PCR is a technique used to amplify specific DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to denature the double-stranded DNA, anneal primers, and extend new strands using a DNA polymerase. This process is repeated many times, exponentially amplifying the target DNA sequence. PCR is widely used in scientific research and forensic analysis.

2. PCR is the technique for
generating large quantities of
specific DNA .
PCR is a cell free amplification
technique to synthesizing multiple
identical copies of the DNA .
It utilizes the principle of DNA
replication.

3. In 1993 Kary Mullis was awarded the
Nobel Prize for the discovery of PCR.
 In 1983 Karry mullis
conceived the idea of PCR.
Developed in 1984.

4. The double stranded DNA of interest is denatured
to separate into two individual strands.
Each strand is then allowed to hybridize with a
primer (renature).
The polymerase enzyme that starts synthesizing new
strands
These three steps are repeated to get more copies of
DNA

5. copies of DNA
synthesis
Renaturat
ion or
annealing
denaturation

6. Target DNA (100-35,000 bp in length)
Primers (synthetic oligonucleotides of 17-30 nucleotides in length )
Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP)
Thermo stable DNA polymerase that can withstand
at a temperature upto 95 C (derived from Thermus
aquaticus)

7. •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.

8. Major processes in PCR
Denaturation:
The temperature is raised at 94–98 C for 1minute to separate
the double stranded DNA.
Renaturation:
decrease the temperature at 55 C . This helps the primer to
bind with target DNA . This step is also known as annealing.
Synthesis :
The initiation of DNA synthesis occurs at 3’- hydroxyl end of
each primer. The primers are extended by joining the bases
complementary to DNA at 75 C.
Note : This whole steps are considered as one cycle

9. Initializationstep: This step consists of heating the reaction to a
temperature of 94–96 C (or 98 C if extremely thermostable polymerases are
used), which is held for 1–9 minutes. It is only required for DNA polymerases
that require heat activation by hot-start PCR.
Denaturation step: This step is the first regular cycling event and
consists of heating the reaction to 94–98 C for 20–30 seconds. It causes DNA
melting of the DNA template by disrupting the hydrogen bonds between
complementary bases, yielding single-stranded DNA molecules.
Annealing step: The reaction temperature is lowered to 50–65 C for 20–
40 seconds allowing annealing of the primers to the single-stranded DNA
template. Typically the annealing temperature is about 3-5 degrees Celsius
below the Tm 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 synthesis.

10. Extension/elongation step: The temperature at this step depends on the
DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–
80 C,and commonly a temperature of 72 C is used with this enzyme. 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, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl
group at the end of the nascent (extending) DNA strand. The extension time
depends both on the DNA polymerase used and on the length of the DNA fragment
to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA
polymerase will polymerize a thousand bases per minute. Under optimum
conditions, i.e., if there are no limitations due to limiting substrates or reagents, at
each extension step, the amount of DNA target is doubled, leading to exponential
(geometric) amplification of the specific DNA fragment.
Final elongation: This single step is occasionally performed at a temperature
of 70–74 C for 5–15 minutes after the last PCR cycle to ensure that any remaining
single-stranded DNA is fully extended.
Final hold: This step at 4–15 C for an indefinite time may be employed for
short-term storage of the reaction.

12. Thermo cycler
This whole process was done by using an automated machine called as
thermo cycler. It can raises and lowers the temperature automatically.
The PCR is commonly carried out in 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 to achieve the
temperatures required at each step of the reaction
Modern thermo cycler
An older model three-temperature
thermal cycler for PCR

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temperature
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temperature vs time

14. Nested PCR
Nested primers increases the
specificity and selectively amplifies
the target DNA.
Inverse PCR
Can be study the unknown
sequences using known sequence.
Anchored PCR
this is particularly useful when the
sequence surrounding the target is
not known. It can be done by using

15. Reverse transcription PCR
The mRNA converted to cDNA by reverse
transcriptase , this cDNA serve as the
template for PCR
Real time PCR
Commonly used technique for measuring
the quantity of
DNA by employing fluorescence
compound ethidium bromide.
Asymmetric PCR
This technique can be used for the
synthesis of single stranded DNA
, particularly used for DNA sequencing.

16. PCR in clinical diagnosis:
Prenatal diagnosis of inherited
diseases:
Using chorionic villus samples or
cell from amniocentesis. Thus
diseases like sickle-cell
anemia, β- thalassemia can be
detected by PCR.
Diagnosis of retroviral and
bacterial infections:
PCR from cDNA is valuable tool for

17. PCR in comparative study of
genomes:
The differences in the genomes
can be measured after
electrophoresis. The closely
related organisms can give
similar bands.
PCR is very useful in the study of
evolutionary biology, more
specifically referred as
phylogenetics.

18. PCR IN FORENSICS:
A single molecule of DNA from any
sources like blood ,hair, small tissue
etc can be amplified by PCR.
The PCR is useful in the DNA finger
printing technology.