1. PCR
The Polymerase Chain Reaction

2. Polymerase Chain Reaction
The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention.
A special DNA polymerase (Taq) is used to make multiple identical copies of a short length of DNA (100-10,000 bp) defined by primers.
Kary Mullis (mid 1980’s), the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry.

3. What is PCR? : Why “Polymerase”?
It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
Why “Chain”?
It is called “chain” because the products of the first reaction become substrates of the following one and so on.
The “Reaction” Components
Target DNA - contains the sequence to be amplified.
Pair of Primers - oligonucleotides that define the sequence to be amplified.
3) dNTPs – deoxy-nucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH & ionic strength of the reaction solution suitable for the activity of the enzyme

4. Required a thermostable DNA polymerase
Initially PCR used the Klenow fragment of E. coli DNA polymerase - inactivated by high temperatures.
Required a thermostable DNA polymerase
Taq DNA polymerase from Thermus aquaticus
A thermophilic eubacterial µ-org isolated from a hot spring in Yellowstone National Park
Kcat = 150 nucleotides/sec/enzyme (at Topt)
Taq1/2 =
Klenow Fragment (3´→ 5´ exo-) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity but has lost the 5´→ 3´ exonuclease activity
Kcat is the "turnover number" and can be calculated as Vmax/[E concentration] and its units are 1/seconds. It basically tells you how much substrate is converted to product in one second.
5 min
97.5 oC
40 min
95.0 oC
130 min
92.5 oC

5. PCR - before the Thermocycler
8 BORING hours per PCR!
95º C (5 min)
35 times
55º C (3 min)
72º C (5 min)

6. Development….
PCR work was first published (1985) using Klenow polymerase – unstable with heat
New enzyme had to be added manually at each step
Maximum length 400bp
Great idea – not very practical
First reports using DNA polymerase from Thermus aquaticus (1988)
Taq-polymerase from Yellow stone National Park hot springs
Developed automatic “thermocycler” programmable heat block

7. Thermocyclers Heated lids Adjustable ramping times
Single/Multiple blocks
Gradient thermocycler blocks

8. Standard Tube
 Volume
 Cost & Evaporation
Thin Walled Tube
Volume
Cost & Evaporation

9. “Xeroxing” DNA (Xerography, Photocopying)
Cycle 1
PLUS dNTPs, buffer,
salts,Taq pol,
primers
Cycle 35
n36 = 68,719,476,736 copies in ~ 2 hrs
2 copies
Cycle 2
4 copies
Cycle 3
8 copies

10. A simple Thermocycling Protocol
annealing
94ºC
55ºC
72ºC
4ºC
3 min
1 min
45 sec
∞ hold
Initial denaturation
of DNA 1X
35X
extension
denaturation

11. Step 1
Denaturation
dsDNA to ssDNA
Step 2
Annealing
Primers onto template
Step 3
Extension
dNTPs extend 2nd strand
Extension products in one cycle serve as template in the next

12. Basic Components of PCR
Template DNA ( ng)
Oligonucleotide primers (0.1 – 2.0 μM)
dNTP’s (20 –250 μM)
Thermostable DNA pol (0.5 – 2.5 U/rxn)
MgCl2 (1 – 5 mM) affects primer annealing and Taq activity
Buffer (usually supplied as 10X)
Working concentrations
KCL (10 – 50 mM)
Tris-HCl (10 mM, pH 8.3)
NaCl2 (sometimes)
Primers
DNA template
Buffer
+ +
A C T G
MgCl2
dNTPs
Taq polymerase

13. Magnesium ions have a variety of effects
MgCl2 (mM)
Magnesium Chloride
(MgCl2 - usually mM)
Magnesium ions have a variety of effects
It acts as cofactor for Taq polymerase
Required for Taq to function
Mg2+ binds with DNA and affects primer/template interactions
Mg2+ influences the ability of Taq pol to interact with primer / template sequences
More magnesium leads to less stringency in binding
Strength

14. Taq Pol is active at low temperatures
PCR Problems
Taq Pol is active at low temperatures
At low temperatures mis-priming is likely
Temp
Extension Rate
55o C
24 nt/sec
37o C
1.5 nt/sec
22o C
0.25 nt/sec
150 nucleotides in 10 min

