PRESENTATION REPORT ON PCR 201

Introduction
Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and
inexpensive technique used to "amplify" - copy - small segments of DNA. Because significant
amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of
isolated pieces of DNA are nearly impossible without PCR amplification.

The polymerase chain reaction (PCR) is a technique widely used in molecular biology,
microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental
science, hereditary studies, paternity testing, and many other applications.

Brief history
The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that
is used by the bacterium Thermus auquaticus that was discovered in hot springs by Kary Mullis
in 1984. This DNA polymerase is stable at the high temperatures need to perform the
amplification, whereas other DNA polymerases become denatured.

Often heralded as one of the most important scientific advances in molecular biology, PCR
revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded
the Nobel Prize for Chemistry in 1993.

Basic theme
PCR is performed after region of target DNA to be amplified (Target Sequence) has been
specified. Target Sequence, as its name implies, is a specific part of DNA that is needed for
further study.The method relies on thermal cycling, consisting of cycles of repeated heating and
cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short
DNA fragments) containing sequences complementary to the target region along with a DNA
polymerase (after which the method is named) are key components to enable selective and
repeated amplification. As PCR progresses, the DNA generated is itself used as a template for
replication, setting in motion a chain reaction in which the DNA template is exponentially
amplified. With PCR it is possible to amplify a single piece of DNA, or a very small number of
pieces of DNA, over many cycles, generating millions of copies of the original DNA molecule.

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Pcr process
1. PREPARATION BEFORE REPETITION OF PCR CYCLE:

The PCR components are added to the solution containing DNA to be amplified. This step is
only performed before the first PCR cycle. Addition of components after the first cycle is not
advised because PCR reaction is temperature sensitive (slight change in temperature can greatly
affect the reaction).

Components to be added are as followed:

 DNA templates to be amplify during this process.

 A pair of primers (usually between 10 - 30 nucleotides long) that
is complementary to the Flanking Sequence (several bases directly left or right of the
target sequence). These two different primers will be referred as primer F (for the forward
primer) and primer R (for the reverse primer).
 The four dNTPs (deoxyribonucleotide triphosphates); they are:
dATP, dGTP, dCTP, and dTTP.
 Magnesium Chloride (MgCl2) in order to stabilize the charge of
phosphate group and activate the replication process.
 Heat stable DNA polymerase; heat stability is very important
because PCR reaction is performed at various temperatures. This heat stable DNA
polymerase is the Taq DNA polymerase. Other polymerases with different properties can
be used depending on the purpose and requirements of the PCR. Some polymerases, such
as Pfu Turbo, for example, have higher fidelity rates but are much more sensitive.
 Buffer solution to stabilize the DNA sample.

To maximize the efficiency of this process, a master mix is made and contained of the primer,
MgCl2, buffer, and Taq polymerase.

These component must be added in excess to ensure that when the two DNA strands are
separated in the second step, the chance of the two DNA to re-hybridize is minimized.

2. PCR CYCLE (PERFORM ABOUT 20-30 CYCLES):

1. Strand Separation
 Strand separation is performed to expose target sequence and flanking
sequence to primer. This step is done by heating the solution, causing the hydrogen
bonds maintaining the double helix structure to break.
 Parameter:
Temperature: about 95oC
Time: 15 to 30 seconds (to separate the DNA to 2 single strands)

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2. Hybridization of Primers (Also known as the annealing step)

 At this step, the solution is cooled to around 54oC. By cooling down the
solution, primers are allowed to hybridize with the flanking sequences via hydrogen
bonding.
 It is very possible that the two main strands will re-hybridize because both
strands are complimentary to each other. Fortunately, as explained earlier, it can be
easily avoided by having excess primers in solution. Excess primers are expected to
hybridize with the flanking sequence before any two complimentary DNA can
hybridize.

3. DNA Synthesis (also known as elongation step)

 Solution heated to 72oC. At this optimal temperature, the Taq polymerase
will start elongates both primers.
 There are two types of primer; each is the complimentary oligonucleotides
of the flanking sequence.
 Elongation happens not unlike the regular polymerization of DNA. The
primers are elongated from the 5' to the 3' ends (which is the opposite direction of the
actual DNA strand; strand that have target sequence, flanking sequence, and un-
amplified sequence).

