AMPLIFICATION:
The Polymerase Chain Reaction
Adapted from a presentation written for
Principles of Gene Manipulation · November 6, 2000
Jennifer Cooper · America Madrigal · Laleña Vellanoweth
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AMPLIFICATION: PCR and Its Applications
I.
II.
III.
IV.
Definition of PCR
Requirements for PCR
PCR Process
A. Denaturation
B. Annealing
C. Extension
D. Cycling (repeat A-C)
PCR for HLA DQ-alpha
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PCR What is it?
The Polymerase Chain Reaction (PCR) is an
in vitro method to amplify a specific region of DNA.
PCR is extremely sensitive, with the capability
of amplifying minuscule quantities of DNA.
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PCR REQUIREMENTS

In the reaction







Sample – template
Primers
High temperature resistant polymerase; e.g., Taq
Deoxynucleotide triphosphates – dNTPs
(dATP, dGTP, dCTP, dTTP)
Buffer
Mg++, KCl
Thermocycler – instrument programmed to change
samples rapidly from one set temperature to another
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PCR METHOD
There are three basic steps in PCR
1. Denaturation (~95oC)
2. Annealing (~55oC, but varies)
3. Extension (~72oC)
Cycling repeats Steps 1-3 up to 35 times.
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PCR METHOD – DENATURATION STAGE
High temperature separates the two strands.
(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)
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PCR METHOD – ANNEALING STAGE
Primer length is usually ~20 nucleotides.
(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)
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PCR METHOD – EXTENSION STAGE
Thermostable polymerase adds dNTPs one at a time at this stage.
(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)
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PCR METHOD - CYCLING
The average number of cycles = 30 for efficiency reasons.
230 = 1.07 X 109 copies
(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)
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PCR METHOD - CYCLING
DNA
Cycle
Products
1
2
2
3
4
4
8
5
16
6 …
32 …
After the first two cycles, fragments with the correct length begin to be amplified.
(dictated by the placement of the primers)
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PCR for HLA DQ-alpha


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Biotinylated primers amplify a region of ~250 bp.
 Sequence of primers is common to all individuals.
 Sequence between primers is polymorphic – may differ between
chromosomes and among individuals.
 PCR picks out the 250 bp HLA DQ- α region from 3.3 X 109 bp of DNA
present in 23 chromosomes.
Note that in the kit we are using there are actually six pairs of biotinylated
primers (12 primers total).
 Only 1 pair will amplify the HLA DQ- α region
 The other five pairs of primers amplify genes that we are not interested
in for this class. The analysis for these genes employs a different probe
strip than the HLA DQ- α probe strip that we will be using.
 After agarose gel electrophoresis six bands will be seen, only one of
which is the band of interest for this class.
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The human male karyotype: 22 homologous chromosomes + X and Y
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PCR for HLA DQ-alpha
Products of PCR reaction
 >106 double strand fragments ~ 250 bp in length.
All are biotinylated (biotinylated primers).
1/2 = DQ-α allele on one copy of your Chromosome 6;
1/2 = DQ-α allele on the other copy.
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PCR REFERENCES
1. Gene Cloning: an introduction. T.A. Brown
2. The world wide web:
http://sunsite.berkeley.edu/PCR/whatisPCR.html
http://www.accessexcellence.org/AB/GG/polymerase.html
http://www.sciam.com/1998/0598issue/0598working.html
http://www.faseb.org/opar/bloodsupply/pcr.html
http://faculty.plattsburgh.edu/donald.slish/PCR.html
http://library.thinkquest.org/24355/data/light/details/media/polymeras
eanim.html
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After a long process, this PowerPoint presentation has now
ended. We hope it is of good help in your studies.
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PCR Requirements
The Details
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PCR REQUIREMENTS: sample - template

Amount
 Needed
 very small; intact DNA from one cell
 to see on a gel, need 1011 final copies;
need 104 starting copies
 Concern –competition with primers for annealing by
 Too much starting template
 Too much product from excessive cycling
Remember, the association rate of two strands increases with
the square root of the length of the DNA.
Longer strands anneal more quickly than shorter. So . . .
Templates and products are longer than primers. In high
concentration, they reanneal before primers can anneal.
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PCR REQUIREMENTS: sample - template
 Even degraded DNA is OK if sample is large enough
 Fossils
 Remains
 Old samples from crime scenes
PCR REQUIREMENTS - primers
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
Two primers of known sequence
 flank region you are interested in
 anneal to opposite strands of template
 prime toward the region between them
 non-complementary to each other
 lack internal complementarity
 of sufficient length to anneal to unique site in the genome
(~20 nt)
 1/420 = 1 site of identical sequence/1 X 1012 bp



