PCR

(POLYMERASE CHAIN REACTION)

What is a thermocycler
A thermocycler raises and lowers temperature. For
molecular or DNA applications it needs to heat up
to about 90-95 degrees centigrade. DNA which
usually exists in double strands in a helix (like a
spiral staircase), at that temperature DNA strands
fall apart. Then the thermocycler needs to cool
down to the point where ―bases‖ can connect to the
separated DNA strands(like a template) to form
two new double helix strands of DNA. If this
process is cycled through this temperature range
from 20-30 times millions of copies of the original
DNA can be produced.
 Calibration and Maintenance

Similar to other laboratory instrumentation, thermal

cyclers must be calibrated and maintained.
Routine calibration and maintenance may include:
• Cleaning
• Temperature calibration verification test
• Temperature non-uniformity test
• Performance diagnostics tests
 Cleaning
Prior to using a thermal cycler, sample wells should be
examined to ensure that no debris (Waste) has settled into
the wells. Debris in the wells can prevent reaction tubes
from achieving an optimal fit and may prevent the
required heat transfer from the block to the
tube. Changes in heat transfer may adversely affect the
PCR.
The block and sample wells, trays, and the heat cover
can be cleaned with a 10% bleach solution. This should
be followed by the use of isopropanol or other
appropriate alcohol to remove any sodium hypochlorite
residue. Bleach can be corrosive and damaging to the
thermal cycler. Cotton swabs work well for the cleaning
process.
 Performance Diagnostics Test

The performance diagnostic tests may include a cycle

and rate test. These tests are used to verify the integrity
of the cooling and heating system, incorporated into the
instrument's software, and described in detail in the
thermal cycler user manual(s).
PCR is a process in which a DNA sequence is artificially
amplified by repeated cycles of replication and strand
separation, generating thousands to millions of copies of
a particular DNA sequence
It is a genetic technique that occurs in vitro which allows
the enzymatic synthesis of large quantities
(amplification)of a targeted region of DNA .

The DNA is synthesized in the same manner as that seen

in vivo (in the cells ) using a DNA polymerase (the
enzymes that cells use to replicate their DNA).
Principle of PCR
The basic PCR principle is simple. As the name implies, it is
a chain reaction: One DNA molecule is used to produce two
copies, then four, then eight and so forth. This continuous
doubling is accomplished by specific proteins known as
polymerases, enzymes that are able to string together
individual DNA building blocks to form long molecular
strands. To do their job polymerases require a supply of
DNA building blocks, i.e. the nucleotides consisting of the
four bases adenine (A), thymine (T), cytosine (C) and
guanine (G). They also need a small fragment of DNA,
known as the primer, to which they attach the building
blocks as well as a longer DNA molecule to serve as a
template for constructing the new strand. (AT-GC)
 PCR is a technique, in which within a minute
amounts of DNA can be replicated very rapidly and
thereby amplified to such an extent that the DNA
becomes easy to detect, study and use for any given
purpose. The potential of this technique in
medicine has long been known, and ever more
applications are being developed. Wherever genes
provide clues to the cause or natural history of a
disease, PCR is the method of choice .
This technique has become a ever-present and powerful
tool in diagnostics, forensics and research biology. Once
amplified, PCR products can simply be visualized by
agarose gel electrophoresis or can subsequently be
digested with restriction enzymes for DNA
fingerprinting, cloned or sequenced.
PCR works by using a thermostable DNA polymerase and
short DNA fragments, called ‗primers‘, to direct the
synthesis of a specifically-targeted region of a template
DNA. The synthesis reaction is repeated numerous times
called ‗cycles‘. The products of previous synthesis cycles
serve as template for the next cycle. This results in an
exponential amplification of the targeted region of DNA.
A basic PCR components and reagents

1) DNA template
2) Two primers
3) Taq polymerase
4) Deoxynucleoside triphosphates

1) Buffer solution
2) Divalent cations
3) Monovalent cation
DNA template: is the DNA molecules that contains the
DNA region (segment) to be amplified, the segment that
we are concered with is the target sequence.

Two primers: a short segment of DNA that are

complementary to the ends of each of the sense and anti-
sense strand of the DNA target, they are needed to get
DNA synthesis started.

