Polymerase chain reaction

1. POLYMERASE CHAIN REACTION
(PCR)
Prepared by:
Ibrahim mussa
B.pharmacy 6th semester

2. POLYMERASE CHAIN REACTION
(PCR)
• Polymerase chain reaction (PCR) is technique for
generating large quantities of a specified DNA.
• PCR is a cell free amplification technique for
synthesizing multiple identical copies of any DNA of
interest

3. PRINCIPLE OF PCR
 The double-stranded DNA of interest is denatured to
separate into 2 individual strands.
 Each strand is allowed to hybridize with a primer
(renaturation).
 The primer-template duplex is used for DNA
synthesis (DNA polymerase).
 Denaturation, renaturation & synthesis are repeated
again & again to generate multiple forms of target
DNA.

4. TECHNIQUE FOR PCR
Requirements for PCR:
a. A target DNA (100-35,000 bp in length).
b. Two primers (synthetic oligonucleotides of 17-30
nucleotides length) that are complementary to
regions flanking the target DNA.
c. Four deoxyribonucleotides (dATP, dCTP, dGTP,
dTTP).
d. A DNA polymerase that can withstand at a
temperature up to 95˚C.

5. STAGES OF PCR
1. Denaturation
2. Renaturation or annealing
3. Synthesis
 PCR involves repeated cycles for amplification of target
DNA.

6. Denaturation
• On increasing the temperature to about 95˚C
for 1 minute, the DNA gets denatured & two
strands separate.

7. Renaturation or annealing
As the temperature of mixture is slowly cooled to
about 55˚C, the primers base pair with
complementary regions flanking target DNA strands.

8. 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 strands.
 The synthetic process is comparable to DNA replication of
leading strand.
 Optimum temperature has to be maintained as required by
DNA polymerases.

9. GRAPH OF PCR

10. example
• Taq DNA polymerase, optimum temperature is
around 75˚C & E.coli DNA polymerase around
37˚C.
• The reaction can be stopped by raising the
temperature (about 95˚C).

11. Sources of dna polymarase
• Klenow fragment of E.coli DNA polymerase is used
in original technique. This enzyme, gets denatured at
higher temperature, therefore, fresh enzyme had to be
added for each cycle.
• Taq DNA polymerase (Thermus aquaticus) is heat
resistant, not necessary to freshly add this enzyme for
each cycle.

12. APPLICATIONS
• Prenatal diagnosis of inherited diseases: PCR is used
for prenatal diagnosis of various diseases by using
chorionic villus samples or cells from amniocentesis.
• Sickle-cell anemia, β-thelassema & PKU can be
detected.
• Diagnosis of retroviral diseases: Used for diagnosis
of HIV infection

13.  Diagnosis of bacterial infections: Used for the
detection of bacterial infections like tuberculosis.
 Diagnosis of cancers:Used for the detection of
cervical cancer.
 PCR in forensic medicine: Used for the identification
of criminals.