
temperature and amplify the wrong sequence). Usually, hot start PCR is not necessary if
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the primers are well designed and the target DNA is abundant. Hot start PCR is useful
when template DNA is limiting, when performing multiplex PCR, and whenever you are
amplifying high molecular weight DNA (due to its sequence complexity). A third form of
PCR is called touchdown PCR. This is not just for sports enthusiasts. It is a good method
for optimization of the PCR reaction when the melting point of the primers is not known
(for example, when amplifying multigene families). In this technique, one sets up a
reaction where the annealing temperature is very high at first (about 3C above the
melting temperature). The temperature of annealing is then reduced one degree for each
subsequent cycle. As the temperature is decreased, it reaches a point where only perfectly
matched primers can bind to the target DNA and make a product. As the temperature is
decreased further in subsequent cycles, the primers can also start to bind nonspecifically.
However, the beauty of the touchdown PCR reaction is that the first products made from
perfect matches are in excess at this point. They effectively prevent the formation of the
poorly matched hybrids because there are so many well-matched templates from the first
several cycles. Finally, there is a new method called real time PCR. In real time PCR, the
results of the reaction are viewed in real time (after each cycle, its possible to monitor the
amount of product). This is particularly useful for experiments that require quantitation of
the PCR product. It allows one to observe the reaction product at early stages, before any
component becomes limiting, and the reaction is still linear. The only drawback is that
real time thermocyclers are very expensive, and many labs can’t afford them.
Primer design:
Specificity is the most important factor in primer design for PCR. The primer should only
hybridize with the target sequence. Primers that are longer than 20 base pairs are usually
unique for just one sequence in the genome. However, it is very important to check to
confirm that your primer does not cross hybridize to other sequences. Primer design is
often performed using commercial or web based software. These programs allow one to
select several different parameters (such as primer length, product length, melting
temperature, etc) to optimize their PCR. The first step is to down load the DNA sequence
of the gene from a data base such as UniGene http://www.ncbi.nlm.nih.gov/UniGene/ .
You can copy and paste this sequence into one of the primer design software packages
such as Primer 3 http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi from
the Whitehead Institute for Biomedical Research. This program allows selection of
specific primer properties that can optimize your reaction. It will furnish several potential
primer pairs in order of best fit. Once you have obtained your primers, the final step is to
check whether they might cross hybridize to other unrelated genes. This can be done by
copying the primer sequence and performing a BLAST search with the BLAST web site
http://www.ncbi.nlm.nih.gov/BLAST/ . Use the Standard nucleotide-nucleotide BLAST.
This may take a minute or two because you are checking your sequence against a very
large number of known sequences. The program will return your query with a set of
potentially matching genes. Many of these are actually different clones of the same gene
or the same gene in a different species.
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Objectives:
The goal of this lab is to introduce students to basic PCR methods and to perform the
PCR reaction with an automated thermocycler. The reaction products will be analyzed by
minigel. The lab will also provide an introduction to primer design using several web
based tools for sequence analysis.
Materials:
Ice water bath
PCR reaction tubes
Forward and reverse primers at 10 nanomoles/ml
PCR Supermix (Taq polymersae at 22 U/ml, 22 mM Tris pH 8.4, 1.65 mM MgCl2, 220
M each of dCTP, dGTP, dATP, and dCTP).
Template DNA (high molecular weight cell DNA)
Micropipetters and yellow barrier tips
Thermocycler
Minigel boxes, combs, and power packs
1X TAE buffer with ethidium bromide (0.5g/ml)
2% agarose in TAE buffer with ethidium bromide (0.5 g/ml)
100 base pair molecular weight markers
6X gel loading buffer
UV box with camera for photography
Computer with Internet access
Methods:
PCR Reaction:
1. Assemble the PCR reaction components in PCR tubes on ice. Add 1 l of
template DNA (high molecular weight cell DNA), 2 l of each forward and
reverse primer, and 45 l of PCR Supermix for a final volume of about 50 l. The
supermix contains premixed reaction components including buffer, Taq
polymerase, deoxynucleotide triphosphates, Mg++ and K+.
2. Include a negative control tube that contains both primers, the PCR Supermix, but
no template DNA.
3. Mix the reaction contents briefly by pipetting.
4. Close the tubes and place in the aluminum block of the thermocycler. Wait for the
other groups to finish loading samples.
5. Turn on the thermocycler by pushing the main power switch. Push the run button
and choose the program craig 001. Push start.
6. Allow the program to finish (approximately 2 hours). The lab instructor will
remove samples and place at 4ºC for analysis during the next lab period.
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Primer Design:
1. Go to the NCI UniGene homepage http://www.ncbi.nlm.nih.gov/UniGene/. Type
in the identifier of the gene for which you need sequence information. Hit go and
then click on the appropriate listing for the gene.
2. Look for the longest and full length mRNA clone. Click on the UniGene number.
3. When the sequence information is displayed, copy the sequence and close
UniGene.
4. Open the program for Primer 3 http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi and paste the sequence information into the box. Hit
the pick primers button and wait for the program to list the best primers.
5. Copy the forward primer first and open the web page for BLAST search
http://www.ncbi.nlm.nih.gov/BLAST/ . Click on the Standard nucleotidenucleotide BLAST. Paste the first primer directly into the box and hit the submit
button. The program may take a few seconds to return your answer because it
searches large data bases. Scroll down to the output and look at the chart listing
similar gene sequences. Pay attention to any genes that are listed in blue (40), as
these might cross hybridize with your primers. Usually you will pick up genes
that are simply clones of the same gene or clones in another species. Repeat this
process with the second primer.
Minigel analysis:
1. Use the microwave to heat the 2% agarose in 1X TAE buffer containing 0.5
g/ml ethidium bromide.
2. Pour the minigel when the agarose is not hot to the touch (after about 10 min in
the 37ºC water bath).
3. Load 20 l of the PCR reaction plus 4 l of 6X gel loading buffer. Load 15 l of
the 100 kb ladder in the last lane.
4. Hook up the electrodes and run the gel at 70 V for 1-2 hours. Stop the gel when
the first blue marker has migrated ½ of the way to the end of the gel.
5.
Examine the gel under UV light to visualize and photograph bands.
6. Clean up your work area and put equipment away!
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