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Introduction

Off all techniques In molecular biology, the polymerase
chain reaction (PCR), has been by for the most useful.

Haifa Almubarak and Amani Alghamdi

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.

The principle of PCR Definition of PCR

The PCR technique copies the target DNA by performing
repeated cycles each containing the following three main
steps : It is a genetic technique that occurs in vitro which allows
the enzymatic synthesis of large quantities
1- A denaturation or melting step to separate the two strands (amplification)of a targeted region of DNA .
of DNA, this step requires very high temp 95C for 10-20
seconds.

2-The Annealing step, allowing the primers to bind to the

complementary sequences on the template DNA, this step
requires the temp to be dropped to 50-60 C. The DNA is synthesized in the same manner as that seen
in vivo (in the cells ) using a DNA polymerase (the
3- The Elongation step, once the primers are bound to the
template the synthesis of DNA can start, the temperature enzymes that cells use to replicate their DNA).
should be increased to 70c which is the optimum
temperature for the polymerase enzyme.

A basic PCR components and reagents A basic PCR components and reagents

DNA template: is the DNA molecules that contains the DNA
region (segment) to be amplified, the segment that we are 1) DNA template
concered with is the target sequence.

Two primers: a short segment of DNA that are complementary to 2) Two primers
the 3' ends of each of the sense and anti-sense strand of the DNA
target, they are needed to get DNA synthesis started. 3) Taq polymerase

4) Deoxynucleoside

triphosphates

Taq polymerase: is the enzyme to manufacture the DNA copies.
The PCR involves a couple of high temperature steps so we use a 1) Buffer solution
heat resistant DNA polymerase, this is extracted from heat
resistant bacteria living in a hot springs at temperature up to 80C, 2) Divalent cations
or another DNA polymerase with a temperature optimum at
around 70 C. 3) Monovalent cation

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A basic PCR components and reagents

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.

Divalent cations, magnesium or manganese ions; generally
Mg2+ is used, but Mn2+ can be utilized for PCR-mediated
DNA mutagenesis, as higher Mn2+ concentration increases
the error rate during DNA synthesis

Monovalent cation potassium ions.

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 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
RNAsynthesized by enzymes called primasesto 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.

PCR Procedure
PCR Procedure (Denaturation)
The DNA to be amplified is mixes with

The mixture is heated to break the hydrogen bonds in Deoxynucleoside triphosphates, a thermal stable DNA
the DNA, forming single stranded molecules. polymerase and DNA primers.

PCR Procedure (Extension) PCR Procedure (Primer Annealing)

Tag polymerase then synthesizes the complementary
The mixture is then cooled
strand of DNA. Using the primer as the starting point sufficiently to allow the
DNA primer to anneal to
each end of the segment
to be copied

The DNA primers

hybridize to the end of the
gene to be amplified and
provide a starting point for
the tag polymerase

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Denaturation PCR Procedure

Denaturation: During the denaturation step, the reaction
Each synthesis cycle Is composed of Three steps
cocktail (reaction mixture) is exposed to high temperature,
usually 94-95C. This high temperature will denature the
DNAmeaning the hydrogen bond between the two
complementary strands melt, unraveling the DNA
Denaturation
molecule and exposing the nucleotide bases.

The high temperature of the denaturing step has the added

advantage of denaturing proteins (inactivating them) and
Primer Annealing
disrupting cells so that you dont have to always start with
purified DNA as your amplification template. You can often
amplify DNA directly from cell lysatesor even whole
cells.
Extension

Extension: Primer Annealing

The reaction cocktail is now brought to the optimum
reaction temperature for Taq polymerase (68 to 72C).
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).

During the second step of each cycle, the temperature
is lowered to an annealing temperature, 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.

PCR Procedure (new cycle)

The temperature is raised again to separate the DNA
strands and the lowered sufficiently to allow the
primers to attach. Tag polymerase now synthesizes
another set of new complementary strands

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PCR Procedure

This amount of amplification can be achieved by
running the reaction overnight in a thermal cycler, an
instrument that automatically raises and lowers the
temperature at appropriate time intervals
Applications of PCR
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.
PCR Optimization:
In practice, PCR can fail for many reasons that is why it is
important to ensure successful PCR conditions of the
reactions and technique should be optimized:
Contamination can cause the amplification of unwanted
DNAs, thus there should be spatial separation of PCR set
up areas from areas of purification and analysis of the PCR
product. Add to that all precautions should be taken to
minimize contamination as much as possible.
PCR Procedure

This process is repeated until enough DNA has been
produced to be identified or used for further research.
After twenty-one cycles, one molecule of DNA can be
amplified to over a million copies
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,
Applications of PCR
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)

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General Precautions:

There should be a spatial separation of PCR set up areas PCR Optimization:
from areas of purification and analysis of PCR.

The primer designing is very important in improving
the product yield and avoiding formation of spurious
products.

Use of pipette tips with filters (to minimize
contamination).

The choice of buffer or polymerase enzyme can help in
the amplification of long or otherwise problematic

storage of materials used in PCR should be separated regions of the DNA.
from all other reagents and should be added to the
reaction mixes in a sterile spatially separated facility.

General Precautions:

Use of PCR grade distilled water which has been heat and
UV sterilized .

The use of non powdered gloves

What is DNA Profiling?

A technique used by scientists to distinguish between

individuals of the same species using only samples of (DNA fingerprinting)
their DNA

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Stages of DNA Profiling Stages of DNA Profiling

Step 2:
Stage 1:
The DNA is cut into fragments using restriction enzymes. Cells are broken down
to release DNA
Each restriction enzyme cuts DNA at a specific base
sequence.
If only a small amount of
DNA is available it can be
amplified using the
polymerase chain reaction
(PCR)

Stages of DNA Profiling

Stage 3:
Stages of DNA Profiling

Fragments are
The sections of DNA that are cut out are called
separated on the basis restriction fragments.
of size using a process
called gel
This yields thousands of restriction fragments of all
electrophoresis. different sizes because the base sequences being cut

DNA fragments are may be far apart (long fragment) or close together
injected into wells and (short fragment).
an electric current is
applied along the gel.

Stages of DNA Profiling Stages of DNA Profiling

A radioactive material DNA is negatively
is added which charged so it is
combines with the attracted to the
DNA fragments to positive end of the gel.
produce a fluorescent The shorter DNA
image. fragments move faster
than the longer

A photographic copy of fragments.
the DNA bands is DNA is separated on
obtained. basis of size.

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Uses of DNA Profiling

DNA profiling is used to
Stages of DNA Profiling
solve crimes and Stage 4:
medical problems
The pattern of fragment distribution is then analysed.

Biological materials used for DNA profiling Crime

Blood
Forensic science is the use of scientific knowledge in legal

Hair situations.

Saliva
The DNA profile of each individual is highly specific.

Semen
The chances of two people having exactly the same DNA

Body tissue cells profile is 30,000 million to 1 (except for identical twins).

DNA samples have been
obtained from vaginal cells
transferred to the outside
of a condom during sexual
intercourse.

Example: A Paternity Test Solving Medical Problems

By comparing the DNA profile of a mother and her DNA profiles can be used to determine whether a
child it is possible to identify DNA fragments in the particular person is the parent of a child.
child which are absent from the mother and must
A childs paternity (father) and maternity(mother)
therefore have been inherited from the biological
can be determined.
father.
This information can be used in
Paternity suits
Inheritance cases
Immigration cases

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Is this man the father of the child?

Mother Child Man