PCR
Polymerase chain reaction
Revolution in the work with DNA
Doc. RNDr. Jan Hosek, Ph.D. hosek@mail.muni.cz
Department of Molecular Pharmacy FaF MU
Who is responsible ?
Kary Mullis 1985 Nobel prize in 1993
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The origin of PCR by Kary Mullis
• "Sometimes a good idea comes to you when you are not looking for it. Through an improbable combination of coincidences, naivete, and luck mistakes, such a revelation came to me one Friday night in April, 1983, as I gripped the steering wheel of my car and snaked along a moonlit mountain road into northern California's redwood country." Sci. Am. 1990 262:56-61, 64-5.
Principle of PCR
Polymerase chain reaction (PCR) enables selective amplification of specific region of
DNA in vitro:
by the process which resembles DNA
synthesis in vivo
newly synthesized strand
-► 3'
- 5'
DNA primer
I I I I I
5'
3 _.....
DNA template
Basic prerequisites of amplification
Template DNA Amplification proceeds only on a DNA sequence -
template
Primers
Represent beginning of amplification
Complementarity
Growing of DNA chains are performed according the rules of base pairing on the level primer/template
Direction of polymerisation
New nucleotides are done only in the 5 - 3' direction
PCR = cyclic changes of temperature in the reaction mixture
Denaturation
a
Annealing Extension
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The components of PCR
/-\
Thermus aquaticus, Thermococcus, Thermophilics,
Pyrococcus
TAQ
buffer MgCI2
DNA primers
I I I I I
I I I I I
.............................. ssDNA
Denaturation 92-96°C
The 1st PCR cycle
1. denaturation (92-96°C) i i i i i i I I I I I I I I I I I I I I I I I I I I I I I I . _...
jji | | | | | | | | | | i i i i i i i i i i i i i i i i i CISDNA
2. annealing (45-72°C)
Primary products
3. extension (72°C)
i i i i i i i i i i i i i i i i i i i i i i i i ^
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The 2nd PCR cycle
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Secondary products
....................
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..............................
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I I I I I I I I I I I I I I I I I I I I I I I I I
....................
I I I I I I I I I I I I I I I I I I I I
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The other cycles
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Number of amplicons is growing geometrically
PCR - cycles
wanted gene
template DNA
number of double strands with the right length
The first 4 cycles of PCR in detail
_«
4th cycle
And\ V -
2001
Secondary product
Amplicon
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Amounts and types of the PCR
products
Cycle No.	Primary	Secondary	Amplicons	Total (for X =1)
0	0	0	0	1
1	2	0	0	2
2	2	2	0	4
3	2	4	2	8
4	2	6	8	16
5	2	8	22	32
Generally	2x	x(2n-2)	(2n-2n)x	(2n)x
X = number of template at the beginning, n = number of cycles
Yield and numbers of the PCR cycles
Cycle No.	Primary	Secondary	Amplicons	Total
10	2	18	1 004	1 024
20	2	38	10 485 438	1 0242
30	2	58	~ 1.1 x109	1 0243
40	2	78	~ 1.1 x 1012	1 0244
50	2	98	~ 1.1 x 1015	1 0245
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Conclusions
> Each amplicon is formed from two strands = two templates that are doubled in each PCR cycle
> The number of amplicons is extremely growing in comparison with primary and secondary products
> After several cycles the amplicons absolutely dominate as products of the PCR reaction
> Primary and secondary products form only minor part of the resulting products
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Parameters of PCR
Quality of PCR reaction and result of amplification are influenced by several predictable factors which are
Chemical factors
Physical factors
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Chemical parameters of PCR
Amount of Taq polymerase Reaction buffer Amount of dNTPs Primers
Volume of the PCR reaction Quality of DNA
Amount of Taq polymerase
> 0,5-2,5u correspond to 25-125 fmol of enzyme
> A lot of different Taq polymerases and their mixtures is available at the present time
> High termostability and accuracy to include the correct nucleotide to the DNA strand is called fidelity
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Reaction buffer
> It contains a cofactor of Taq polymerase -Mg2+ ions in the form of MgCI2 or MgS04
> Concentration of Mg2+ varies between 0,5-5,0 mM (1,5 mM)
> The ions influence activity of the enzyme, increase Ta of dsDNA, and form soluble complexes with nucleotides, which is necessary to incorporation of nucleotides to DNA
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Influence of Mg2+ ions on PCR
http://www.thermoscientificbio.com/
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Amount of dNTP
> In vitro the nucleotides are sufficiently incorporated to the DNA structure in concentrations about 10 uM, that are lower than the concentrations used in PCR (100-200 uM)
The correct concentration (amount) of dNTPs depends on:
> Length of the products of amplification
> Concentration of Mg2+
> Concentration of the primers
> Temperature profile of the reaction
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Primers
> Responsible for the specificity of PCR
> Long from 14 to 40 nucleotides
> Content of G+C from 40% to 75%
> 3- end of one of the primers should avoid complementarity to prevent „primer dimer" formation
> Avoid imbalanced distribution of G/C- and A/T- rich domains. But oligo (dT) and poly (dC) work well.
