polymerase chain reaction

1. Polymerase Chain Reaction (PCR)
Done by PhD students:
Farah Abdulhaleem Abbas Hameed Mustafa Hussein
Supervised by:
Dr. Mayssaa Essam

2. INTRODUCTION
developed in 1983 by Kary Mullis.
In 1993, Mullis was awarded the Nobel prize in Chemistry along with Michael Smith
molecular biology technique for enzymatically replicating DNA without using a living
organism, such as E. coli or yeast.
the DNA molecule is amplified in an exponential manner.
PCR is commonly used in medical and biological research labs for a variety of tasks, such as
the detection of hereditary diseases,
the identification of genetic fingerprints,
the diagnosis of infectious diseases,
the cloning of genes,
paternity testing, and
DNA computing.

3. INTRODUCTION
a reaction volume of 10-200 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal
cycler.
The thermal cycler heats and cools the reaction tubes
Many modern thermal cyclers make use of the Peltier effect
Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal
equilibration.
Most thermal cyclers have heated lids to prevent condensation at the top of the reaction
tube.

4. INTRODUCTION
PCR Assay Components
DNA template, or cDNA which contains the region of the DNA fragment to be amplified
Two primers, which determine the beginning and end of the region to be amplified
Taq polymerase, which copies the region to be amplified
Nucleotides, from which the DNA-Polymerase for new DNA
Buffer, which provides a suitable chemical environment for the DNA-Polymerase.
MgCl2 Concentration: Free Mg2+ ions are required as a co-factor for DNA polymerase activity;
however Mg2+ ions form complexes with dNTPs, primers and DNA templates.
Fluorescent Labels: Fluorescent dyes are incorporated into the reaction when the amplicon is
to be detected directly, such as when using a qPCR or digital PCR (dPCR) set up.

5. INTRODUCTION
STAGES OF PCR
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100%
reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be
present.
Leveling off: The reaction slows as the DNA polymerase loses activity and as consumption of
reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

6. INTRODUCTION
Advantages of PCR
PCR can be used for diagnosis of many human diseases, broad variety of experiments and
analysis.
PCR is very important confirmatory diagnostic aid in infectious disease like tuberculosis,
HIV,CMV, cancers specially leukaemia and lymphoma
" PCR is also important for genetic fingering and paternity test.
" PCR has high sensitivity(95-100%) and specificity

7. INTRODUCTION
Disadvantages of PCR
Requires costly instruments like thermal cycler, agargel diffusion tray, DNA separation kit,
other chemicals & reagents which not all laboratories can afford to buy.
Requires trained, experienced, qualified manpower and technologists,
Adequate space with air-condition, dehumidifier, laminar flow facilities,
Limited scope for diagnosis of diseases,
Costly and not all people can afford to do the test,
False positive and false negative results may lower specificity & sensitivity,

8. Real-time PcR
Traditional PCR is not useful for quantifying the amount of original RNA or DNA template
since the amount of PCR product synthesized is determined more by the amount of primers
and other reagents in the reaction mixture than the amount of template. However,
quantitation is possible using real-time PCR.
There are two main methods for carrying out real-time PCR:
a dye in the PCR reaction mixture that binds only to double-stranded DNA and which
fluoresces strongly when bound, e.g. SYBR Green I. However, because the dye binds to any
double-stranded DNA irrespective of its sequence, it is a nonspecific quantitation method.
the primers used in the PCR reaction mixture is labeled at one end with a fluorescent
reporter molecule and at the other end with a fluorescence quencher molecule.

9. PCR is a cyclic process
• Consists of a series of 30-35
cycles
• Each cycle is a 3 steps:
• 1. Denaturation
• 2. Annealing
• 3. Extension

10. 1. Denaturation
• The reaction mixture is heated to 94∞C for a short time period
(about 15–30 sec.) to denature the target DNA into single strands that
can act as templates for DNA synthesis.

11. 2. Primer annealing
• The mixture is rapidly cooled to a defined temperature (50–60∞C), which allows the two
primers to bind to the sequences on each of the two strands flanking the target DNA.
• This annealing temperature is calculated carefully to ensure that the primers bind only to
the desired DNA sequences.
• One primer binds to each strand .
• The two parental strands do not re-anneal with each other because the primers are in
large excess over parental DNA.

12. 3. Elongation
• The temperature of the mixture is raised to 72∞C (the optimum
temperature for Taq polymerase) .
• At the end of this incubation, both single-stranded template strands
have been made partially double stranded.