Cycle details
 By the end of first cycle, we obtain 2 types of new strand in addition to the
original DNA strands. These strands are the elongation of each type of primer. They
contain:
 One of the primer (exp: primer A)
 The target sequence
 A complementary of a flanking sequence (the one that is not
hybridized to the primer contained in this strand; e.g: complementary of flanking
sequence of primer B)
 The rest of the non-target sequence after this complimentary of
flanking sequence.
 On the second cycle, some of the primers will hybridize with the new type
of sequence obtained on first cycle (exp: on the shorter strand elongated from primer
A, primer B will attach to its flanking sequence and start elongates the cycle until it
reaches the end of the shorter strand, the flanking sequence of primer A).
 On the second cycle, the new type of strand formed, Short Strand consist
of:
 One of the primer (exp: Primer B)
 The target sequence
 The flanking sequence that can hybridize with Primer A
 On the second cycle, besides forming the short strand, the same strands as
the one formed on the first cycle can also form.

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 On the second to the nth cycle, the short strand is amplified exponentially
while the slightly longer strand (the only strand that form on the first cycle) is
amplified arithmetically.
 This cycle is repeated n times. At the nth repetition, there will be 2n of
desired sequence.

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PCR process with detailed steps can be repeated in multiple cycle to amplify original DNA
structure.

Finally, the reaction is usually held at 4 degrees Celsius after completion to stabilize the DNA,
which is temperature sensitive.

Additional Steps
A final elongation step is performed at 70-74°C for at least 5 minutes after the last PCR cycle has
been done. This step will guarantee that any remaining DNA strand is fully extended. In
addition, a hold step at 4°C for an indefinite time can be utilized for storage of the DNA product.

pcr machine: the thermal cycle

The thermal cycler (also known as a Thermocycler, PCR

Machine or DNA Amplifier) is a laboratory apparatus used
to amplify segments of DNA via the polymerase chain
reaction (PCR) process. The device has a thermal block with
holes where tubes holding the PCR reaction mixtures can be
inserted. The cycler then raises and lowers the temperature
of the block in discrete, pre-programmed steps.

The earliest thermal cyclers were designed for use with the
Klenow fragment of DNA Polymerase I. Since this enzyme
is destroyed during each heating step of the amplification
process, new enzyme had to be added every cycle. This led
to a cumbersome machine based on an automated pipettor,
with open reaction tubes. Later, the PCR process was adapted to the use of thermostable DNA
polymerase from Thermus aquaticus, which greatly simplified the design of the thermal cycler.
While in some old machines the block is submerged in oil bath to control temperature, in modern
PCR machines a Peltier element is commonly used. Quality thermal cyclers often contain silver
blocks to achieve fast temperature changes and uniform temperature throughout the block. The
Peltier effect allows both heating and cooling of the block holding the PCR tubes simply by
reversing the electric current. Thin-walled reaction tubes with no RNAase and DNAase are
suitable for PCR. Due to their thickness, they can create thermal conductivity to allow for rapid
thermal equilibration.

Modern thermal cyclers are equipped with a heated lid, a heated plate that presses against the
lids of the reaction tubes. This prevents condensation of water from the reaction mixtures on the
insides of the lids and makes it unnecessary to use PCR oil to cover the reaction mixture. Some
thermal cyclers are equipped with multiple blocks allowing several different PCR reactions to be
carried out simultaneously. Also some apparatus have a gradient function, which allows different

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temperatures in different parts of the block. This is particularly useful when testing suitable
annealing temperatures for primers.
PCR PHASES

To understand why end-point PCR is limiting, it is important to understand what happens during
a PCR reaction.
A basic PCR run can be broken up into three phases:

1. Exponential: Exact doubling of product is accumulating at every cycle (assuming 100%

reaction efficiency). The reaction is very specific and precise.

2. Linear (High Variability): The reaction components are being consumed, the reaction is
slowing, and products are starting to degrade.