chance that any one of the 4 bases will be at a given site = 1/4
have similar annealing temperatures
present in excess (0.1 – 1.0 uM each)
 favor annealing of primer over reannealing of strands
 sufficient for amplification through 25-30 cycles
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PCR REQUIREMENTS - dNTPs
• must be present in sufficient excess to complete
extension through all cycles (~200 M in each
dNTP)
• must not be present in such high excess that Mg++
ions are complexed and unavailable as cofactors
for polymerase activity
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PCR REQUIREMENTS - “buffer” components
• The buffer, e.g., Tris base adjusted to a
specific pH with HCl
– maintains pH
• pH 8.3 is optimum for Taq.
• pH optimum keeps protein folded in a conformation
at which it is enzymatically active.
• Different temperature-insensitive polymerases have
different pH optima
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PCR REQUIREMENTS - “buffer” components
• Monovalent salt, e.g., KCl
– to contribute to correct folding of enzyme and
thereby
– to contribute to optimum activity of enzyme
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PCR REQUIREMENTS - “buffer” components
• Mg++ (MgCl2, MgSO4)
– Mg++ is a cofactor for DNA polymerases
– for Taq, required free [Mg++] = ~2mM
– calculated [Mg++] may differ from the actual
free [Mg++]
• Positively charged Mg++ is complexed by ionic
bonding with the negative charges on primers,
template, and dNTPs
• Mg++ must be uncomplexed (free) to act as a
cofactor for the polymerase
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PCR REQUIREMENTS - “buffer” components
• Mg++ (cont’d)
– Determining the optimal concentration of Mg++
is the most important step in setting up PCR
conditions
• too little - polymerase can’t work
• too much - favors annealing of primers to
mismatched locations on the template
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PCR REQUIREMENTS - polymerase
• 1-2 units of enzyme/100 l reaction
• High temperature resistant
– able to remain active through up to 35 cycles
with DNA denaturation at 95oC
– Example: Taq polymerase
• isolated from Thermophilus aquaticus
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PCR REQUIREMENTS - polymerase (cont’d)
• Many different
polymerases
available
– some have both
polymerase and
editing (exonuclease)
activities
Pfu polymerase can edit. Taq polymerase can not.
Taq polymerase is more likely to misincorporate a dNTP.
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PCR REQUIREMENTS - polymerase (cont’d)
• Many different polymerases available
– leave different types of ends
• blunt
• single A 3’overhang
– each isolated from a different organism which
has evolved to survive at high temperatures
• deep water vents
• hot springs
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PCR REQUIREMENTS
- denaturation of dsDNA
• Denaturation temperature
– high enough to overcome attractive energy of Hbonds between bases of the complementary template
and product strands
– 95oC provides sufficient energy to separate even
long strands
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PCR REQUIREMENTS - annealing
• The lower the temperature, the easier it is
for 2 strands of DNA to pair with each other
• So the chosen annealing temperature must
be
– High enough to prevent hybridization of
primers to imperfectly complementary template
sequences (i.e., non-specific annealing)
– Not so high that the primers can’t anneal to
template DNA at all
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PCR REQUIREMENTS - annealing
– What determines the optimum annealing temp
• length of primer - the longer, the higher the optimum
annealing temp will be
– longer the primer, the more H-bonds
– So, the more H-bonds, more likely 2 strands are to anneal or stay
annealed
• % GC - the more GC, the higher the optimum annealing temp
will be
– GC base pairs have 3 H-bonds; AT base pairs have only 2
– So, the more GC, more likely 2 strands are to anneal or stay annealed
• the  the [salt], the  the optimum annealing temp will be
– positive ions in salt are counterions to the negatively charged sugarphosphate backbone of the ds DNA
– positive counterions prevent repulsive forces of negative charges
from pushing the strands apart
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PCR REQUIREMENTS - annealing
• Annealing temperature for PCR is often set
at 5oC below the Tm
• Tm = temperature at which 50% of the
possible correct primer/template complexes
are unformed
• Estimate Tm for primers 10-23 nt long in
1M salt
Tm (oC) = 4 (G+C) + 2 (A+T)
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Hot start
• Way to minimize early non-specific annealing that
causes
– primer dimers
– amplification of incorrect product
Why might early annealing be expected to be non-specific?
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Hot start
• Enzyme is not mixed with reaction until sample has
reached denaturation temperature
– manual addition of enzyme at 95C
– polymerase separated from other reagents by layer
of solid wax
• wax melts at denaturation temperature, polymerase mixes
with reagents, wax rises to top and prevents evaporation