> 5 - end is less sensitive to mutations presented on the template DNA
> Concentration in the range of 0,1 -1,0 |tiM
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Primers
The length and specificity of PCR products are done by the position of primers on target sequences of the DNA template
TGATGCGTTCGTATCATT.......................................AATCGATGGTTTCGTATC
T TAGC T AC CAAAGC AT AG
forward 3 reverse
3
TGATGCGTTCGTATCATT lCTACGCAAGCATAGTAA.......................................T TAGC T AC CAAAGC AT AG
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Design of primers - example
Design the primers sequences that could amplify the selected part of dsDNA
- You know the sequence of only one DNA strand
- Write orientation in 5'- 3' direction
5- TGA TGC AAA GTT CGC TCA GGT ACG ATT CCC AAA TGT GGA GCT TAG TCG ATG ATG GGC AAA TCT GTG ATT ATC CGA CGT CCC ATG TGC GTC AAA TGC CGT AGG ACC CTA TTT TGA CGT CCT GCT GGT ACG CAT CAT CCC TGG TGA CGT CCT ACG TGC TGC GCT CGC ACG ATG CGT ACG AAC GCT CGT CGG - 3'
How long will be the resulting amplicons ?
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Design of primers - resolution
Primer forward
5- AAA GTT CGC TCA GGT ACG - 3
Primer reverse
5- CGT ACG CAT CGT GCG AGC - 3
Amplicon will be 171 bp long
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Volume of the PCR reaction
> Influence the result less than the previously described factors
> May influence sensitivity of reaction (detection limit of rare template)
> Reaction volumes are usually 20-100 ul
> PCR in capillaries - reaction volume slow down to 10 Ml
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DNA quality
> One of the most important factor to receive good results
> Purity and quality of DNA influence especially sensitivity of PCR reaction => Rubbish in, rubbish out.
> PCR is completely inhibited by compounds such are heparin, porphins and similar; and HxP04n- ions
27
Physical parameters of PCR
1) Initial denaturation
2) Primer annealing
3) Primer extension
4) Cycle number
5) Final extension
28
Initial denaturation
> Initial heating of the PCR mixture for 3 to 5 min at 94-96°C
> It is enough to complete denaturing complex genomic DNA so that primers can anneal to their target sequences after cooling
> It has not be very long - heat damages DNA strands and Taq polymerase
29
Primer annealing
> Critical for specificity of PCR reaction
> Very high temperature = primers are not able to anneal
> Very low temperature = non specific products are formed
30
Influence of Ta on PCR
7
Optimal temperature
Temperature for primer annealing
Tm (melting temperature)
Temperature during which about 50% primers
will bind to template
Tm = (number of G+C) x 4 + (number of
Ta (annealing temperature)
Temperature during which the most of the primers will bind to template
Ta = Tm - (3-5°C)
32
Calculation of Ta - example
What are the Tm and Ta of the following
primers?
