familarize students how to perform PCR

1. University of Gondar
Institute of Biotechnology
Techniques in Biotechnology (Biot.602)
Lecture 5
Polymerase Chain Reaction (PCR)

2. DNA Replication vs. PCR
• PCR is a laboratory version of DNA Replication.
• laboratory version is commonly called “in vitro” since it
occurs in a test tube while “in vivo” signifies occurring in a
living cell.
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in vitro in vivo

3. DNA Replication enzymes:
• DNA Polymerase- builds DNA strand
• DNA Ligase- joins DNA strand together
• Primase- short RNA sequence that serves as a starting point for
DNA synthesis
• Helicase untwists the two parallel DNA strands
• Topoisomerase relieves the stress of this twisting
• Single-strand binding protein binds to and stabilizes the
unpaired DNA strands.
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4. Con…
• DNA Replication occurs with very few errors (on average
there is one error per 1 billion nucleotides copied).
• It typically takes a cell just a few hours to copy all of its DNA
• DNA replication is semi-conservative (i.e. one strand of the
DNA is used as the template for the growth of a new DNA
strand)
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5. Polymerase Chain Reaction (PCR)
• Polymerase: DNA polymerase
– DNA polymerase duplicates DNA
– Before a cell divides, its DNA must be
duplicated
– Chain Reaction: The product of a reaction is
used to amplify the same reaction
– Results in rapid increase in the product
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6. PCR Con’t
Discovery
• PCR was discovered by Kary Mullis
– On a long motorcycle drive
– Mentally visualized the process
• Nobel Prize in Chemistry
– 1993
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7. DNA polymerase
• Duplicates DNA
• Necessary for reproduction of new cells
• More than one DNA polymerases exist in different organisms
 Taq DNA polymerase
• Derived from Thermus aquaticus
• Heat stable DNA polymerase
• Ideal temperature 72C
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8. Properties of DNA polymearse
• Needs a pre-existing DNA to duplicate
– Cannot assemble a new strand from template DNA
• Can only extend an existing piece of DNA
Called primers
3’ 5’
5’ 3’
• DNA polymerase needs Mg++ as cofactor
• Each DNA polymerase works best under optimal
temperature, pH and salt concentration
PCR buffer provides optimal pH and salt condition
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9. Properties of DNA polymearse
• DNA strands are anti-parallel
– One strand goes in 5’  3’
– The complementary strand is opposite
• DNA polymerase always moves in one
direction (from 5’  3’)
3’ 5’
5’ 3’
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10. Properties of DNA polymearse
• DNA polymerase incorporates the four
nucleotides (A, T, G, C) to the growing chain
• dNTP follow standard base pairing rule
3’ 5’
5’ 3’
dCTP
dTTP
dCTP
dGTPdATP
dGTP
dCTP
dTTP
dATP
dGTP
dCTP
dTTP
dATP
dATP
dGTPdCTP dTTPdATP dGTP
dATP
dGTP
dTTP
dATP
dCTP
dTTP
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11. The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building
blocks.
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
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12. 1- DNA template
• DNA containing
region to be
sequenced
• Size of target DNA
to be amplified : up
to 3 Kb
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13. • A primer is a short synthetic oligonucleotide which is used in
many molecular techniques from PCR to DNA sequencing.
• Are designed to have a sequence which is the reverse
complement of a region of template or target DNA
2. Primer
 18-24 bp best for general applications
• 2 sets of primers
• Generally 20-30 nucleotides long
• Synthetically produced
• complimentary to the 3’ ends of
target DNA
• Not complimentary to each other
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14. 3-Enzyme
• Usually Taq Polymerase or anyone of the
natural or Recombinant thermostable
polymerases
• Stable at T0 up to 950 C
• High procesivity
• Taq Pol has 5’-3’ exo only, no proofreading
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15. Typical PCR mix
In a thin wall Eppendorf tube assemble the following
PCR components Amount
Template DNA (5-200 ng)
1 mM dNTPs (200 uM final)
10 X PCR buffer
25 mM MgCl2 (1.5 mM final)
20 uM forward primer (20 pmoles final)
20 uM reverse primer (20 pmoles final)
5 units/uL Taq DNA polymerase (1.5 units)
Water
Final Volume
variable
10 uL
5 uL
3 uL
1 uL
1 uL
0.3 uL
Variable
50 uL
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16. Prepare a master mix for four rxns with a rxn volume of
25ul
component Stock sol Final sol Single PCR in
25ul
For 5 rxn
DNTP mix 1oomM 0.2mM
PCR Buffer 10x 1x
FP 100mM 10mM
RP 100mM 10mM
MgCl2 25mM 1.5mM
Taq Poly 5u/ul 2.5
PCR Grade
H2O
- -
Tem. DNA 2ug/ul 2ug/ul
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17. The PCR Cycle
• A PCR machine controls temperature
• Typical PCR go through three steps
1- Denaturation: double stranded DNA seprated into two single
strands 90-95c
2- Annealing: Cooling at 40-60c Primers attached
complementary region of target DNA
3- Extention: Heating at 70c. The primers extended to form a
new strand of DNA
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18. Denaturation
• Heating separates the double
stranded DNA
– Denaturation
• Slow cooling anneals the two
strands
– Renaturation
Heat Cool
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19. Annealing
• Typical temperature from 50 to 60 C
• Primers attached complementary region of target DNA
• Optimal temperature varies based on primer length.
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20. Annealing Con’t…
• The annealing temp at which the primers
attach to template, can be calculated by
determining the melting temp (Tm) of primer
template hybrid. What is the TM of the ff
sequence?
5’ AGACTCAGAGAAACC 3’
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21. Extension
 Optimal temperature 72C
 DNA polymerase catalyses primer extension as
complementary nucleotides are incorporated.
 The newly generated DNA strands serve as template
DNA for the next cycle
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22. 2/26/2019 22

