PCR Rt- PCR QPCR

1. POLYMERASE
CHAIN
REACTION
(PCR)
Chethan N
M. Pharm
JSSCP
Mysuru

2. INTRODUCTION
 PCR, polymerase chain reaction, is an in-vitro
technique for amplification of a region of DNA
whose sequence is known or which lies between
two regions of known sequence
 PCR can be regarded as a form of molecular
cloning since it is a technique similar to DNA
replication that occurs in the cells

3. REACTION COMPONENTS
 DNA template
 Primers
 Enzyme
 dNTPs
 Buffers

4. 1- DNA TEMPLATE
• DNA containing
region to be
sequenced
• Size of target DNA
to be amplified :
up to 3 Kb

5. 2- PRIMERS
• 2 sets of primers
• Generally 20-30
nucleotides long
• Synthetically
produced
• complimentary to
the 3’ ends of target
DNA
• not complimentary
to each other

6. 3-ENZYME
• Usually Taq Polymerase or anyone of
the natural or Recombinant
thermostable polymerases
• Stable at T0 up to 950 C
• High processivity

7. THE PCR CYCLE
Contains of 3 steps:
- Denaturation of DNA at 950C
- Primer hybridization ( annealing) at 40-
500C
- DNA synthesis ( Primer extension) at
720C

12. TARGET AMPLIFICATION
 The target DNA is amplified into multiple copies in a
exponential pattern with repeated cycles of PCR
and multiple copies of the DNA are produced

13. REVERSE TRANSCRIPTION PCR
• Reverse Transcriptase PCR
• Uses RNA as the initial template
• RNA-directed DNA polymerase (rTth)
• Yields ds cDNA

17. DETECTION OF AMPLIFICATION PRODUCTS
• Gel electrophoresis
• Sequencing of amplified fragment
• Southern blot
• etc...

18. APPLICATIONS
• Amplification of small amounts of DNA for further
analysis by DNA fingerprinting.
• The analysis of ancient DNA from fossils.
• Mapping the human (and other species) genome.
• The isolation of a particular gene of interest from a
tissue sample.
• Generation of probes: large amount of probes can
be synthesized by this technique.
• Production of DNA for sequencing: Target DNA in
clone is amplified using appropriate primers and
then its sequence determined. Helpful in
conditions where amount of DNA is small.

19. • Analysis of mutations: Deletions and insertions in
a gene can be detected by differences in size of
amplified product.
• Diagnosis of monogenic diseases (single gene
disorders): For pre-natal diagnosis, PCR is used
to amplify DNA from foetal cells obtained from
amniotic fluid. PCR has also proved very
important in carrier testing.
• Detection of microorganisms: Especially of
organisms and viruses that are difficult to culture
or take long time to culture or dangerous to
culture.

20.  The PCR has even made it possible to analyze DNA
from microscope slides of tissue preserved years
before.
 Detection of microbial genes responsible for some
aspect of pathogenesis or antibiotic resistance.
 Crucial forensic evidence may often be present in very
small quantities, e.g. one human hair, body fluid stain
(blood, saliva, semen). PCR can generate sufficient
DNA from a single cell.

21. ADVANTAGES
• Automated, fast, reliable (reproducible)
results
• Contained :(less chances of
contamination)
• High output
• Sensitive
• Broad uses
• Defined, easy to follow protocols

22. • Real-time polymerase chain reaction,
also called quantitative real time
polymerase chain reaction is a
laboratory technique based on the PCR,
which is used to amplify and
simultaneously quantify a targeted DNA
molecule.
Real-time PCR

23. • Real-Time PCR permits the analysis of the
products while the reaction is actually in
progress. This is achieved by using various
fluorescent dyes which react with the
amplified product and can be measured by
an instrument. This also facilitates the
quantitation of the DNA.
• Quantitative PCR (Q-PCR), as this
technique is known, is used to measure the
quantity of a PCR product

24.  It is a refinement of conventional polymerase chain
reaction methods that can be used to directly quantify
and clonally amplify nucleic acids including DNA, cDNA
or RNA.
 The main advantage being the rapidity of the complete
process, as there is non requirement of any further
quantitationas both the processess occur simultaneously

25. PROCESS OF QUANTIFICATION OF DNA
 A DNA binding dye such as SYBR green is
introduced into the reaction mixture of PCR.
 Then the PCR cycles are started.
 As amplicons accumulate, SYBR green binds to the
ds DNA proportionally.
 Fluorescence emission of the dye is detected
following excitation.

26.  The binding SYBR green is non specific. Therefore, in
order to detect specific amplicons an oligonucleotide
probe labeled with a fluorescent reporter and quencher
molecule at either end is included in the reaction in the
place of SYBR green.
 When the oligonucleotide probe binds to the target
sequence, the 5’ exonuclease activity of Taq polymerase
degrades and releases the reporter from the quencher.

27.  A signal is generated which increases in direct
proportion in no. of starting molecule.
 Hence the detection system is able to induce and
detect fluorescence in real time as the PCR
proceeds.

28. APPLICATIONS
 Gene expression analysis
 Cancer research
 Drug research
 Disease diagnosis and management
 Viral quantification
 Food testing
 Percent GMO food
 Animal and plant breeding
 Gene copy number

29. REFERENCES
 Molecular Biology and Biotechnology by, Walker
and Rapley, 5th edition, pg no. – 122-
128
 Pharmaceutical Biotechnology by , Chandrakant
Kokare, Pg no. -10.15
 A textbook of biotechnology by R. C. Dubey
 International Journal of Biomedical Research,
review article “POLYMERASE CHAIN
REACTION: METHODS, PRINCIPLES AND
APPLICATION” by, Dr. Mohini Joshi & Dr.
Deshpande. J. D

30. THANK YOU