15. Primer Design Typically 20 to 30 bases in length
Annealing temperature dependent upon primer sequence (~ 50% GC content)
Avoid primer complementarity (primer dimer)
The last 3 nucleotides at the 3` end is the substrate for DNA polymerase - G or C

16. Rules for PCR conditions
Add an extra 3-5 minute to your cycle profile to ensure everything is denatured prior to starting the PCR reaction
Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x (G+C))]ºC
If GC content is < 50% start 5ºC beneath Tm for annealing Temp.
If GC content ≥ 50% start at Tm for annealing temperature
72ºC: rule of thumb is ~500 nucleotide per minute.

17. Common PCR additives
BSA (Usually at µg/µL final concentration)
Stabilize Taq polymerase & overcome PCR inhibitors
DMSO (Usually at 2-5% v/v, inhibitory at ≤ 10% v/v)
Acts as denaturant - good at keeping GC rich template/primer strands from forming secondary structures.
Glycerol (Usually at 5-10% v/v)
increases PCR efficiency at high temperatures.
Stringency enhancers (Formamide, Betaine, TMAC)
Concentrations used vary by type
Enhances yield and reduces non-specific priming

18. Typical PCR Temps/Times
Initial denaturation
90o – 95o C
1 – 3 min
Denature
90o – 95o C
0.5 – 1 min
25 – 40 cycles
Primer annealing
45o – 65o C
0.5 – 1 min
Primer extension
70o – 75o C
0.5 – 2 min
Final extension
70o – 75o C
5 – 10 min
Stop reaction
4o C or 10 mM EDTA
hold

20. PROCEDURE …..

22. PCR
Temperature
100 50
T i m e
Melting
94 oC 5’ 3’

23. PCR
Temperature
100 50
T i m e
Melting
94 oC 5’ 3’
Heat 5’ 3’

24. PCR T i m e Temperature Melting Melting 94 oC 94 oC Extension
100 50
T i m e
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC 5’ 3’ 5’ 3’ 5’

25. PCR T i m e Heat Heat 30x Temperature Melting 94 oC Extension
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100 50
T i m e
30x 5’ 3’
Heat 5’ 5’
Heat 5’ 3’ 5’

26. PCR T i m e 30x Temperature Melting 94 oC Extension Annealing Primers
100 50
T i m e
30x 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’

27. PCR T i m e Heat Heat 30x Temperature Melting 94 oC Extension
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100 50
T i m e
30x 3’ 5’ 5’
Heat 5’ 5’
Heat 5’

28. PCR T i m e 30x Temperature Melting 94 oC Extension Annealing Primers
100 50
T i m e
30x 3’ 5’ 5’ 5’

29. PCR T i m e 30x Temperature Fragments of defined length Melting 94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100 50
T i m e
30x 3’ 5’
Fragments of defined length 5’ 5’

30. More Cycles = More DNA
Number of cycles
Size
Marker

31. PCR Optimisation 1: Buffers
Most buffers have only KCl (50mM) and Tris (10mM)
Concentrations of these can be altered
KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq
DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P-40, Triton X-100 can be added to aid in the PCR reaction
Enhance specificity, but also can be inhibitory
Pre-mixed buffers are available

32. PCR Optimisation 2: MgCl2
MgCl2: required for primer binding
MgCl2 affects primer binding, Tm of template DNA, product- and primer-template associations, enzyme activity & fidelity (accuracy).
dNTPs, primers and template, chelate the Mg ion, therefore concentration should be higher than dNTPs.
Excess magnesium gives non-specific binding.
Too little magnesium gives reduced yield.