3. Plateau (End-Point: Gel detection for traditional methods): The reaction has stopped,
no more products are being made and if left long enough, the PCR products will begin to
degrade.

Figure shows three replicates of a sample. The replicates have the same starting quantity. As the
PCR reaction progresses, the samples begin to amplify in a very precise manner. Amplification
occurs exponentially, that is a doubling of product (amplicon) occurs every cycle. This type of
amplification occurs in the exponential phase. Exponential amplification occurs because all of
the reagents are fresh and available, the kinetics of the reaction push the reaction to favor
doubling of amplicon.

However, as the reaction progresses, some of the reagents are being consumed as a result of
amplification. This depletion will occur at different rates for each replicate. The reactions start to
slow down and the PCR product is no longer being doubled at each cycle. This linear

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amplification can be seen in the linear phase of the reaction. The three samples begin to diverge
in their quantities during the linear phase.

Eventually the reactions begin to slow down and stop all together or plateau. Each tube or
reaction will plateau at a different point, due to the different reaction kinetics for each sample.
These differences can be seen in the plateau phase. The plateau phase is where traditional PCR
takes its measurement, also known as end-point detection.

Result interpretation
To check whether the PCR generated the anticipated DNA fragment, agarose gel electrophoresis
is employed for size separation of the PCR products. The size(s) of PCR products is determined
by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments
of known size, run on the gel alongside the PCR products.

The bands that appeared on the agarose

gels provide the following information
about the PCR product:

1. There is a product formed.

Though biochemistry is an
exact science, not every PCR is
successful. There is for example
a possibility that the quality of
the DNA is poor, that one of the
primers doesn't fit, or that there
is too much starting template.
2. The product is of the right size. It is possible that there is a product, for example a
band of 500 bases, but the expected gene should be 1800 bases long. In that case, one
of the primers probably fits on a part of the gene closer to the other primer. It is also
possible that both primers fit on a totally different gene.
3. Only one band is formed. As in the description above, it is possible that the
primers fit on the desired locations, and also on other locations. In that case, different
bands appear in one lane on a gel.

PCR OPTIMIZATION
The most important consideration in PCR is contamination. If the sample that is being tested has
even the smallest contamination with DNA from the target, the reaction could amplify this DNA
and report a falsely positive identification. Thus to avoid contamination DNA sample
preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent

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reaction product analysis, should be performed in separate areas. For the preparation of reaction
mixture, a laminar flow cabinet with UV lamp is recommended. Fresh gloves should be used for
each PCR step as well as displacement pipettes with aerosol filters. The reagents for PCR should
be prepared separately and used solely for this purpose. Aliquots should be stored separately
from other DNA samples. A control reaction (inner control), omitting template DNA, should
always be performed, to confirm the absence of contamination or primer multimer formation.

Difficulties with pcr

While a very powerful technique, PCR can also be very tricky. The polymerase reaction is very
sensitive to the levels of divalent cations (especially Mg2+) and nucleotides, and the conditions
for each particular application must be worked out. As Polymerase chain reaction is not perfect,
errors and mistakes can occur.
These are some common errors
and problems that may occur.

PRIMING ERROR

Primer design is extremely

important for effective
amplification. The primers for the
reaction must be very specific for
the template to be amplified. Cross
reactivity with non-target DNA
sequences results in non-specific
amplification of DNA. The non-
specific binding of primers is
always a possibility due to
sequence duplications, non-
specific binding and partial primer
binding, leaving the 5' end
unattached. This increased by the use of degenerate sequences or bases in the primer.
Manipulation of annealing temperature and magnesium ion (which stabilize DNA and RNA
interactions) concentrations can increase specificity. Non-specific priming can be prevented
during the low temperatures of reaction preparation by use of hot-start polymerase enzymes
where the active site is blocked by an antibody or chemical that only dislodges once the reaction
is heated to 95˚C during the denaturation step of the first cycle.

Also, the primers must not be capable of annealing to themselves or each other, as this will result
in the very efficient amplification of short nonsense DNAs.

