Polymerase chain reaction is a method used widely in molecular biology to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail.

1. BY
OMKAR ABADHESH MISHRA
MSC ( BIOTECHNOLOGY)

2.  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.
 The method of PCR is designed to permit of
a selective amplification of a specific target
DNA sequence within a heterogenous DNA
molecule..

3.  Taq polymerase is first used in PCR in 1986
 Dr. Kary Mullis developed PCR in 1983
 Purified plification of and detection of specific
DNA sequence using a flourescent DNA
binding dye,laying the foundation for future
“real time” or “kinetic” PCR in1990.

4. Purpose:
To amplify a lot of double stranded DNA
molecule{fragments}with same or identical
size and sequence by enzymatic method
and cycling condition.

5. Condition:
 Denaturation of ds DNA
 Annealing of primers
 Extension of ds DNA

6.  Denaturation of ds DNA Template
• Temperature 92-94 c
• Double stranded DNA melts to single stranded
DNA

7. Annealing of primer:
• Tempreture 50-70 c [dependent on the melting
temperature of the expected duplex]
• Primers bind to their complementary
sequences.

8. Extension of ds DNA:
• Temperature 72 c.
• Time 0.5-3 min
• DNA polymerase binds to the annealed primers
and extends DNA at the 3’ end chain

9.  For example the widely used Taq
polymerase is obtained from Thermus
aquaticus and is thermostable upto 94 c with
an optimum working temperature of 75-80 c.
 It has an extension rate of 35 to 100
nucleotides per second at 72 c.
 Taq polymerase lacks 3’-5’ exonuclease
activity.
 When the Taq polymerase incorporates a
wrong dNTP, subsequent extension of the
strand either proceeds very slowly or stops
completely.

10.  This problem can be overcome by using
other thermostable DNA polymerase with 3’-
5’ proofreading exonuclease activity.

11. Nested PCR-:
 Nested PCR increases the specificity of DNA
amplification ,by reducing non specific
amplification of DNA.
Quantitative real time PCR-:
 Quantitative real time PCR is based on the
general principle of PCR, which is used to
amplify a target DNA molecule.

12. RT-PCR-:
 RT-PCR (Reverse Transcription PCR) is a
method used to amplify, isolate or identify a
known sequence from a cellular or tissue RNA.
Inverse PCR-:
 Standard PCR is used to amplify a segment of
DNA lies between two inward pointing primers.
 In contrast inverse PCR also known as inverted
PCR is used to amplify unknown DNA
sequences that flank one end of a known DNA
sequence and for which no primers are
available.

13. Some common application of PCR in various
fields are-:
Medical application:
 Genetic testing for presence of genetic
disease mutations e.g:
heamoglobinopathies, cystic fibrosis, other
inborn errors of metabolism.
 Detection of diseases causing genes in
suspected parents who act as carrier.

14.  Study of alternation to oncogenes may help
in coustemization therapy.
 Can also be used as part of a sensitive test
for tissue typing,vital to organ transplantation
genotype of embryo.
 Helps to monitor the gene in gene therapy.
Infectious Diseases application-:
 Analyzing clinical speciemens for the
presence of infectious agents including
HIV,Hepatitis,Malaria,Tuberoculosis etc.

15. Forensic Applications-:
 Can be used as a tool in genetic
fingerprinting.
 This technology can be identified any one
person from millions of other in case of crime
scenes, police investigation, paternity testing
etc.
Research & Molecular Genetics-:
 In genomic studies
 In phylogenetic analysis
 In gene expression analysis
 In HGP {Human Genome Project}

16. THANK YOU
OMKAR ABADHESH MISHRA
MSC ( BIOTECHNOLOGY)