this ppt contain about pcr technique and its three process,primers in pcr,dna polymerase in pcr,melting temp of dna in pcr and applications of pcr technology

1. Polymerase Chain Reaction
(PCR)
BY
RAJU

2. What is the PCR?
PCR is the technique that take specific sequence of DNA and
amplifies it to be used for further testing.
Purpose of the PCR is to amplify the lot of double stranded
DNA molecules(fragments) with same size and sequence by
enzymatic method and cycling condition.

3. PCR reaction :-
Essential components required:
DNA template
DNA polymerase
Primer
dNTPs
Buffer to maintain PH
Divalent cations (Mg+2)

4. principle :-
PCR contain three process
1. Denaturation
2. Annealing
3. Extension
cycle repeated according to requirement

5. Principle :-
Denaturation
Denaturation is the first step of PCR , in which the DNA strands are separated by
heating to 94–98 °C for 20–30 seconds.
It causes DNA melting of the DNA template by breaking the hydrogen bonds
between two strands and resulting single-stranded DNA molecules.

6. Principle :-
Annealing
The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing
annealing of the primers to the single-stranded DNA template.
This temperature must be low enough to allow for hybridization of the primer to the
strand, but high enough for the hybridization to be specific, i.e., the primer should
only bind to a perfectly complementary part of the template. If the temperature is
too low, the primer could bind imperfectly. If it is too high, the primer might not
bind.

7. Principle :-
Annealing
Typically the annealing temperature is about 3–5 °C below the melting
temperature of the primers used.
Stable DNA–DNA hydrogen bonds are only formed when the primer sequence
very closely matches the template sequence.
The polymerase binds to the primer-template hybrid and begins DNA formation.

8. Principle :-
Extension
The temperature at this step depends on the DNA polymerase used. Taq DNA
polymerase has its optimum activity temperature at 75–80 °C, and commonly a
temperature of 72 °C is used with this enzyme in this step.
The extension time depends both on the DNA polymerase used and on the length
of the DNA fragment to amplify.

9. Principle :-
Extension
At this step the DNA polymerase synthesizes a new DNA strand complementary
to the DNA template strand by adding dNTPs that are complementary to the
template in 5' to 3' direction.
Primer are extended by adding nucleotides (dNTPs) complementary to the DNA
template strand.
Now the first cycle over and second cycle continued, as PCR machine is
automated thermocycler the same cycle repeated up to 30-40 times.

10. PCR reaction steps :-
Step-1
Denaturation
Step-2
Annealing
Step-3
Extension

12. PCR (contd…)
Primer in PCR
Primer are usually short, chemically synthesized oligonucleotides, with a length of
about 18-30 bases, Ideal G-C content 40-60% but play around the length.
Melting temperature should be between 42-65°C
Primers serve as a starting point for DNA synthesis and they are hybridized to the
target DNA, which is then copied by DNA polymerase.
Avoid primer-primer hybridization and pick the primer that do not form hairpins.

13. PCR (contd…)
Taq DNA polymerase in PCR
Taq polymerase is a thermostable DNA polymerase named after thermophilic
bacterium Thermus Aquaticus from which it was isolated.
Taq’s optimum activity between 75-80°C and in extension step applied
temperature is 72°C in PCR.
Drawbacks of Taq is relatively low replication fidelity, it is because lacks of
exonuclease proofreading activity.
Extension rate - 50 nucleotides/second.It has error rate measured at about 1 in
9,000 nucleotides.

14. PCR (contd…)
Pfu DNA polymerase in PCR
Pfu polymerase is a thermostable DNA polymerase named after hyperthermophilic
archaeon pyrococcus furiosus from which it was isolated.
Optimum activity of Pfu is between 72°C .
It has 3’ to 5’ exonuclease proofreading activity, this means Pfu polymerase
generate few errors than Taq polymerase.
Extension rate - 1000 nucleotides/minute.It has error rate measured at about 1 in
1.3 million nucleotides.

15. PCR (contd…)
DNA melting
The melting temperature defined as the temperature at which half of the DNA
strands are in the single-stranded (ssDNA) state.
A=T less energy required to break AT bonds
G=C more energy required to break GC bonds

16. APPLICATIONS OF PCR
1. Diagnosis of genetic diseases
The use of PCR in diagnosing genetic disease, whether inherited genetic changes or as a
result of a spontaneous genetic mutations, is becoming more common. Diseases can be
diagnosed even before birth. Examples include:
Genetic counseling-screening the parents for genetic diseases before deciding on having
children.

17. APPLICATIONS OF PCR (contd…)
2. Genetic fingerprints
One of the most famous uses for PCR is in the creation of a genetic fingerprint (also known
as DNA profiling) from a sample of blood or semen, or from a hair root. Much beloved by
writers of detective fiction, genetic fingerprints are profiles of specific stretches of DNA that
vary from person to person. PCR also plays a role in mitochondrial DNA analysis, used for
samples from hair shafts and bones when other samples are not available.
Genetic analysis based on PCR is also used in paternity testing, and in tissue typing for
organ transplantation.

18. APPLICATIONS OF PCR (contd…)
3. Detection and diagnosis of infectious diseases
PCR can detect infectious disease before standard serological laboratory tests (tests to
detect the presence of antibodies), so allowing treatment to start much earlier. Because of
this, PCR is also useful for screening donated blood for infections, and is especially useful for
infections that are difficult to culture in the laboratory, such as tuberculosis.

19. APPLICATIONS OF PCR (contd…)
4. Detection of infection in the environment
PCR is used to monitor and track the spread of infectious disease within an animal or human
population. PCR can also be used to detect bacterial and viral DNA in the environment, for
example looking at pathogens in water supplies.
5. Personalised medicine
PCR is used in personalised medicine to select patients for certain treatments, for example
in cancer when patients have a genetic change that makes a patient more or less likely to
respond to a certain treatment.

20. APPLICATIONS OF PCR (contd…)
6. PCR in research
Escherichia coli in genetic engineering, and to amplify stretches of genetic material for
Sanger sequencing – the Human Genome Project used PCR.
PCR can be used in analysis of gene expression, for example looking at levels of expression
and when genes are switched on and off in physiological processes, including in health and
disease.