polymerase chain reaction

1. POLYMERASE CHAIN REACTION
(PCR) AND ENZYME-LINKED
IMMUNOSORBANT ASSAY (ELISA)
SUBMITTED BY: ZAMANN WAHEED (70125359)
SUBMITTED TO: DR SHANEEL

2. POLYMERASE CHAIN REACTION (PCR)
 PCR is a technique used in molecular biology to amplify a single copy or a few
copies of DNA segment to millions of copies of a particular DNA sequence.
 PCR is developed in 1983 by Kary Mullis.
 In 1993, Mullis was awarded the Nobel prize in chemistry for his work on PCR.
 It is in-vitro enzymatic DNA Replication techniques.

3. COMPONENTS OF PCR
The following components are required for PCR
1. DNA template
1. DNA template is a target DNA sequence which is to amplified or sample
DNA that contains the target sequence.
2. It can be isolated from blood, tissue, hair, cultured cell and so on.
2. Primers
Short oligonucleotides
It is synthetic DNA stand of about 18-25 nucleotides complementary to 3’
end of template stand.
GC content of primer should be 40-60%
Two types of primers
Forward primers- complementary to 3’end of Anti sense stand
Reverse primers- complementary to 3’end of sense stand

5. 3. Nucleotides
• All nucleotides i-e Adenine (A) , Thymine( T), Guanine (G), and
cytosine( C ) are essentially "building blocks" for new DNA
strands.
4. Taq polymerase
• Taq polymerase isolated from Thermus Aquaticus ( Thermophilic
Bacterium).
5. Magnesium ion
• Magnesium act as Cofactor for DNA polymerase and hence
required for reaction.
6. Temperature Regulatory Machine (PCR machine)
• Used to Regulate the temperatures thus help in automation of
process
7. PCR tubes
• Thin walled plastic tubes or plates and used in DNA extraction.

6. PROCEDURE
• There are three Major steps in PCR. It can be done on an automated
cycler.
1. DENATURATION
During Denaturation, Reaction mixture is heated to 94°C for 1min cause
separation of DNA double stranded. Now each stand act as template for
synthesis of complementary stand.

7. 2. ANEALING
• During Annealing, reaction mixture is cooled at 54°C for 1.5min which
cause hybridization of primers to separate stand of DNA. The length
and GC binding content of the primer should be sufficient for stable
binding with template.

8. 3.EXTENSION
The reaction mixture is heated to 72 °C which is ideal working
temperature for Taq polymerase. Taq polymerase add nucleotides (dNTP)
complementary to template on 3’-OH of primers thereby extending new
stand.

10. It is used for the confirmation of test results of ELISA.
PCR APPLICATION
Biomedical
Research
Cancer Biology
(gene expression/
mutation)
Crime scene
investigation
Forensic
Paternity testing
Evolutionary
studies
Diagnose
Genetic and
viral diseases
(TB, Sickle cell
anemia)

11. ELISA
(ENZYME-LINKED IMMUNOSORBENT ASSAY)
 It is an immunological assay commonly used to measure antibodies,
antigens, proteins, hormones and glycoproteins in biological samples.
 ELISA is a distinguished analysis compared to other antibody-assays as it
yields quantitative results.
 Commonly used analytical biochemistry assay, first described by Eva
Engvall and Peter Perlmann in 1971.
 The assay uses a solid-phase type of enzyme immunoassay to detect the
presence of a ligand in a liquid sample using antibodies directed against
the protein to be measured.
 Used in diagnosis of HIV infection, pregnancy tests, and measurement of
cytokines or soluble receptors in cell supernatant or serum.

12. PRINCIPLE:
 ELISA works on the principle that specific antibodies bind the target
antigen and detect the presence and quantity of antigens binding. In order
to increase the sensitivity and precision of the assay, the plate must be
coated with antibodies/antigen with high affinity.
 ELISA can provide a useful measurement of antigen-antibody
concentration.
 ENZYMES used;
• The most commonly used enzyme labels are horseradish peroxidase (HRP)
and alkaline phosphatase (AP). Other enzymes have been used as well;
these include β-galactosidase, acetylcholinesterase, and catalase.

13. APPARATUS
 Generally the following apparatus is used for ELISA :
• ELISA Kits
• Pipettes
• Pipette tips
• Microplates
• Spectrophotometer
• Well wash microplate washer
 ELISA assays are generally carried out in 96 well plates, allowing multiple
samples to be measured in a single experiment.
 These plates need to be special absorbent plates (e.g. NUNC
Immunoplates) to ensure the antibody or antigen sticks to the surface.
 Each ELISA measures a specific antigen, and kits for a variety ofantigens
are widely available.

14. PROCEDURE
• The substrate is converted by the enzyme to form a coloured product, which can be
measured by spectrophotometry.

15. TYPES OF ELISA
ELISA tests can be classified into Four types depending upon the different methods used
for binding between antigen and antibodies, namely:
 Direct ELISA - (antigen-coated plate; screening antibody)
 Indirect ELISA – Antigen is coated to the microtiter well
 Sandwich ELISA – Antibody is coated on the microtiter well
 Competitive ELISA – Microtiter well which is antigen-coated is filled
with the antigen-antibody mixture

17. EXAMPLES
• Antibodies against bacterial, viral or fungal infections: Examples of
bacterial infections include Lyme disease and syphilis. Examples of viral
infections include HIV and hepatitis A, B and C.
APPLICATION
• The presence of antibodies and antigens in a sample can be
determined.
• It is used in the food industry to detect any food allergens present.
• To determine the concentration of serum antibody in a virus test.
• During a disease outbreak, to evaluate the spread of the disease.
For Example, during recent COVID-19 outbreak, rapid testing
kits are being used to determine presence of antibodies in the
blood sample.

18.  REFERENCE
https://www.immunology.org/public-information/bitesizedimmunology/
experimental-techniques/enzyme-linked-immunosorbent-assay
https://byjus.com/biology/elisa-technique/
THANK YOU.