Taq polymerase: is the enzyme to manufacture the DNA

copies. The PCR involves a couple of high temperature
steps so we use a heat resistant DNA polymerase, this is
extracted from heat resistant bacteria living in a hot
springs at temperature up to 80°C, or another DNA
polymerase with a temperature optimum at around 70 °C.
PCR Primers
All DNA polymerases require short segment of double—
stranded nucleic acid to initiate DNA synthesis.

During DNA replication cells use short stretches of

complementary RNA—synthesized by enzymes called
‗primases‘—to initiate polymerization.

In the laboratory, short, complementary segments of DNA

called ―primers‖ are used in PCR to initiate DNA synthesis
and to designate the specific target region to be amplified.

.
The primers (also called oligonucleotides— meaning small
number of nucleotides) are easily synthesized in the
laboratory and can be designed to be complementary to
any known DNA sequence. They can range in size from 10
to 100 nucleotides in length, but typically they range from
15 to 30 bases for PCR. It Is The Amplification Primers
That Determine Target Specificity (i.e., Which segment Of
The Template DNA will get amplified) in the PCR reaction
Deoxynucleoside triphosphates: the building blocks from which
the DNA polymerases synthesizes a new DNA strand.

Buffer solution, providing a suitable chemical environment for

optimum activity and stability of the DNA polymerase.

PCR machine: Also called thermal cyclers, modern PCR machines

rapidly changes temperatures for PCR reactions, allowing PCR to
cycle between primer annealing, DNA amplification, and strand
melting cycles.
PCR Procedure
Each synthesis cycle Is composed of Three steps

Denaturation

Primer Annealing

Extension
Denaturation
Denaturation: During the denaturation step, the reaction
cocktail (reaction mixture) is exposed to high
temperature, usually 94-95°C. This high temperature will
denature the DNA—meaning the hydrogen bond
between the two complementary strands melt, unraveling
the DNA molecule and exposing the nucleotide bases.

The high temperature of the denaturing step has the

added advantage of denaturing proteins (inactivating
them) and disrupting cells so that you don‘t have to
always start with purified DNA as your amplification
template. You can often amplify DNA directly from cell
lysates—or even whole cells.
 Primer Annealing
During the second step of each cycle, the temperature is
lowered to an annealing temperature,(550C) allowing
binding (annealing) of the primers to their complementary
targets on the DNA template (one for each DNA strand).
These are designed to flank the desired target region of
your DNA template and serve as the starting points for
DNA synthesis by the Taq polymerase.

Each pair of primers will have a particular annealing

temperature determined by the length of the primers and
their G+C content. Using the proper annealing
temperature for your primer set is essential for efficient
and accurate amplification.
Extension:
The reaction cocktail is now brought to the optimum
reaction temperature for Taq polymerase (68 to 72°C).
During this step, the Taq will bind to each DNA strand
and ―extend‖ from the priming sites (add nucleotides to
synthesize a complementary strand of the targeted
DNA).
 When PCR is used only for detecting a specific
DNA segment, the method is referred to as qualitative
PCR. Qualitative PCR is an extremely sensitive method
which is theoretically able to detect a single DNA
molecule in a sample solution. In many cases specific
genes are copied in this way, e.g. in order to identify
pathological changes. As the first gene identified by
PCR was the gene responsible for sickle cell anemia.
Countless other gene tests have meanwhile been
devised. Qualitative PCR is also used around the world
in forensic medicine to identify individuals.
PCR can of course be used to detect not only human
genes but also genes of bacteria and viruses. One of the
most important medical applications of the classical
PCR method is therefore the detection of
pathogens(harmful orgnasims). Here PCR is replacing
immunological methods, in which antibodies against a
pathogen are used to identify the pathogen in a patient‘s
blood. Antibodies are not detectable until several weeks
after the onset of an infection, whereas PCR is able to
detect the DNA or RNA of the pathogens much more
quickly. Moreover, antibodies can remain in the
bloodstream long after an infectious disease has resolved.
Hence, only qualitative PCR can determine whether an
infection
 PCR method can detect the DNA of
microorganisms in any sample, whether of body fluids,
foodstuffs or drinking water. PCR is therefore used in
all these areas. One of the most urgent problems PCR
is helping to solve is to determine if donated blood is
contaminated. Blood banks are one of the major
transmission sources of hepatitis C, for example, and
sometimes of HIV. Fast, simple and above all
inexpensive testing is essential – and PCR ideally
meets all these criteria.
Applications of PCR

PCR is useful when small initial amounts of DNA are

available (e.g., forensics or diagnostic samples) or
anytime large quantities of a particular region of the
genome are needed (e.g., for DNA sequencing or finger
printing).

