PCR is a technique used to amplify specific DNA sequences. It requires a DNA sample, two primers that bind to the ends of the target sequence, nucleotides, and a heat-stable DNA polymerase like Taq polymerase. The reaction involves cycles of heating and cooling: denaturation separates DNA strands, annealing allows primers to bind, and extension synthesizes new strands. Automated thermal cyclers control the cycling process. PCR has many applications, including diagnosing genetic diseases from small DNA samples, DNA fingerprinting for forensics, and studying DNA polymorphisms.

1. POLYMERASE CHAIN REACTION(PCR)
Saji Mariam George
Associate Professor
Assumption College Autonomous
Changanacherry

2. POLYMERASE CHAIN REACTION
Karry Mullis (1985)
 Used for selective amplification of DNA sequences in a genome.
Requirements
 DNA sample containing the desired segment to be amplified.
 Two nucleotide primers (20 bp long) specific for the two 3’ ends of
the desired segment.
 Four deoxyribonucleoside triphosphates-
d ATP, d GTP, d CTP, d TTP
 A heat stable DNA polymerase
Taq polymerase ( isolated from the bacterium Thermus aquaticus)
Pfu ( from Pyrococcus furiosus)
Vent (from Thermococcus litoralis )

3. STEPS
1.DENATURATION – The reaction mixture is heated to a
temperature (usually 90 – 98⁰C→ denaturation of
DNA
2.ANNEALING – The mixture is cooled to 40 ⁰C- 60⁰C - →
annealing of the primer to the complementary
sequences in the DNA
3.EXTENSION OR POLYMERIZATION – The temperature is
adjusted (For Taq polymerase – optimum 70⁰C - 75
⁰C) – DNA polymerase synthesizes the
complementary strands – first cycle is completed.
Repeat 20 – 30 times.
Automated PCR machines – Thermal Cyclers – specify
the Number, duration of cycles etc. after placing the
complete reaction mixture for incubation.

4. PCR
Image: https://www.xxpresspcr.com/

6. APPLICATIONS OF PCR
• In vitro amplification of genes and other
DNA sequences.
• To obtain definitive structural data on genes
and DNA sequences when very small
amounts of DNA are available.

7. • Diagnosis of inherited human diseases,
especially in case of prenatal diagnosis,
where limited amounts of fetal DNA are
available.
E.g. Sickle cell anemia
Tay – Sachs disease ( a lethal autosomal
recessive disorder- normal at birth but
undergo rapid neurological degeneration).
Cystic fibrosis – due to autosomal recessive
mutation – salty sweat, infections of
lungs,pancreas, liver etc.

8. • In Forensics – PCR DNA Finger Printing
experiments - important role in legal cases
involving uncertain identity - identification
of individuals by using minute amounts of
DNA isolated from very small samples - a
few drops of blood, semen, hair follicle etc.

9. • To study DNA polymorphism(an inheritable
mutation observed in a population at high
frequency) in the genome using known
sequences as primers which generates RANDOM
AMPLIFIED POLYMORPHIC DNA (RAPD -
Molecular marker), which is detected as bands
after electrophoresis. RAPD bands of different
strains or species can be compared. The data
can be used to construct RAPD maps (e.g. Maize,
Soy bean, Mouse, Man etc.)

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