1. The Polymerase Chain Reaction
AMPLIFICATION:
The Polymerase Chain Reaction
Adapted from a presentation written for
Principles of Gene Manipulation · November 6, 2000
Jennifer Cooper · America Madrigal · Laleña Vellanoweth

2. AMPLIFICATION: PCR and Its Applications
AMPLIFICATION: PCR and Its Applications
Definition of PCR
Requirements for PCR
PCR Process
A. Denaturation
B. Annealing
C. Extension
D. Cycling (repeat A-C)
PCR for HLA DQ-alpha

3. PCR What is it? The Polymerase Chain Reaction (PCR) is an
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.

4. PCR REQUIREMENTS In the reaction
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

5. PCR METHOD There are three basic steps in PCR 1. Denaturation (~95oC)
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.

6. High temperature separates the two strands.
PCR METHOD – DENATURATION STAGE
High temperature separates the two strands.
(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

7. Primer length is usually ~20 nucleotides.
PCR METHOD – ANNEALING STAGE
Primer length is usually ~20 nucleotides.
(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

8. Thermostable polymerase adds dNTPs one at a time at this stage.
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)

9.
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)

10. (dictated by the placement of the primers)
PCR METHOD - CYCLING
DNA
Cycle …
Products …
After the first two cycles, fragments with the correct length begin to be amplified.
(dictated by the placement of the primers)

11. PCR for HLA DQ-alpha Biotinylated primers amplify a region of ~250 bp.
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.

12. The human male karyotype: 22 homologous chromosomes + X and Y
The human male karyotype: 22 homologous chromosomes + X and Y

14. PCR for HLA DQ-alpha Products of PCR reaction
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.

15. PCR REFERENCES 1. Gene Cloning: an introduction. T.A. Brown
PCR REFERENCES
1. Gene Cloning: an introduction. T.A. Brown
2. The world wide web:

16.
After a long process, this PowerPoint presentation has now ended. We hope it is of good help in your studies.

17. PCR Requirements The Details
PCR Requirements The Details

18. PCR REQUIREMENTS: sample - template
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.

19. PCR REQUIREMENTS: sample - template
PCR REQUIREMENTS: sample - template
Even degraded DNA is OK if sample is large enough
Fossils
Remains
Old samples from crime scenes

20. PCR REQUIREMENTS - primers
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 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 cycles

21. PCR REQUIREMENTS - dNTPs
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

22. PCR REQUIREMENTS - “buffer” components
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

23. PCR REQUIREMENTS - “buffer” components
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

24. PCR REQUIREMENTS - “buffer” components
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

25. PCR REQUIREMENTS - “buffer” components
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

26. PCR REQUIREMENTS - polymerase
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

27. PCR REQUIREMENTS - polymerase (cont’d)
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.

28. PCR REQUIREMENTS - polymerase (cont’d)
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

29. PCR REQUIREMENTS - denaturation of dsDNA
PCR REQUIREMENTS - denaturation of dsDNA
Denaturation temperature
high enough to overcome attractive energy of H-bonds between bases of the complementary template and product strands
95oC provides sufficient energy to separate even long strands

30. PCR REQUIREMENTS - annealing
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

31. 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 sugar-phosphate backbone of the ds DNA
positive counterions prevent repulsive forces of negative charges from pushing the strands apart

32. PCR REQUIREMENTS - annealing
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 nt long in 1M salt
Tm (oC) = 4 (G+C) + 2 (A+T)

33. Why might early annealing be expected to be non-specific?
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?

34.
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

35. PCR REQUIREMENTS - extension
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

36.
PCR REQUIREMENTS
Thermocycler - instrument programmed to change samples rapidly from one set temperature to another

37.
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

38. PCR REQUIREMENTS - thermocycler
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

39. 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

40. PCR REQUIREMENTS - controls (cont’d)
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

41. PCR REQUIREMENTS - Minimizing Contamination
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

42. PCR REQUIREMENTS - Minimizing Contamination (cont’d)
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

43. PCR REQUIREMENTS Minimizing Contamination (cont’d)
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

44. PCR REQUIREMENTS - Troubleshooting
PCR REQUIREMENTS - Troubleshooting
No yield
Extra or incorrect products
Primer dimers
Misincorporation

45. PCR REQUIREMENTS -Troubleshooting
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

46. 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

47. PCR REQUIREMENTS -Troubleshooting
PCR REQUIREMENTS -Troubleshooting
Primer dimers
What are they?
5’ AT3’
3’TA ’
Visible below the 100 bp marker on gel
Can appear even when 3’ ends are not complementary

48. PCR REQUIREMENTS - Troubleshooting
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)

49. PCR REQUIREMENTS - Troubleshooting
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

50. Effects of [template] and [Mg++]
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