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SIZE LIMITATIONS

The reaction is limited in the size of the DNAs to be amplified (i.e., the distance apart that the
primers can be placed). The most efficient amplification is in the 300 - 1000 bp range; however
amplification of products up to 4 Kb has been reported.

POLYMERASE ERRORS

Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to check the base it
has inserted and remove it if it is incorrect, a process common in higher organisms. This in turn results in
a high error rate of approximately 1 in 10000 bases, which, if an error occurs early, can alter large
proportions of the final product. As a result other polymerases are available for accuracy in vital uses
such as amplification for sequencing.

Examples of polymerases with 3' to 5' exonuclease activity include: Vent, which is extracted from
Thermococcus litoralis, Pfu which is extracted from Pyrococcus furiosus and Pwo which is extracted
from Pyrococcus woesii.

APPLICATIONS OF PCR
The Polymerase Chain Reaction (PCR) has found widespread application in many areas of
genetic analysis. This is a list of some of these applications:

1. FORENSIC APPLICATIONS

The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread
application in forensics:

• GENETIC FINGERPRINTING
In its most discriminating form, Genetic fingerprinting can uniquely discriminate any
one person from the entire population of the world. Minute samples of DNA can be
isolated from a crime scene, and compared to that from suspects, or from a DNA
database of earlier evidence or convicts. Theoretically, just a single strand is needed.
First, one breaks the DNA sample into fragments, then amplifies them using PCR. The
amplified fragments are then separated using gel electrophoresis. The overall layout of
the DNA fragments is called a DNA fingerprint. Simpler versions of these tests are often
used to rapidly rule out suspects during a criminal investigation. Evidence from decades-
old crimes can be tested, confirming or exonerating the people originally convicted.

• PARENTAL TESTING
Less discriminating forms of DNA fingerprinting can help in Parental testing, where an
individual is matched with their close relatives. DNA from unidentified human remains

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can be tested, and compared with that from possible parents, siblings, or children. Similar
testing can be used to confirm the biological parents of an adopted (or kidnapped) child.
The actual biological father of a newborn can also be confirmed (or ruled out).

2. INFECTIOUS DISEASE APPLICATIONS

Characterization and detection of infectious disease organisms have been revolutionized by PCR:

• The Human Immunodeficiency Virus (or HIV), responsible for AIDS, is a difficult
target to find and eradicate. The earliest tests for infection relied on the presence of
antibodies to the virus circulating in the bloodstream. However, antibodies don't appear
until many weeks after infection, maternal antibodies mask the infection of a newborn,
and therapeutic agents to fight the infection don't affect the antibodies. PCR tests have
been developed that can detect as little as one viral genome among the DNA of over
50,000 host cells . Infections can be detected earlier, donated blood can be screened
directly for the virus, newborns can be immediately tested for infection, and the effects of
antiviral treatments can be quantified.

• Some disease organisms, such as that for Tuberculosis, are difficult to sample from
patients and slow to be grown in the laboratory. PCR-based tests have allowed detection
of small numbers of disease organisms (both live or dead), in convenient samples.
Detailed genetic analysis can also be used to detect antibiotic resistance, allowing
immediate and effective therapy. The effects of therapy can also be immediately
evaluated.

• The spread of a disease organism through populations of domestic or wild animals can
be monitored by PCR testing. In many cases, the appearance of new virulent sub-types
can be detected and monitored. The sub-types of an organism that were responsible for
earlier epidemics can also be determined by PCR analysis.

3. DETECTION OF HEREDITARY DISEASES

The detection of hereditary diseases in a given genome is a long and difficult process, which can
be shortened significantly by using PCR. Each gene in question can easily be amplified through
PCR by using the appropriate primers and then sequenced to detect mutations.

Viral diseases, too, can be detected using PCR through amplification of the viral DNA. This
analysis is possible right after infection, which can be from several days to several months before
actual symptoms occur. Such early diagnoses give physicians a significant lead in treatment.

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4. RESEARCH APPLICATIONS

PCR has been applied to many areas of research in molecular genetics:

• PCR allows rapid production of short pieces of DNA, even when nothing more than the
sequence of the two primers is known. This ability of PCR augments many methods, such
as generating hybridization probes for Southern or northern blot hybridization. PCR
supplies these techniques with large amounts of pure DNA, sometimes as a single strand,
enabling analysis even from very small amounts of starting material.

