PCR 2

Polymerase Chain Reaction ( PCR )
What is PCR?
Is an ingenious technology in molecular biology to amplify a single or
a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA sequence
in vitro. This PCR process, invented by Kary Mullis in 1984 in
California. The purpose of a PCR is to make a huge number of copies of
a gene. The invention of the PCR (Polymerase Chain Reaction) technique
has resulted in a revolution for evolutionary biologists interested in
genetic questions. Suddenly there was a fast, robust and relatively
inexpensive technique to get hold of genetic information from small
samples of e.g. skin, blood or faeces. An advantage with PCR based
molecular studies of DNA is that, once DNA is extracted and purified, the
techniques are very similar regardless of the taxonomy of the study
organisms.
The technique was made possible by the discovery of Taq polymerase,

the DNA polymerase that is used by the bacterium (Thermus aquaticus)
that was discovered from Yellow Stone National Park hot springs. This
DNA polymerase is stable at the high temperatures need to perform the
amplification, whereas other DNA polymerases become denatured.
The method relies on thermal cycling instrument(thermal cycler),

which provide program consisting of cycles of repeated heating and
cooling of the reaction for DNA melting and enzymatic replication of the
DNA.
Applications of PCR:
1- Medicine: The PCR technique enables early diagnosis of malignant
diseases.
2- Classification of organisms .
3- Mutation detection .
4- Detection of pathogens .
5- Gene therapy.
6- Finger print .
7- Forensic science: PCR is very important for the identification of criminal
1
8-Evolutionary studies: It plays an important role in phylogenetic
analysis.
Requirements of PCR Technology
1- The thermal cycler heats and cools the reaction tubes to achieve the
temperatures required at each step of the reaction.
2- DNA template that contains the DNA region (target) to be amplified, the
source of DNA for the PCR amplification. We use a standard
concentration at 25ng/μl. DNA purity is very important, because template
contaminants (i.e. excesses of phenolic compounds, EDTA) may leads to
PCR inhibition and give false-negative results.
3- Pair of primers: short artificial DNA fragments containing sequences
complementary to the target region, that are complementary to the 3'
(three prime) ends of each of the sense(forward) and the 5' end of the
anti-sense (reveres) strand of the DNA target, usually the length of 18-30
nt.
4- Master Mix Which Contains:
 Taq DNA polymerase the enzyme that puts the free nucleotides together.
It starts at the 3’end of the primer, and uses the complementary DNA strain
as a template.
 Deoxynucleoside triphosphates (dNTPs, sometimes called
"deoxynucleotide triphosphates"; nucleotides containing triphosphate
groups), Free nucleotides (G, A, T,C) of which the artificial DNA copies
are made, the building-blocks from which the DNA polymerase synthesizes
a new DNA strand.
 Buffer solution, maintains pH and ionic strength of the reaction solution
suitable for the activity of the enzyme.
 Mg++ ions - cofactor of the enzyme.
5- free nuclease water: must be present for the reaction to work.
Procedure:
Typically, PCR consists of a series of 25-40 repeated temperature
changes, called cycles, each cycle of PCR includes steps for template
denaturation, primer annealing and primer extension:
2
 Initialization step: This step consists of heating the reaction to a
temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases
are used), which is held for 1–9 minutes. It is only required for DNA
polymerases that require heat activation by hot-start PCR.
 Denaturation step: This step is the first regular cycling event and consists
of heating the reaction to 94–98 °C for 30sec.-1min. It causes DNA
melting of the DNA template by disrupting the hydrogen bonds between
complementary bases, yielding single-stranded DNA molecules.
 Annealing step: The reaction temperature is lowered to 50–65 °C for 30
sec-1min allowing annealing of the primers to the single-stranded DNA
template. Typically, the annealing temperature is about 3-5 degrees
Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen
bonds are only formed when the primer sequence very closely matches the
template sequence. The polymerase binds to the primer-template hybrid
and begins DNA formation.
 Extension/elongation step: The temperature at this step depends on the
DNA polymerase used; Taq polymerase has its optimum activity
temperature at 75–80 °C, and commonly a temperature of 72 °C is used
with this enzyme. At this step the DNA polymerase synthesizes a new
DNA strand complementary to the DNA template strand by adding dNTPs
that are complementary to the template in 5' to 3' direction, condensing the
5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of
the nascent (extending) DNA strand. The extension time depends both on
the DNA polymerase used and on the length of the DNA fragment to be
amplified. As a rule-of-thumb, at its optimum temperature, the DNA
polymerase will polymerize a thousand bases per minute. Under optimum
conditions, i.e., if there are no limitations due to limiting substrates or
reagents, at each extension step, the amount of DNA target is doubled,
leading to exponential (geometric) amplification of the specific DNA
fragment.
 Final elongation: This single step is occasionally performed at a
temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to
ensure that any remaining single-stranded DNA is fully extended.
3
PCR Steps ：
• Initialization step ：94℃ 10min

• 35 Cycles：
Denaturation step 94℃ 30s
Annealing step 55℃ 30s 1 cycle
Extension step 72℃ 30s
• Final elongation ： 72℃ 10min
IMPORTANT REMINDERS:
 ALWAYS keep PCR reagents (master mix, primers, DNA template on

ICE.
 Gloves are good to use to keep your tubes from getting contaminated.
 Keep your tubes closed and make sure you do not cross contaminate with
the tips of the micropipettes.
 VERY IMPORTANT when using the micropipettes be careful and avoid
contaminating the micropipettes by slowly releasing the plunger.
 PCR product(amplicon) is separated in agarose and the result is examined
via gel electrophoresis
4
Why “Polymerase”? It is called “polymerase” because the only enzyme
used in this reaction is DNA polymerase.
Calculating annealing temperature (Tm) of a primer:
Tm = 4(G + C) + 2(A + T)
where G,C,A,T are the number of respective nucleotides. This formula can
be used for primers that are no longer than 25 nt long
Preparation of primers
Primers were provided in lyophilized form, dissolved in de ionized distilled

water as recommended by provider to give a final concentration of 100
pmol/μl, this is stock solution. Working solution was prepared by diluting
the stock to 1/10 to obtain 10 pmol/μl, and stored in a deep freezer until used
in PCR amplification.

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Polymerase Chain Reaction ( PCR )

The technique was made possible by the discovery of Taq polymerase,

The method relies on thermal cycling instrument(thermal cycler),

Requirements of PCR Technology

5- free nuclease water: must be present for the reaction to work.

• Initialization step ：94℃ 10min

Denaturation step 94℃ 30s

Annealing step 55℃ 30s 1 cycle

Extension step 72℃ 30s

• Final elongation ： 72℃ 10min

 ALWAYS keep PCR reagents (master mix, primers, DNA template on

Calculating annealing temperature (Tm) of a primer:

Primers were provided in lyophilized form, dissolved in de ionized distilled

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