Is a Molecular, Biochemical technique, Used to amplipy a piece of DNA for further testing.and cure for many disease.

1. By:- Vinita Jagat
B.Sc (Biotechnology)
Polymerase Chain
Reaction (PCR)

2. Contents
 What is PCR ?
 History of PCR
 Principle of PCR
 PCR Machine:- Thermocycler
 PCR Components
 Basic requirementsfor PCR reaction
 Mechanism of PCR
 Advantage and Disadvantages of PCR
 Application and it’s limitations

3. What is PCR ?
 PCR is a technique that takes specific sequence of
DNA of small amount and amplifies it to be used
for further testing .
 In-vitro technique.
 Is a Molecular, Biochemical technology, Used to
amplify a single piece of DNA, By a series of
Heating and Cooling cycles, generating millions
of copies of a particular DNA sequence In Vitro.

4.  DNA template:- contains the DNA region (target)
to be amplified.
 DNA Primer:- Complementary to the 3’ (three prime) ends
of each of the sentences and anti-sence
strand of the DNA target.

5. History of PCR
In 1983, Dr.Karry Mullis described the technique of
in vitro gene amplification and named it as a Polymer
- ase Chain Reaction. Later on, he was awarded the
Noble prize for his findings.
 1985:- First publication of PCR by Cetus Corporation
appears in science.
 1986:- purified Taq polymerase is first used in PCR.
 1988:- PerkinElmer introduces the automated thermal cycler.
 1989:- Science declares Taq polymerase “molecules” of the
year.

6.  1990:- Amplification and detection of specific DNA
sequence using a fluorescent DNA- binding dye,
laying the foundation for future “real-time “ or
“kinetic” PCR.
 1993:- Dr.Karry Mullis shares Noble prize in Chemistry for
conceiving PCR technology.

7. Principle of PCR
Purpose
To amplify a lot of double – stranded DNA molecules
(fragments) with same (identical) size and sequence
enzymatic method and cycling condition.
The principle of the PCR is based on the
temperature variations of heating and cooling –
thermocyclling reaction divided into three
steps :-

8.  Denaturation:- The dsDNA becomes single stranded at a higher
temperature during denaturation. Here hydrogen
bonds betweentwo DNA strands break.
 Annealing:- In the primer binds or anneals to its exact complem
-entry sequence on a DNA during the annealing steps.
The primer provides a site for the initiation of synthesis.
 Extension:- Taq DNA polymerase uses the 3’end of the primer and
starts DNA synthesis by adding nucleotides to the growing
DNA strand.
All three steps are repeated for 25 to 40 cycles and in each
cycle the DNA becomes double.

9. PCR Machine:- Thermocycler
The machine thermocycler provides
various temperature for each step to
complete Denaturation, annealing and
extension of DNA occur at different
temperature thus the machine is known
as Thermocycler.

10. PCR Components
 Chemical:- dNTPs, distill water, PCR reaction buffer, enzyme Taq
DNA polymerase, primers and template DNA.
 Instruments:- Thermocycler, spinner and agarose gel electrophoresis
unit.
 Other utilities:- PCR tubes, stands, pipettes , tips.
 PCR reagents:- Template DNA, PCR primers, dNTPs, Taq DNA polyme
rase and PCR buffer are the major reagents of PCR
reaction. The composition and quantity of each reagent
are very important. A single ųL variation in any
of the reagents leads to reaction failure.

11. Basic Requirements for PCR
reaction
 DNA sequence of target region must be known.
 Primer:- typically 20 – 30 bases in size. These can be readily
produced by commercial companies.
 Thermo – stable DNA polymerase:- Taq polymerase
which is not inactivatedby heating to 95°C.
 DNA thermocycler:- machine which can be programmed
to carry out heating and cooling of sample
overa number of cycles.

12. Mechanism of PCR
Various temperature zone governs each PCR steps, viz
Denaturation, annealing and extension followed by a single
initial denaturation and final extension steps.
In each step, different reactions occurs.
 Denaturation:- Tem: 90°C to 95°C.
Time: 30 seconds to 90 sec.

13. In a denaturation two single – stranded DNA forms from the double –
stranded one. At 94°C temperature,the double – stranded DNA opens up
by breaking hydrogen bonds. The process of denaturationis followed by
the initial denaturation for 5 to 7 minutes at the same temperature.
 Annealing:- Temperature:- 55°C to 65°C.
Time:- 30 to 60 sec.
Primer bind to their complimentary sequences.

14. After the denaturationprimer anneals to ssDNA at its exact
annealing temperature. Base on the GC content of primer, every primer
has its own annealing temperature.
The annealing temperature is usually raging from 55°C to 65°C .
Annealing temperature lower than that leads to non-specific binding
while highertemperature leads to amplification failure. 45 seconds to 1
minute are enough for the second step, annealing for more than 1minute
causes non-specific amplification.
 Extension:- Temperature: 70°C to 72°C
Time: 45 sec

15. After the binding of the primer, it’s time to expand the DNA
strand. Here in extension step the Taq DNA polymerase comes in action
and add dNTPs to the DNA strand. The temperaturefor the extensionis
72°C for 45 seconds.
After completingall steps one more time the final extension is
performed for 7 minutes. The graphical representation of each PCR steps is
explained in the figure below:

17. Advantage of PCR
 Small amount of DNA is required for test.
 Result obtained more quickly- usually within 1day for PCR.
 Usually not necessary to use radioactivematerial (32P) for PCR.
 PCR is much more precise in determining the size of alleles – essential for same disorders.
 PCR can be used to detect point mutation.
 Robust , making it possible to amplify DNA from degraded samples.
Disadvantages of PCR
 Prior sequence knowledge.
 Short size range of amplification products 100bp – 5000 bp.
 Chance of Contamination.
 Novel mutation can not be found using PCR, we have to do sequencing for that.

18. Application of PCR
The PCR has numerous applications in biological research as well as
diagnostics.
 Diagnosis of inherited disease: the PCR is most routinelyused in the
diagnosis of some inheriteddisease such as
sickle cell anemia, thalassemia, gene mutation, etc.
This technique is appropriate for single – gene disorders.
 Microbial identification: the microbial culture technique is traditional and
time consumingand the chance of infection is also high
in the case of culturing. In modern days, PCR is used in the
identificationof microbes.

19.  DNA Fingerprinting and genetic imprinting:The PCR is first choice
for DNA Fingerprinting. Criminal
verification, identification of a person, and material
cell contaminationcan be detected using DNA
Fingerprinting.
 The PCR is one of the best techniques for marker assistant selection. RFLP,
AFLP, RAPD ,VNTR and STR are some of the PCR based techniques.
 PCR helps in detecting cancer genes and infections.
 Further PCR is applicable to sex determination and sex identification.
The PCR is used in:
 Gene editing.
 Geneticengineering.
 DNA and RNA quantification.

20.  cDNA and gDNA library preparation.
 Developingnew assays.
Limitations of PCR
 Also Multigenic disorders cannot be
detected using PCR.
 We cannot identify structural and numerical
chromosomal anomalies through PCR.