• The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can
easily be produced from a patient with a genetic disease mutation. Modifications to the
amplification technique can extract segments from a completely unknown genome, or can
generate just a single strand of an area of interest.

• An exciting application of PCR is the phylogenic analysis of DNA from ancient sources,
such as that found in the recovered bones of Neanderthals, or from frozen tissues of
Mammoths. In some cases the highly degraded DNA from these sources might be
reassembled during the early stages of amplification.

5. CLONING GENES

Cloning a gene, not to be confused with cloning a whole organism, describes the process of
isolating a gene from one organism and then inserting it into another organism (now termed a
genetically modified organism (GMO)). PCR is often used to amplify the gene, which can then
be inserted into a vector (a vector is a piece of DNA which 'carries' the gene into the GMO) such
as a plasmid (a circular DNA molecule). The DNA can then be transferred into an organism (the
GMO) where the gene and its product can be studied more closely. Expressing a cloned gene
(when a gene is expressed the gene product (usually protein or RNA) is produced by the GMO)
can also be a way of mass-producing useful proteins, for example medicines or the enzymes in
biological washing powders. The incorporation of an affinity tag on a recombinant protein will
generate a fusion protein which can be more easily purified by affinity chromatography.

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Cloning a gene using a plasmid.

(1) Chromosomal DNA of organism A. (2) PCR. (3) Multiple copies of a single gene from
organism A. (4) Insertion of the gene into a plasmid. (5) Plasmid with gene from organism A. (6)
Insertion of the plasmid in organism B. (7) Multiplication or expression of the gene, originally
from organism A, occurring in organism B.

6. MUTAGENESIS
Mutagenesis is a way of making changes to the sequence of nucleotides in the DNA. There are
situations in which one is interested in mutated (changed) copies of a given DNA strand, for
example, when trying to assess the function of a gene or in in-vitro protein evolution. Mutations
can be introduced into copied DNA sequences in two fundamentally different ways in the PCR
process. Site-directed mutagenesis allows the experimenter to introduce a mutation at a specific
location on the DNA strand. Usually, the desired mutation is incorporated in the primers used for
the PCR program. Random mutagenesis, on the other hand, is based on the use of error-prone
polymerases in the PCR process. In the case of random mutagenesis, the location and nature of
the mutations cannot be controlled. One application of random mutagenesis is to analyze
structure-function relationships of a protein. By randomly altering a DNA sequence, one can
compare the resulting protein with the original and determine the function of each part of the
protein.
7. COMPARISON OF GENE EXPRESSION
Researchers have used traditional PCR as a way to estimate changes in the amount of a gene's
expression. Ribonucleic acid (RNA) is the molecule into which DNA is transcribed prior to
making a protein, and those strands of RNA that hold the instructions for protein sequence are
known as messenger RNA (mRNA). Once RNA is isolated it can be reverse transcribed back
into DNA (complementary DNA to be precise, known as cDNA), at which point traditional PCR
can be applied to amplify the gene, this methodology is called RT-PCR. In most cases if there is
more starting material (mRNA) of a gene then during PCR more copies of the gene will be
generated.
When the products of the PCR reaction are run on an agarose gel a band, corresponding to a
gene, will appear larger on the gel (note that the band remains in the same location relative to the
ladder, it will just appear fatter or brighter). By running samples of amplified cDNA from

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differently treated organisms one can get a general idea of which sample expressed more of the
gene of interest.

conclusion
One of PCR's distinctive characteristics is unquestionably its extraordinary versatility. That
versatility is more than its "applicability" to many different situations. PCR is a tool that has the
power to create new situations for its use and those required to use it.
PCR's versatility has been astounding, scientists have produced new contexts and new uses with
stunning regularity. These uses have opened new avenues of research, which have in turned
proved amenable to new uses of PCR. In less than a decade, PCR has become simultaneously an
absolutely routine component of practically every molecular biology laboratory and a constantly
changing tool whose potential has shown no signs of leveling off.

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