1- Selective DNA isolation; PCR allows selective

amplification of a specific region of the DNA which can
be used later for many other investigations such as DNA
sequencing ,genetic finger printing, investing evolutionary
relationships among organisms,
2- For quantitification of DNA : this allows to estimate
the amount of DNA present in a sample which is used to
quantitatively determine the level of gene expression .
Real -Time PCR is used then which measures the
accumulation of the DNA product after each PCR round.

3-PCR In diagnosis of disease; It permits early diagnosis

of malignant diseases or can establish whether the person
is at risk or not (by investigated the presence of a certain
gene that is associated with a certain type of cancer)
It is used in forensic medicine where amplification of
the DNA is very useful especially when only trace
amounts of DNA is available as evidence, may also be
used in paternity testing, can also be used in the
analysis of ancient DNA like the DNA analysis of the
remains of Egyptian mummies.
Applications of PCR

Quantitative PCR provides additional information

beyond mere detection of DNA. It indicates not just
whether a specific DNA segment is present in a
sample, but also how much of it is there. This
information is required in a number of applications
ranging from medical diagnostic testing through target
searches to basic research.
Quantitative PCR is used to help search for and evaluate
targets, i.e. the sites in the body at which new drugs can
act. This primarily relates to the discovery of new genes, a
task in which PCR is basically used as a DNA copying
tool.

Quantitative PCR is used in molecular diagnosis, i.e. the

diagnosis of diseases based on molecular findings rather
than on physiological symptoms. In this connection the
diagnosis of viral diseases is an area that is gaining
increasing importance. For simple diagnostic testing, i.e. to
determine if a pathogen is present in the patient‘s body,
qualitative PCR is sufficient.
 Quantitative PCR used in the field of infectious
diseases is AIDS. Those infected with the causative
agent of the disease, the human immunodeficiency
virus (HIV), have to take a cocktail of usually three
drugs indefinitely, this being the only way to keep the
virus in check, if only for a while.

PCR can identify genes that have been

implicated in the development of cancer. Identifying
the genes that play a role in the development of cancer
is therefore an important step towards improving
treatment. Both qualitative and quantitative PCR play a
crucial role in the fight against cancer.
PCR-based diagnostics tests are available for detecting
and/or quantifying several pathogens, including:

 HIV-1, which causes AIDS

 Hepatitis B and C viruses, which can lead to liver

cancer

 Human Papillomavirus, which can cause cervical

cancer

 Chlamydia trachomatis, which can lead to infertility

in women
Neisseria gonorrhoeae, which can lead to pelvic
inflammatory disease in women

 Cytomegalovirus, which can cause life threatening

disease in transplant patients and other
immunocompromised people, including HIV-1/AIDS
patients

 Mycobacterium tuberculosis, which in its active state

causes cough and fatigue and can lead to tissue
damage of infected organs
DNA deoxyribonucleic acid; the chemical substance
of our genes

RNA ribonucleic acid; the chemical substance that

makes up the working copies of genes (mRNA), among
other things

Nucleic acids a chemical term that covers both DNA

and RNA; nucleic acids are molecules consisting of long
chains of nucleotides linked together
Nucleotides the building blocks of DNA; they comprise
the four bases adenine, thymine, cytosine and guanine (A,
T, C, G; in RNA thymine is replaced by uracil [U]), a
sugar and at least one phosphate group; without the
phosphate group these building blocks are referred to as
nucleosides

Sequence the order of the nucleotides in DNA (DNA

sequence) or RNA (RNA sequence)

Primer a short DNA fragment with a defined sequence

that serves as an extension point for polymerases
Polymerases enzymes that link individual nucleotides
together to form long DNA or RNA chains

Hybridisation (annealing) the joining of two

complementary DNA (or RNA) strands to form a double
strand

Complementary DNA The building blocks of DNA

and RNA form specific pairings. Two strands whose
building blocks form a sequence of perfect pairings are
able to form a stable double strand and are referred to as
complementary strands