Biotechnology Engineering

1. INTRODUCTION
● PCR is a technique that results in exponential
amplification of a selected region of a DNA molecule.
● PCR is widely held as one of the most important
inventions of the 20th century in molecular biology.
● The idea of PCR is credited to Kary Mullis, a research
scientist at California Biotech Company, Cetus, in 1983.
● For this work, Mullis received the Nobel Prize in
Chemistry jointly with Michael Smith in 1993.
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2. developed the PCR
(polymerase chain reaction)
Kary Banks Mullis
(Dec 28, 1944 - )
Nobel Prize in Chemistry 1993
1985 1986 1987 1988
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3. Kary Mullis received the Nobel Prize in Chemistry jointly with
Michael Smith in 1993. 4

4. CONSTITUENTS OF PCR
REACTION
One or more molecules of target DNA
Two oligo-nucleotide primers(forward and reverse primers)
All the four (dNTPs)
Thermostable DNA polymerase
PCR Buffer to maintain pH
Divalent cation (Mg++).
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5. STEPS IN PCR REACTION
Denaturation of Double Stranded DNA (ds DNA)
Annealing of Primers to Single Stranded DNA template
Extension of primer or Synthesis of ds DNA.
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6. Denaturation of ds DNA
Convertion of the ds DNA molecule to ss DNA.
This reaction is usually performed at 94oC .
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7. Annealing of Primers to ss DNA
Template
 The base pairing of a single stranded primer to its
complementary region of the ss DNA molecule is known
as annealing.
 The common choice of temperature range for this reaction
is 55-60oC.
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8. Extension of primers or Synthesis of ds DNA
 The annealing of a primer provides a free 3’-OH
group for synthesis of ds DNA by thermostable
DNA polymerase using ss DNA as a template.
 Extension is the synthesis of DNA by a thermostable
DNA polymerase using 3’-OH end of a primer.
 It is done at 72oC, the optimal working temperature
for thermostable DNA polymerase.
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9. PCR procedure: cycle 1
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10. PCR procedure: cycle 2
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11. PCR procedure: cycle 3
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12. Temperature profile of PCR
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13. THEMOSTABLE DNA
POLYMERASES
 Originally thermostable polymerase was purified from the hot
spring bacterium Thermus aquaticus .
 The thermophilic DNA polymerases catalyze template directed
synthesis of DNA using nucleotide triphosphates.
 A primer having a free 3’-OH is required to initiate synthesis
and Mg++ ion is necessary.
 These enzymes have maximum catalytic activity in the
temperature range of 75oC to 80oC.
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14. Thermostable DNA Polymerase
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15. Properties of Different
Thermostable DNA Polymerases
Enzyme Source M.W.(KDa) Optimum
temperature
(oc)
Exonuclease
activity
Fidelity Stability(Half
–Life)
Remarks
Taq DNA
polymeras
e(natural)
Thermus
aquaticus 94 74 3-5
Low 40 min at
90oC
Use in
routine
PCR exp.
AmpliTaq Thermus
aquticus
- 75-80 None Low 21 min.at
97.5oC
Processivit
y is lower.
Tli DNA
polymerase(
Recombinan
t
Thermococc
us litoris
90 74 3-5 High 400min at
95oC
Primer
extension
and high
fidelity .
Pfu DNA
polymerase(
natural)
Pyrococcus
furiosus
90 75 3-5 High 240 min.at
95oC
High
fidelity and
primer
extension
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16. PRIMER DESIGNING PARAMETERS
 Primer Length
 Melting Temperature (Tm)
 Primer Dimer
 GC Content
 Runs and Repeats
 Distance Between Primers
 Secondary Structures
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17. Primer Length
 Optimal length of PCR primer is 18-30 nucleotides.
 This length is sufficient for adequate specificity and
short enough for the primer to bind to the template at
the annealing temperature.
 Shorter primers lead to amplification of nonspecific
PCR products.
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18. Melting Temperature (Tm)
 Tm of a DNA molecule is defined as the temperature
at which one-half of the duplex DNA will dissociate to
become single stranded.
 Tm indicates the duplex stability.
 The specificity of PCR depends strongly on the
melting temperature of the primers.
 Tm of primer hybridization can be calculated using
various formulae:
(i) Tm =4 (G+C) + 2 (A+T) o C (commonly used)
(ii) Tm =22 +1.46 [2 x (G+C) +(A+T)]
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19. Primer Dimer
 Primer dimers are generally two types:
(i) Self dimer: It is formed by intermolecular
interaction between the two same primers, where the
primer is homologous itself ;
(ii) Cross dimer : It is formed by intermolecular
interaction between complementary regions of two
different primers, i. e., sense and antisense.
The formation of primer dimers prevents the
hybridization of primers to the template DNA, there
by reducing the product yield.
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20. GC Content
 The GC content of a primer should be 40-60%.
 The presence of G or C bases within the last five bases
from the 3’-end of a primer helps to promote specific
binding at 3’-end due to the stronger bonding of G and C
bases.
 More than three G or C should be avoided in last 5 bases
at the 3’-end of a primer.
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21. Runs and Repeats
 Primer should lack stretches of polynucleotide sequences,
i.e., runs (e.g., poly dA) or repeating motifs, because these
can hybridize at wrong places on the template.
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22. Distance between Primers
 Theoretically, the least distance between the primers on
template DNA should be 150 bp and utmost 10 kbp.
 Typically, yield is reduced when the primers extend from
each other beyond 3 kbp approx.
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23. Secondary Structure
 Presence of the secondary structures produced by
intermolecular or intramolecular interactions can lead to
poor or no yield of the product.
 These adversely affect primer template annealing and thus
the amplification.
 These greatly reduce the availability of the primers to the
reaction.
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24. CYCLE NUMBER
 The number of amplification cycles necessary to produce
a band visible on a gel depends largely on the starting
concentration of the template DNA.
 It is recommended that to amplify 50 target molecules 40-
45 cycles are needed,while 25-30 cycles are required to
amplify 3 x 105 molecules to the same concentration. This
non- proportionality is called Plateau effect which is the
decrease in the exponential rate of product accumulation
in late stages of a PCR.
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25. 26