13. PCR cycles:
• The new strand of each double-stranded DNA extends for a variable
distance downstream.
• The three steps of the PCR cycle are repeated.
• Thus in the second cycle, the four strands denature, bind primers and
are extended. No other reactants need to be added.
• The three steps are repeated once more for a third cycle (Figure 1)
and so on for a set number of additional cycles.
• By the third cycle, some of the PCR products (indicated by asterisks in
Figure 1) represent DNA sequence only between the two primer sites
and the sequence does not extend beyond these sites.

14. PCR cycles:
• As more and more reaction cycles are carried out, this type of double-
stranded DNA molecule becomes the majority species present.
• After 20 cycles, the original DNA has been amplified a million-fold and
this rises to a billion-fold (1000 million) after 30 cycles.
• At this point, the vast majority of the products are identical in that
the DNA amplified is only that between the two primer sites.
• Automated thermocyclers are now routinely used to cycle the
reaction without manual interference so that a billion-fold
amplification of the DNA sequence between the two primer sites (30
cycles) can take less than 1 hour!

18. Applications of PCR
:1- Detecting infectious agents
PCR is extensively used in analyzing clinical specimens for the
presence of infectious agents, including HIV, hepatitis, human
papillomavirus (the causative agent of genital warts and cervical
cancer), Epstein-Barr virus (glandular fever), malaria and anthrax.
PCR is particularly invaluable in the early detection of HIV as it
can identify the DNA of the virus within human cells immediately
following infection, as opposed to the antibodies that are produced
weeks or months after infection.
PCR can also be used to determine the viral load (i.e. how much
virus is circulating around the body), which is a useful measure of
prognosis.

19. Malaria is traditionally diagnosed by identifying malarial
parasites (Plasmodium falciparum) through microscopic
analysis of the blood.
However, PCR technology has been useful in that it can
rapidly identify the species of malaria present. This is
important in cases of mixed infection, and also in
determining the type of drug treatment to use. Currently,
PCR is used to complement microscopic examination.
PCR can be used to identify the bacterium Bacillus
anthracis, the causative agent of anthrax.
Because of the need to rapidly diagnose such infections, PCR
has become an important tool in detecting the presence of
anthrax in clinical specimens. It replaces conventional
methods of using a specimen to grow the bacteria in the
laboratory, which take at least 24 hours. PCR provides a
rapid, sensitive and specific alternative.

20. :2- Genetic diseases
PCR is used in the analysis of mutations that occur in many
genetic diseases (e.g. cystic fibrosis, sickle cell anemia,
phenylketonuria, muscular dystrophy).
Because of the sensitivity of PCR, this can be done from a single
cell taken from an embryo before birth.

21. 3- paternity testing:
The paternity test is essentially carried out by PCR.
A cheek swab is taken from inside the mouth of both parents and
the child. The DNA is extracted from the cells obtained and is
analyzed by PCR.
By amplifying specific DNA fragments from parents or close
relatives, it is possible to reconstruct relatedness between
individuals.
PCR can not only identify relationships between people today, but
can also be used to identify historical family relationships.

22. 4-Forensics:
PCR’s ability to amplify even the smallest amount of DNA from
samples collected at a crime scene gives the method great power
when used in criminal forensics.
The DNA from body fluid, hair, or other tissue samples is
amplified to create a nearly unique pattern for each individual.
This pattern can then be compared to suspects in the case.

23. 5-The role of PCR in cancer diagnostics:
Many cancers are characterized by small mutations in
certain genes, and this is what PCR is employed to identify.
For example, in acute myeloid leukemia (AML) the
presence of a mutation known as t(8:21) can indicate a good
prognosis, as certain drugs are known to be successful in
patients who carry this mutation.
The presence of a mutation in a gene called Flt3 can identify
those patients who are less likely to fully respond to
treatment with chemotherapy, and who have a high risk of
relapsing following treatment.
PCR can also be applied in monitoring leukemia patients
following treatment, by counting the number of cancerous
cells that are still circulating in their bodies.

24. :6- Cloning procedure
PCR is an essential technique in cloning procedure which allows
generation of large amounts of pure DNA from tiny amount of
template strand and further study of a particular gene.
PCR cloning differs from traditional cloning in that the DNA
fragment of interest, and even the vector, can be amplified by the
Polymerase Chain Reaction (PCR) and ligated together, without
the use of restriction enzymes.
PCR cloning is a rapid method for cloning genes, and is often used
for projects that require higher throughput than traditional
cloning methods can accommodate.
It allows for the cloning of DNA fragments that are not available in
large amounts.