33. PCR Optimisation 3: Primer Design
Specific to sequence of interest
Length nucleotides
Annealing temperature 50oC-70oC
Ideally 58oC-63oC
GC content 40-60%
3’ end critical (new strand extends from here)
GC clamp (G or C at 3’ terminus)
Inner self complementarity:
Hairpins <5bp, dimers <9bp

34. PCR Optimisation 4: Cycling Conditions
Denaturation:
Some Taq polymerases require initial denaturation (hot start)
Annealing temperature:
~ 5oC less than Tm of primers
Tm = 4(G + C) + 2(A + T)oC

35. PCR Optimisation 5: Cycle Number
25-40 cycles
Half-life of Taq is 30 minutes at 95oC
Therefore if you use more than 30 cycles at denaturation times of 1 min, the Taq will not be very efficient at this point

36. Types of PCR

37. Colony PCR
Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products.
Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of Tris-EDTA buffer autoclaved dH2O in an microfuge tube.
Heat the mix in a boiling water bath (90-100C) for 2 minutes
Spin sample for 2 minutes high speed in centrifuge.
Transfer 20 μl of the supernatant into a new microfuge tube
Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.

38. Hot Start PCR
This is a technique that reduces non-specific amplification during the initial set up stages of the PCR
The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase
DNA Polymerase- Eubacterial type I DNA polymerase, Pfu
These thermophilic DNA polymerases show a very small polymerase activity at room temperature.

39. Nested PCR
Nested PCR is a modification of PCR intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites.
PCR itself is the process used to amplify DNA samples, via a temperature-mediated DNA polymerase. The products can be used for sequencing or analysis, and this process is a key part of many genetics research laboratories, along with uses in DNA fingerprinting for forensics and other human genetic cases.
Conventional PCR requires primers complementary to the termini of the target DNA. A commonly occurring problem is primers binding to incorrect regions of the DNA, giving unexpected products.
Nested PCR involves two sets of primers, used in two successive runs of PCR, the second set intended to amplify a secondary target within the first run product.

40. Processes
The target DNA undergoes the first run of PCR with the first set of primers. The selection of alternative & similar primer binding sites gives a selection of products, only one containing the intended sequence.
The product from the first reaction undergoes a second run with the second set of primers. It is very unlikely that any of the unwanted PCR products contain binding sites for both the new primers, ensuring the product from the second PCR has little contamination from unwanted products of primer dimers, hairpins & alternative primer target sequences.

41. Multiplex PCR
Multiplex PCR is a modification of PCR in order to rapidly detect deletions or duplications in a large gene. This process amplifies genomic DNA samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler.
Multiplex-PCR was first described in 1988 as a method to detect deletions in the dystrophin gene. It has also been used with the steroid sulfatase gene. In 2008, multiplex-PCR was used for analysis of microsatellites and SNPs.
Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences.

42. Types of Multiplex PCR
Multiplexing reactions can be broadly divided in two categories:
Single Template PCR Reaction.
This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template.
2. Multiple Template PCR Reaction.
It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates.

43. Primer Design Parameters for Multiplex PCR
Design of specific primer sets is essential for a successful multiplex reaction. The important primer design considerations are;
Primer Length
Multiplex PCR assays involve designing of large number of primers, hence it is required that the designed primer should be of appropriate length. Usually, primers of short length, in the range of bases are used.
2. Melting Temperature
Primers with similar Tm, preferably between 55°C - 60°C are used. For sequences with high GC content, primers with a higher Tm (preferably 75°C-80°C) are recommended. A Tm variation of between 3°-5° C is acceptable for primers used in a pool.
3. Specificity
It is important to consider the specificity of designed primers to the target sequences, while preparing a multiplex assay, especially since competition exists when multiple target sequences are in a single reaction vessel..

44. 4. Avoid Primer Dimer Formation
The designed primers should be checked for formation of primer dimers, with all the primers present in the reaction mixture. Dimerization leads to unspecific amplification.
All other parameters are similar to standard PCR primer design guidelines
Applications of Multiplex PCR
Pathogen Identification
High Throughput SNP Genotyping
Mutation Analysis
Gene Deletion Analysis
Template Quantitation
Linkage Analysis
RNA Detection
Forensic Studies

45. Reverse Transcriptase PCR
Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses.
First step of RT-PCR - "first strand reaction“-Synthesis of cDNA using oligo dT primers (37°C) 1 hr.
“Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)-
Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.

47. Applications of PCR Classification of organisms Genotyping
Molecular archaeology
Mutagenesis
Mutation detection
Sequencing
Cancer research
Detection of pathogens
DNA fingerprinting
Drug discovery
Genetic matching
Genetic engineering
Pre-natal diagnosis