– start with antibody/polymerase complex
• antibody denatured and releases enzyme at 95C
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PCR REQUIREMENTS - extension
• Note: the longer the expected product, the
longer the extension time required
– exact time depends on rate of progression of the
specific polymerase
• Extension temperature
– optimal temperature for enzyme
– determined for each enzyme empirically
– usually around 72oC
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PCR REQUIREMENTS
• Thermocycler - instrument programmed to change
samples rapidly from one set temperature to
another
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PCR REQUIREMENTS
• Way to prevent evaporation of water from reaction
at high temperatures
– Why?
• evaporation raises concentrations of reaction solutes
 inhibition of reaction
– How?
• Thermocycler applies heat to the top of the reaction
tube, thereby preventing condensation
• or, overlayer the reaction with mineral oil,
preventing evaporation
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PCR REQUIREMENTS - thermocycler
• Way to heat and cool the sample
– Solid heating/cooling block that holds samples
• Efficient conduction of heat between
heating/cooling block and sample
– pressure applied from top pushes walls of tube
directly against block, eliminating air space, or
– mineral oil is used to fill in air space between
heating block and sample
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PCR REQUIREMENTS - controls
• No template control
– should be no product
– if there is, contaminating DNA is present
• Known positive (if possible)
– should be a product
– tells you all reaction components are working
– may tell you what your product should look like
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PCR REQUIREMENTS - controls (cont’d)
• Size markers
– show
• what size your product is
• and if you know what size to expect, whether you are
getting the expected product
• Results usually analyzed by gel electrophoresis
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PCR REQUIREMENTS Minimizing Contamination
• Contamination of pipettors
– Use aerosol barrier pipet tips
• Contamination of supplies and reagents
– UV irradiation
– base/acid treatment of reusable supplies
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PCR REQUIREMENTS Minimizing Contamination (cont’d)
• Contamination of work area with sample or product
– Perform steps at separated benches or rooms
•
•
•
•
sample prep
reaction set up
thermocycling
product analysis
– Prevent aerosols containing PCR products
• centrifuge reagents and products before opening tube
– also prevents contamination of reagents from gloves
• uncap tubes carefully
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PCR REQUIREMENTS
Minimizing Contamination (cont’d)
• Contamination of
reaction mix
– Use aerosol barrier
pipettor tips
– Use distilled
deionized water
– Add DNA to the
reaction last
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PCR REQUIREMENTS Troubleshooting
•
•
•
•
No yield
Extra or incorrect products
Primer dimers
Misincorporation
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PCR REQUIREMENTS -Troubleshooting
• No yield
– Were all reagents included?
– Insufficient denaturation?
• Higher temp
• Check conditions for transfer of heat from block to tube
– Active nucleases or proteases present in rx?
– Insufficient free Mg++?
– Bad primers?
• Degraded
• Wrong sequence
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PCR REQUIREMENTS -Troubleshooting
• Extra or incorrect products
– Mispriming
•
•
•
•
–
–
–
–
annealing temp too low
may need Hot Start
too much Mg++; facilitates misannealing
primer sequence insufficiently specific
[dNTP] too high
Too much polymerase
Annealing and/or extension time too long
Too many cycles; rare misprimed products
become amplified
– Template contamination
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PCR REQUIREMENTS -Troubleshooting
• Primer dimers
– What are they?
5’---------------------AT3’
3’TA----------------------5’
- Visible below the 100 bp marker on gel
- Can appear even when 3’ ends are not
complementary
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PCR REQUIREMENTS Troubleshooting
• Primer dimers
– Causes
•
•
•
•
•
Primer excess too great
3’ primer complementarity
Insufficient target template
Too many cycles
Annealing temperature too low
– Hot start may be required to avoid initial primer/primer annealing.
• Primers insufficiently specific (too short)
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PCR REQUIREMENTS Troubleshooting
• Misincorporation
– Excess of dNTPs too great
– dNTPs present in unequal concentrations; one is
exhausted before others
– Polymerase lacking exonuclease (editing) activity
– Polymerase concentration too high
– Extension temperature too low
– [Mg++] too high
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Lane #
1: no template.
2: +  DNA.
3-5: 100X  DNA,
[Mg++]
6,8: hair root lysate
7,9: hair shaft lysate
Rx in lanes 1-5 were performed with
 primers.
Rx in lanes 6-9 were performed with
HLA-DQ alpha primers.
Effects of [template] and [Mg++]
on PCR products