23. PCR Amplification
Exponential Amplification of template DNA
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24. PCR Amplification
• A 200ul of PCR mix has 100 template of DNA
molecule and the rxn was performed for 10
cycle.
A) How many molecule of amplicons will be
generated
B) How many molecule amplicons will present in
0.1ul of rxn?
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25. Applications of PCR
• Classification
of organisms
• Genotyping
• Mutation
detection
• Sequencing
• Cancer research
• Detection of
pathogens
• DNA
fingerprinting
• Drug discovery
• Genetic
matching
• Genetic
engineering
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26. Applications Con’t
Primers can be created that will only bind and
amplify certain alleles of genes or mutations of
genes.
The role of PCR in diagnosis of infectious
diseases.
– Fastidious and slow growing microorganism
– Detect antimicrobial resistance
– Detect microorganism cannot be cultivated
– Measurement value of viral load
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27. Detection Of Pathogens
Sensitivity of detection of PCR-
amplified M. tuberculosis DNA.
(Kaul et al.1994)2/26/2019 27
Detection of ACE gene on 2% agarose gel
electrophoresis. Lane 1. Show 100bp DNA
marker. Lane 2 and 5 show homozygous DD.
Lane 3 and 4-show heterozygous II genotype.
Lane 6 shows control.

28. PCR Vs DNA Replication
PCR is an in vitro method of DNA amplification in which
thousands to millions of copies of DNA are produced.
DNA Replication is a natural process that produces two
identical copies of DNA from one DNA molecule.
Steps
PCR has three steps; denaturation, primer annealing and strand
extension.
DNA Replication has three steps; initiation, elongation and
termination.
Involvement of Primers
PCR needs artificial primers DNA Replication does not need artificial primers. A short
fragment of RNA is involved in DNA replication.
Denaturing of the double-Strands
Double strands are separated by applying a high temperature in
PCR.
Double strands are separated from each other by the
enzyme DNA helicase in DNA Replication.
Enzyme Involved
PCR uses Taq polymerase. DNA Replication uses DNA polymerase.
Temperature
PCR occurs at three different temperatures inside a machine. DNA Replication occurs at body temperature within the
body of the living organism.
In vivo or In vitro
PCR is an in vitro method. DNA Replication is an in vivo method.
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29. Summary
blood, chorionic
villus, amniotic
fluid, semen, hair
root, saliva
68,719,476,736 copies Gel Analysis,
Restriction
Digestion,
Sequencing
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30. Thank you