26. PCR PRODUCT YIELD
The predicted yield of PCR product can be calculated by a
simple ‘invested equation,
PCR product yield =(input target amount) x (1 + %
efficiency) x number of cycles
Thus , ~26 cycles are required to produce 1 mg of PCR
product from 1pg of a target sequence with an efficiency
value of 70%,i.e.,
1 mg PCR product =(1 pg target ) x 1+0.7) x26
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27. Amplification of target gene
30 cycles --- 1 billion copies in theory
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28. VERIFICATION OF PCR PRODUCTS
 After PCR reaction is completed, the agarose gel
electrophoresis is performed to determine the
following:
(i)Whether or Not a Product is formed
(ii)Whether or Not the Product Formed is Right Size
(iii)Whether or Not a Single Band of Right size is
formed
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29. FACTORS AFFECTING PCR
AMPLIFICATION
 Buffer Composition
 Quality of Primers and their Concentrations
 Nucleotides Concentration
 Primer Annealing temperature
 Choice of Polymerases for PCR
 Cycle Number
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30. Buffer Composition
Buffer for PCR reaction generally contains:
(i) Tris-HCl 10-15mM pH 8.3
(ii) KCl 50mM
(iii) MgCl2 1.5mM
(iv) Primers (forward and reverse) 0.2-1 µM each
(v) dNTPs 50-200 µM each
(vi)Gelatin or BSA up to 100µg/ml and Non-ionic
detergents such as Tween-20/Triton X-100
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31. Quality of Primers and Their
Concentrations
 Good primer design is indispensable for successful
reaction.
 The primers anneal to the complementary sequences
on the template DNA and thereby determine the
boundaries of the amplified product.
 Primer concentration is also important .
 It should not go above 1 µM except for degenerate
primers.
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32. Nucleotides Concentration
 Nucleotides concentration should not exceed 50 µM each.
 Long products may need higher concentration.
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33. Primer Annealing Temperature
 Ta of the PCR reaction is determined on the basis of Tm
of primer.
 Too high Ta will produce insufficient primer-template
hybridization resulting in low PCR product yield.
 If the Ta is too low, it may lead to the formation of
nonspecific products because of high number of
mismatches.
 Ta can be calculated by this formula:
Ta =0.3 x Tm(primer) + 0.7 Tm (product) - 14.9
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34. Choice of Polymerases for PCR
 The enzymes used for polymerization also affect the
PCR amplification.
 The polymerases used in synthesis of DNA should be
thermostable up to the temperature 94oC.
 The polymerases lacking 3,-5, exonuclease activity
generally have higher error rates than the polymerases
with exonucleases activity.
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35. Cycle Number
 The number of amplification cycles necessary to produce
a band visible on a gel depends largely on starting
concentration of the target DNA.
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