DETAILS ABOUT PCR

2. What is PCR?
• Polymerase Chain Reaction was first developed by Kary
Mullis in 1983.
• Definition : It is a technique used in molecular biology by
which small samples of DNA can be quickly amplified, that
is increased to quantities that are large enough for
analysis.
• PCR often provides an alternative to gene cloning & gene
libraries as a way of obtaining usable quantities of specific
DNA sequences.
• It is directed by a machine called Thermal Cycler.
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3. A strip of 8 PCR
tubes,each containing a
100ul reaction mixture
Placing a strip of 8 PCR
tubes into a thermalcycler

4. Why PCR?
• The manipulation and analysis of genes requires multiple
copies of the DNA sequences.
• A major problem in working at the molecular level is that
each gene is a tiny fraction of the total cellular DNA.
Because each gene is rare, it must be isolated and amplified
before it can be studied.
• PCR allows DNA fragments to be amplified a billionfold
within just a few hours.
• It can be used with extremely small amounts of original
DNA, even a single molecule.
• The PCR has revolutionized molecular biology & is now one
of the most widely used of all molecular techniques.
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5. Components of PCR
1)PCR tubes(polypropylene material)
2)Sample mixture:
• Target DNA(contains the target fragment to be amplified)
• dNTP mixture(deoxynucleotide triphosphates are single units of
the bases A,T,G & C,which are essentially building blocks for new
DNA strands)
• Taq DNA polymerase(enzyme that helps to catalyze the
polymerization of deoxynucleotide into a DNA,thermostable)
• Primer 1,Primer 2 (short fragments of DNA, 17 to 25 nucleotides
long that are complementary to the known sequences on target
DNA)
• 10X PCR buffer (Tris HCl+KCl;pH=8.4)
• Mg ions (divalent cation,essential cofactor for Taq DNA
polymerase)
3)Thermal Cycler Machine (5)

6. How does a PCR works?
• A starting solution of DNA is heated to between 90° and
100°C for a minute to break the hydrogen bonds between
the strands and thus produce the necessary single-stranded
templates.
• DNA solution is cooled quickly to between 30° and 65°C &
held at this temperature for a minute or less.
• The primers are now able to attach to the target site on the
template strands.
• Now the Taq DNA polymerase comes & attaches to the
primer helping in the extension of the new strand. Here the
temperature is adjusted between 60⁰C to 7O⁰C for a
minute. (6)

7. • At the end of the cycle, two new double-stranded DNA
molecules are produced for each original molecule of target
DNA.
• Hold the sample 4⁰C to limit the Taq DNA polymerase
activity.
• The whole cycle is then repeated. With each cycle, the
amount of target DNA doubles;so the target DNA increases.
• One molecule of DNA increases to more than 1000
molecules in 10 PCR cycles, to more than 1 million
molecules in 20 cycles, and to more than 1 billion
molecules in 30 cycles.
• Each cycle is completed within a few minutes; so a large
amplification of DNA can be achieved within a few hours.
• The no.of target DNA will grow exponentially. (7)

9. Types of PCR
• Colony PCR
• Nested & Semi-nested PCR
• Multiplex PCR
• AFLP PCR
• Hot start PCR
• Insitu PCR
• Inverse PCR
• Asymmetric PCR
• Long PCR
• Long accurate PCR
• RT PCR
• Allele specific PCR
• Real time PCR
• Helicase dependent PCR
• And many more (around 150 types) (9)

10. RT PCR
• In addition to amplifying DNA, PCR can be used to amplify
sequences corresponding to RNA.
• This amplification is accomplished by first converting RNA into
complementary DNA (cDNA) with the use of the enzyme
Reverse Transcriptase.
• The cDNA can then be amplified by the usual PCR reaction.
• This technique is referred to as Reverse-Transcription PCR.
Nested PCR
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11. Limitations
• The polymerase chain reaction is now often used in place
of gene cloning, but it has several limitations.
• First, the use of PCR requires prior knowledge of at least
part of the sequence of the target DNA to allow the
construction of the primers.
• Second, the capacity of PCR to amplify extremely small
amounts of DNA makes contamination a significant
problem. Minute amounts of DNA from the skin of
laboratory workers and even in small particles in the air can
enter a reaction tube and be amplified along with the
target DNA.
• A third limitation of PCR is accuracy. Unlike other DNA
polymerases, Taq polymerase does not have the (11)

12. capacity to proofread & under standard PCR
conditions, it may incorporate an incorrect
nucleotide about once every 20,000 bp.
• A fourth limitation of PCR is that the size of the
fragments that can be amplified by standard Taq
DNA polymerase is usually less than 2000 bp. By
using a combination of Taq DNA polymerase and a
DNA polymerase(newly isolated which are heat
stable & also have proofreading activity)by
modifying the reaction conditions, investigators
have been successful in extending PCR amplification
to larger fragments, but even these larger
fragments are limited in length to 50,000 bp or
smaller. (12)

13. Applications
• Widely used tools within moloecular biology
• The primers used in PCR are specific for known DNA sequences,
and so PCR can be used to detect the presence of a particular
DNA sequence in a sample. For example, PCR is often used to
detect the presence of viruses in blood samples by adding
primers complementary to known viral DNA sequences to the
reaction
• Modern diagnostic tests for infection with HIV, the causative
agent of AIDS use this type of PCR amplification of HIV
sequences
• Also can be used to identify genetic variations
• It is commonly used to amplify small amounts of DNA from
crime scenes,eg: by DNA fingerprinting
• Metagenomics:Genetic analysis of entire microbial community
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14. Yellowstone National Park

15. REFERENCES
• Pierce,B.A.(2012).Genetics:A Conceptual
Approach,4th Edition,W.H.Freeman and
Company,NewYork
• Tortora.G.J.,et.al(2009).Microbiology: An
Introduction,10th Edition,Benjamin
Cummings,San Francisco
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