Polymerase Chain Reaction

1. SCHOOL OF PHARMACEUTICAL SCIENCES
SIKSHA ‘O’ ANUSANDHAN (DEEMED TO BE UNIVERSITY)
PRESENTED BY
RANDEEP PATRO
M.PHARM(1ST SEM)
PHARMACOLOGY

2. The polymerase chain reaction is a laboratory
invitro cellfree amplification technique for generating
large quantities of identical copies of any DNA .
PRINCIPLE :
1) Denaturation :
Double strand of DNA is denatured at 90-98℃ for
1-2min to form single strands by disrupting the
hydrogen bonds between complementary bases .
2) Renaturation :
Each strand are allowed to hybridize with a primer by
cooling the reaction mixture at45-60 ℃ for 1 min .
3) Synthesis :
By shifting temp upt 72℃ for 2 min primers are
extended by joining the bases complemetary to
DNA strand .

3. Requirements :
 Targeted DNA/RNA
 Primers – 17to30 nucleotide are complemetary
to ergions flanking the target DNA.
 PCR buffer
 dNTPS- dATP,dGTP,dCTTP,dTTP
 Polymerase – Taq DNA polymerase,Tma polymerase from
Themotoga maritama, Pfu Polymerase from Pyrococus furiosus
Critera for primer
• Uniqueness-complementary towards DNA template
• Length-not so long and not so small
• Melting temp.- DNA m.p and Primer m.p should not be same
• Internal structure- avoid hairpin sequence i.e loop formation
• Avoid primer primer interaction
• A balanced distribution of G/C and A/T domains

5. Various PCR ;
Nested PCR
It involves two sets of primer,use of
nestsed primer increases specificity
of PCR by binding with targeted
sequence from the 1st PCR product
and the undesired product can’t amplified.
Inverse PCR
Amplification of DNA of the unknown
sequence is carried out from known
sequence and Primers are generated in
the opposite direction to normal.

6. Reverse Transcriptase PCR
• Used to detect RNA expression.
mRNA to cDNA by reverse
transcriptase enzyme.
• Genetic diaseases diagnoised and
study of viruses whose genetic
info. Is stored in RNA .
Asymetric PCR
• The amount of primer used for targeted strand is much more than
that of nontargeted.
• Lower conc. of limited primer is used.
Real-time PCR
Use of fluoresence compound like ehidium bromide binds with
dsDNA emits fluorescence.

7. Advantages
 Simplicity procedure
 Sensitivity of procedure
 Rapid
 Require small amount of DNA
 Involves no radio active assay
Disadvantages
Cross contamination
High sterile environment
High equipment cost
Require technical skill
Low reproducibility
Applications
Parentral diagnosis of inherited diaeases like sickle cell anemia
Diagnosis of retroviral infections like HIV infection
Diagnosis of bacterial infections e.g – tuberoculosis
Diagnosis of cancers(cervical) which occure due to
chromosomal translocation
Sex determination of embryos and sex-linked disorders in
fertilized embryos

8.  PCR in DNA sequencing
By asymmetric PCR process amplification of a single strand is
carried out and strand removal achieved by use of exonuclease
enzyme .
 Gene Expression Studies
Important in the study of mRNAs,the products of gene expression
which is carried out by reverse transcription PCR
 Comparative study of Genomes
Difference in genomes of two organisms can be measued by PCR
with random primers.
 In Forensic Medicine
Minute quantities of DNA from any source (hair,minute
tissues,blood,semen) of an individual is adequate for PCR which
is useful to identify criminals.
 In comparison with Gene Cloning
Due to minute quantities of sample requirement,low cost,minimal
technical skill and less time consuming