Primer forward
5 - AAA GTT CGC TCA GGT ACG - 3'
Primer reverse
5'- CGT ACG CAT CGT GCG AGC - 3
Calculation of Ta - resolution
Primer forward
5'- AAA GTT CGC TCA GGT ACG - 3'
Tm = 54°C, Ta = 50°C Primer reverse
5'- CGT ACG CAT CGT GCG AGC - 3'
Tm = 60°C, Ta = 56°C
Optimal Ta for the both of the primers
will be about 50°C
Primer extension
> Synthesis of new DNA strands
> Proceeds in 72°C
> It is recommended not longer than 20 s for amplicons to 500 bp long
> For fragments to 1,2 kbp about 40s
> Taq polymerase synthesizes about 150 nucleotides Is
35
Cycle number
> Analytical PCR - no more than 40 cycles
> Mostly 25 to 35 cycles
> Higher cycle number = formation of nonspecific products
> exhausting of reaction compounds
> polymerase and/or DNA degradation
36
Cycle number
"Plateau Effect" in PCR Amplification
0 20        30 40
cycle number
http://www.mcb.uct.ac.za/
Final extension
> To promote completion of partial extension products and complete annealing of ss complementary products
> It is hold usually, after the last cycle, for 5-15min. in 72°C
> After PCR completion, the tube with reaction can be stored at -20°C until needed for product analysis
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Technical performance of PCR
- termocyclers
By microprocessor controlled
equipment which contains metal reaction block heated and cooled by semiconductors (Peltier pump), water, air or microwaves
Termocyclers quickly change temperature in reaction blocks between the three temperatures of the PCR cycle
39
Thermocycler evolution
Types of PCR
> PCR-REA, PRA, PCR-RFLP
> nested PCR
> multiplex PCR
> competitive PCR
> RT-PCR
> real-time PCR
41
PCR-REA, PRA, PCR-RFLP
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..............................
amplification
I I I I I I I I I I I I I I I I I I I I I I I I I I i i i i i i i i i i i i i i i i i i i i i i i i i i
/l\
restriction digestion
I I I I I I I I I I I I I I I I I I I I I I ............. .....PPP, .........
.........
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PCR-REA, PRA, PCR-RFLP - example
Uncut
Detection of polymorphism G908R in NOD2 gene
Cut by restriction endonuclease
1 \
Heterozygot x Homozygot 2
Homozygot 1 43
Nested PCR
Arrangement of nested PCR
primer 1    primer 2
primer 3    primer 4
f—:-:
primer 1
double tube
primer
4
one tube
45
One tube nested PCR
Ta = 65°C
Ta = 58°C
Ta = 65°C
20 cycles Ta = 62°C
Ta = 58°C
30 cycles Ta = 52°C
Nested PCR/PCR - example
IVP gag HTLV-2 PCR System Titration
Dilution of Positive HTLV-2 DNA Control First       10'5 10'e 10'7 10s 10s 1010101110121013
Second     io5 ioe io71°8 1°9 io10 io11 io12 io13
Wisconsin Viral Research Group, Ltd. Testing for HHV-6 and EBV by nested PCR
Multiplex PCR
Simultaneous amplification of several loci in
one PCR reaction
Multiplex PCR
Strategy for the detection and differentiation of Mycobacterium avium
species in isolates and heavily infected tissues. Morávkova M, Hlozek P, Beran V, Pavlik I, Preziuso S, Cuteri V, Bartos M
Competitive PCR
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Competitive PCR
Positive Negative Inhibition
Target DNA amplification
Internal standard amplification
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negative
positive
inhibition
Mycobacterium tuberculosis PCR Kit
52
Reverse transcription PCR
ssDNA
Viral ssRNA
					3'
1  3 N Hybrid cDNA/viral ssRNA				RNA dependent DNA polymerase	
cDNA
denaturation
3'
cDNA
amplification
DNA dependent DNA polymerase
IS
Thank you for your attention
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