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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

The PCR technique copies the target DNA by performing
repeated cycles each containing the following three main
steps :

Definition of PCR
It is a genetic technique that occurs in vitro which allows
the enzymatic synthesis of
large quantities
(amplification)of a targeted region of DNA .

1- A denaturation or melting step to separate the two strands

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.
3- The Elongation step, once the primers are bound to the
template the synthesis of DNA can start, the temperature
should be increased to 70c which is the optimum
temperature for the polymerase enzyme.

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

concered with is the target sequence.

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

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).

A basic PCR components and reagents

1)

DNA template

2)

Two primers

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

heat resistant DNA polymerase, this is extracted from heat
resistant bacteria living in a hot springs at temperature up to 80C,
or another DNA polymerase with a temperature optimum at
around 70 C.

1)

Buffer solution

2)

Divalent cations

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 Procedure (Denaturation)

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

The mixture is heated to break the hydrogen bonds in

the DNA, forming single stranded molecules.

The DNA to be amplified is mixes with

Deoxynucleoside triphosphates, a thermal stable DNA
polymerase and DNA primers.

PCR Procedure (Extension)

PCR Procedure (Primer Annealing)

Tag polymerase then synthesizes the complementary

strand of DNA. Using the primer as the starting point

The mixture is then cooled

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

Denaturation: During the denaturation step, the reaction

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
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 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:
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).

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

PCR Procedure

Each synthesis cycle Is composed of Three steps

Denaturation

Primer Annealing

Extension

Primer Annealing

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.

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PCR Procedure

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

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
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
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-

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.

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
from areas of purification and analysis of PCR.

Use of pipette
contamination).

tips

with

filters

(to

PCR Optimization:

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

minimize

storage of materials used in PCR should be separated

from all other reagents and should be added to the
reaction mixes in a sterile spatially separated facility.

The choice of buffer or polymerase enzyme can help in

the amplification of long or otherwise problematic
regions of the DNA.

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
their DNA

(DNA fingerprinting)

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Stages of DNA Profiling

Step 2:

The DNA is cut into fragments using restriction enzymes.

Each restriction enzyme cuts DNA at a specific base
sequence.

Stages of DNA Profiling

Stage 1:

Cells are broken down

to release DNA
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:

Fragments are
separated on the basis
of size using a process
called gel
electrophoresis.

DNA fragments are
injected into wells and
an electric current is
applied along the gel.

The sections of DNA that are cut out are called

restriction fragments.

This yields thousands of restriction fragments of all
different sizes because the base sequences being cut
may be far apart (long fragment) or close together
(short fragment).

Stages of DNA Profiling

Stages of DNA Profiling

A radioactive material
is added which
combines with the
DNA fragments to
produce a fluorescent
image.

DNA is negatively
charged so it is
attracted to the
positive end of the gel.
The shorter DNA
fragments move faster
than the longer
fragments.
DNA is separated on
basis of size.

A photographic copy of
the DNA bands is
obtained.

Stages of DNA Profiling

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Uses of DNA Profiling

DNA profiling is used to
solve crimes and
medical problems

Biological materials used for DNA profiling

Blood
Hair
Saliva
Semen
Body tissue cells
DNA samples have been
obtained from vaginal cells
transferred to the outside
of a condom during sexual
intercourse.

Stages of DNA Profiling

Stage 4:

The pattern of fragment distribution is then analysed.

Crime

Forensic science is the use of scientific knowledge in legal
situations.

The DNA profile of each individual is highly specific.

The chances of two people having exactly the same DNA
profile is 30,000 million to 1 (except for identical twins).

Example: A Paternity Test

Solving Medical Problems

By comparing the DNA profile of a mother and her

child it is possible to identify DNA fragments in the
child which are absent from the mother and must
therefore have been inherited from the biological
father.

DNA profiles can be used to determine whether a

particular person is the parent of a child.
A childs paternity (father) and maternity(mother)
can be determined.
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