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Polymerase chain reaction
1. 1
ANUSHA SHAJI, B.Pharm, M.Pharm
Assistant Professor
Department of Pharmacology
Nirmala College of Pharmacy,
Muvattupuzha, Ernakulam, Kerala
POLYMERASE CHAIN
REACTION
4. 4
INTRODUCTION-PCR
ï In genetic engineering , the availability of sufficient
amount of DNA of interest is indispensable in order to
study its structure , function and manipulation.
ï Until recently the micro-organisms are the chief source
to obtain multiple copies of DNA of interest.
ï Now a new and novel cell free technique are available
to obtain millions of copies, known as polymerase chain
reaction (PCR technique)
5. 5
ï PCR, polymerase chain reaction, is an in-vitro technique
that allows amplification of a particular DNA sequence
and is able to generate innumerable copies of specific
DNA sequence.
ï The technique is so precise and powerful, that even a
single gene sequence can be amplified into a detectable
amount within a short time.
ï The technique was independently designed and
developed by Karymullis in 1980.
7. 7
1- DNA TEMPLATE
ï§ DNA containing region
(target) to be sequenced
or amplified
ï§ Size of target DNA to be
amplified : up to 3 Kb
8. 8
2- PRIMERS
âą 1 sets of primers
âą Generally 20-30
nucleotides long
âą Synthetically produced
âą complimentary to the 3â
ends of target DNA
âą not complimentary to each
other
9. 9
3-ENZYME
ï§ Usually Taq Polymerase or anyone of the natural or
Recombinant thermostable polymerases
ï§ Taq polymerase â obtained from Thermus
aquaticus
ï§ This enzyme,Taq polymerase has an optimum
temperature 72á”C and the enzyme remains stable
at 950 C
10. 10
ïAnother reason for using thermostable DNA polymerase
is that :
ï± DNA synthesised at high temperature is relatively more
error free than the DNA synthesised at ordinary
temperature like 25á” to 30á”C.
11. 11
DEOXYNUCLEOTIDE TRIPHOSPHATES
ï The four nucleosides containing triphosphate groups;
dNTPs
ïThey are the building-blocks from which the DNA
polymerase synthesizes a new DNA strand.
12. 12
BUFFER SOLUTION
ï Providing a suitable chemical environment for
optimum activity and stability of the DNA polymerase
ïTris HCl , KCl , MgCl2 , Taq buffer etc can be used.
13. 13
DIVALENT CATIONS
ï Magnesium or manganese ions
ï Generally Mg2+ is used, but Mn2+ can be utilized for
PCR mediated DNA mutagenesis
ïAs higher Mn2+ concentration increases the error rate
during DNA synthesis
14. 14
ï± PCR, POLYMERASE CHAIN REACTION, is an in-vitro
technique that allows amplification of a particular DNA
sequence and is able to generate innumerable copies
of specific DNA sequence.
15. 15
PRINCIPLE
A characteristic feature of DNA is that bringing it to a
certain high temperature known as melting temperature
(Tm)
â
The double strands fall apart into single strands by
dissolution of the H-bonds
â
When the temperature is lowered , the two strands again
anneal to restore the double helix structure (annealing
temperature)
â
PCR technique utilizes this property of DNA for replication
16. 16
THE PCR MACHINE
ï The PCR machine is a thermal cycler in which the
temperature , time and number of cycles required
for amplification of the sample DNA can be preset
for automatic operation.
ï Normal DNA polymerase is thermo labile â a
thermo stable DNA polymerase obtained from a
thermophilic organism is used.
ï This avoids the addition of the enzyme in each
cyclic operation.
18. 18
THE PCR CYCLE
Comprised of 3 steps:
âą Denaturation of DNA at 950C
âą Primer hybridization ( annealing) at 40-500C
âą DNA synthesis ( Primer extension) at 720C
19. 19
DENATURATION
ïThe first step is the
denaturation of DNA
sample in a reaction
mixture to 95 °C .
ïAt this thermal condition
DNA strand gets
separated.
ïThe temperature is
maintained up to 5
minutes.
20. 20
RENATURATION
ïThe temperature is
lowered to 50 °C to allow
the 2 oligonucleotide
primers to anneal (bind) to
the complementary
sequence in the DNA
molecule.
