Basics of PCR principle, types and applications

1. Polymerase Chain ReactionPolymerase Chain Reaction
(PCR) and Its(PCR) and Its
ApplicationsApplications
S.Karthikumar.,M.Sc.,M.Phil.,M.Tech.,(Ph.D)
Assistant Professor, Department of Biotechnology
Kamaraj College of Engineering and Technology
Virudhunagar-626001, Tamilnadu, INDIA
karthikumarbt@kcetvnr.org 1

2. What is PCR?What is PCR?
PCR is an exponentially
progressing synthesis
of the defined target
DNA sequences in vitro.
It was invented in 1983 by
Dr. Karry Mullis, for
which he received the
Nobel Prize in
Chemistry in 1993.
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3. Did He Really Invent PCR?
• The basic principle of replicating a piece of
DNA using two primers had already been
described by Gobind Khorana in 1971:
– Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.
• Progress was limited by primer synthesis
and polymerase purification issues.
• Mullis properly exploited amplification.
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4. What is PCR? :What is PCR? :
Why “Polymerase”?Why “Polymerase”?
It is called “polymerase” because the only
enzyme used in this reaction is DNA
polymerase.
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5. What is PCR? :What is PCR? :
Why “Chain”?Why “Chain”?
It is called “chain” because the products
of the first reaction become substrates
of the following one, and so on.
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6. What is PCR? :What is PCR? :
The “Reaction” ComponentsThe “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
4) Thermostable DNA Polymerase - enzyme that
catalyzes the reaction
5) Mg++
ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength of
the reaction solution suitable for the activity of the
enzyme karthikumarbt@kcetvnr.org 6

7. The Basics of PCR Cycling
• 30–35 cycles each comprising:
– denaturation (95°C), 30 sec.
– annealing (55–60°C), 30 sec.
– extension (72°C),
time depends on product size.
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8. The ReactionThe Reaction
THERMOCYCLERPCR tube
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9. Taq polymerase
Thermus aquaticus, a
thermophilic bacteria
discovered in 1969 in
hot spring of
Yellowstone National
park . It can tolerate
high temperature. The
DNA polymerase
(Taq polymerase) was
isolated.
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10. Thermostable Polymerases
Polymerase T ½,
95o
C
Extension
Rate (nt/sec)
Type of
ends
Source
Taq pol 40 min 75 3’A T. aquaticus
Amplitaq
(Stoffel
fragment)
80 min >50 3’A T. aquaticus
Vent* 400 min >80 95%
blunt
Thermococcus
litoralis
Deep Vent* 1380 min ? 95%
blunt
Pyrococcus
GB-D
Pfu >120 min 60 Blunt Pyrococcus
furiosus
Tth*
(RT activity)
20 min >33 3’A T.
thermophilus
*Have proof-reading functions and can generate products over
30 kbp karthikumarbt@kcetvnr.org 10

11. • Taq: Thermus aquaticus (most commonly used)
– Sequenase: T. aquaticus YT-1
– Restorase (Taq + repair enzyme)
• Tfl: T. flavus
• Tth: T. thermophilus HB-8
• Tli: Thermococcus litoralis
• Carboysothermus hydrenoformans (RT-PCR)
• P. kodakaraensis (Thermococcus) (rapid
synthesis)
• Pfu: Pyrococcus furiosus (fidelity)
– Fused to DNA binding protein for processivity
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12. A Standard PCR Reaction Mix
0.25 mM each primer
0.2 mM each dATP, dCTP,
dGTP, dTTP
50 mM KCl
10 mM Tris, pH 8.4
1.5 mM MgCl2
2.5 units polymerase
102
- 105
copies of template
50 ml reaction volume
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13. Denature (heat to
95o
C)
Lower temperature to 56o
C
Anneal with primers
Increase temperature to
72o
C DNA polymerase +
dNTPs
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16. PCR Products on Agarose gel
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17. Combinations Of Cycle
Temperatures
TEMP FOR COMMENTS
94-60-72 Perfect, long
primers
Higher temp can be used;
maximum annealling temp
94-55-72 Good or perfectly
matched primers
between 19-24 nt
Standard conditions
94-50-72 Adequate primers Allows 1-3 mismatches/20 nt
94-48-68 Poorly matched
primers
Allows 4-5 mismatches/20 nt
94-45-65 Unknown match,
likely poor
Primers of questionable
quality, long-shot PCR
94-37-65 Hail Mary Uncontrolled results
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18. Do Errors Matter in PCR?
• Yes, if you want to clone the amplified
DNA — an individual molecule may
harbour several mutations.
• Use a proof-reading thermo-stable
enzyme rather than Taq.
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19. How Big A Target?
• Amplification products are typically in the
size range 100-1500 bp.
• Longer targets are amplifiable — >25 kb.
• Requires modified reaction buffer,
cocktails of polymerases, and longer
extension times.
• Limited by the integrity of the starting
target DNA — > 50 kb.
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20. Can I PCR Amplify RNA?
• Not directly — the DNA polymerase
requires a DNA template and will not copy
RNA.
• mRNA can first be copied into cDNA using
reverse transcriptase.
• cDNA is a template for PCR — it need not
be double-stranded.
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21. Designing PCR Primers
• Primers should be ~20 bases long.
• The G/C content should be 45–55%.
• The annealing temperatures should be
within 1°C of one another.
• The 3´-most base should be a G or C.
• The primers must not base pair with each
other or with themselves or form hairpins.
• Primers must avoid repetitive DNA
regions.
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22. Primers That Form Hairpins
• A primer may be self-complementary and
be able to fold into a hairpin:
5´-GTTGACTTGATA
||||| T
3´-GAACTCT
• The 3´ end of the primer is base-paired,
preventing it annealing to the target DNA.
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23. Primers That Form Dimers
• A primer may form a dimer with itself or
with the other primer.
5´-ACCGGTAGCCACGAATTCGT-3´
||||||||||
3´-TGCTTAAGCACCGATGGCCA-5´
• Primer dimers can be an excellent, but
unwanted, substrate for the Taq
polymerase.
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24. Will Other Genes Amplify Too?
• The primers have been designed on the
basis of the DNA sequence of a single
gene.
• Might the primers also amplify other
segments whose sequence we have not
taken into account?
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25. Type of PCR
• Single PCR
• Nested PCR
• Multiplex PCR
• Reverse transcriptase PCR
• Asymmetric PCR
• Quantitative PCR
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26. Nested PCR
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27. Applications of PCRApplications of PCR
Molecular Identification Sequencing Genetic Engineering
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
• Bioinformatics
• Genomic cloning
• Human Genome
Project
• Site-directed
mutagenesis
• Gene expression
studies
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28. MMOLECULAROLECULAR IIDENTIFICATIONDENTIFICATION
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31. Detection Of PathogensDetection Of Pathogens
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32. SSEQUENCINGEQUENCING
Nucleotides (dNTP) are modified
(dideoxynucleotides = ddNTP)
NO polymerisation after a dideoxynucleotide!
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34. Reading Classical Sequencing GelsReading Classical Sequencing Gels
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35. Enzyme linked oligonucleotide sorbent assay
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