ïThe time duration of 30
seconds is maintained for
renaturation.
21. 21
SYNTHESIS OF NEW DNA
ïThe temperature is
raised to 72-75 °C .
ïAt this elevated
temperature , Taq
polymerase shows
optimum activity and
initiates DNA synthesis
at 3 hydroxyl end of each
primer.
25. 25
TYPES OF PCR
ï Several modifications of PCR have been developed for
specific applications
ï Another reason for the modification is to overcome
problems or trouble shooters during amplification.
ï Following are some of the improved version of PCR.
ï¶ Nested PCR
ï¶ Inverse PCR
ï¶ Hot start PCR
26. 26
1. NESTED PCR
ï Nested PCR is a variation of the polymerase chain
reaction , in that 2 pairs ( instead of 1 pair) of PCR primers
are used to amplify a fragment.
ï±TECHNIQUE
ïStep 1 : primers binds to template DNA and PCR start.
ïStep 2 : PCR products from the first PCR reaction are
subjected to a second PCR run.
ïStep 3 : we can get multiple copies
28. 28
2. HOT-START PCR
ïA technique that reduces non-specific amplification
during the initial set up stages of the PCR.
ïIt may be performed manually by heating the reaction
components to the melting temperature (eg.95á”C) before
adding the polymerase.
ï 2 types
a. Mechanical hot start b. non-mechanical hot start
29. 29
ï± MECHANICAL HOT START PCR
ïAll components of PCR are added to the PCR vial
except for the DNA polymerase enzyme which will be
added just at the first denaturation step.
ï± NON-MECHANICAL HOT START PCR
ïThe use of a form of Taq DNA polymerase , for
example , Amplitaq Gold which is activated only if the
reaction mixture is heated at about 94á”C ( the first
denaturation step)
30. 30
ï Other methods depend on covalent linking of the
polymerase enzyme to certain inhibitors.
ïThe enzyme becomes dissociated from these
inhibitors at the first denaturation step.
33. 33
3. INVERSE PCR
ïTarget DNA is lightly cut into smaller fragments of
several kilobases by restriction endonuclease digestion.
ïSelf-ligation is induced under low concentrations causing
the phosphate backbone to reform.
ïThis gives a circular DNA ligation product.
ïTarget DNA is then restriction digested with a known
endonuclease.
34. 34
ïThis generates a cut within the known internal
sequence generating a linear product with known
terminal sequences.
ïThis can now be used for PCR (polymerase chain
reaction)
36. 36
OTHER VARIATIONS OF THE PCR
ï±Colony PCR
ï±Multiplex PCR
ï±Asymmetric PCR
ï±Long PCR
ï±Allele specific PCR
ï±Real time PCR
ï±Reverse transcriptase PCR (RT-PCR)
ï±In situ PCR
ï±Long accurate PCR
ï±Touch down PCR
ï±AFLP PCR
37. 37
âą Automated, fast, reliable (reproducible) results
âą High output
âą Sensitive
âą Broad uses
âą Defined, easy to follow protocols
âą Commercial kits are now available for easy PCR
reaction setup and amplification
ADVANTAGES
38. 38
DISADVANTAGES
âą Extreamly liable to contamination
âą High degree of operator skills required
âą False positive results due to amplifications
âą Expensive to the developing world
39. 39
APPLICATIONS
âą It is extensively used for the amplification of a particular
target DNA sequence
âą Genome mapping and gene function determination
âą Biodiversity studies ( e.g. evolution studies)
âą Detection of drug resistance genes
âą In plant biotechnology, PCR is used in plant disease
identification , presence of any target gene in crop plants
40. 40
âą The PCR is widely employed in disease diagnosis (early
detection of cancer, viral infections...) .
âą The speed and sensitivity of PCR helps in prenatal
diagnosis of inherited disease.
âą Forensic (DNA fingerprinting)
âą PCR has an important role in gene manipulation and
expression studies.
41. 41
REFERENCE
ï Biotechnology-4 by S. Mahesh and A.B Vedamurthy
, Page number : 32-35
ï Ananthanarayanan and Panikerâs text book of
microbiology , 7th edition. C K J Paniker. Page
number : 591
ï Molecular biology and biotechnology , 5th edition.
John M Walker and Ralph Rapley. Page number : 15-
18
ï www.wikipedia.com