1. Polymerase Chain Reaction
What is PCR
History of PCR
How PCR works
Optimizing PCR
Fidelity, errors & cloning
PCR primer design
Application of PCR

2. Polymorphism among 32 wheat samples revealed by AFLP

3. Characteristics of AFLP
dominant marker.
DNA variation is detected by presence/absence of DNA bands due to:
a) presence/absence of restriction sites
b) additional bases (insertion) between two restriction sites are too large

4. Advantages higher reproducibility compared to RAPD.
highly polymorphic.

5. Think … …

6. PCR Requirements Magnesium chloride: .5-2.5mM Buffer: pH 8.3-8.8
dNTPs: µM
Primers: µM
DNA Polymerase: units
Target DNA:  1 µg

7. Can I PCR Amplify RNA?
Not directly — the DNA polymerase requires a DNA template and will not copy RNA.
mRNA can first be copied into cDNA using reverse transcriptase.
cDNA is a template for PCR —it need not be double-stranded.

8. Cloning PCR Products
Products should be ligatable into blunt-ended restriction enzyme site.
Lower than expected efficiency.
Products are not truly blunt-ended.
Taq polymerase adds a single nontemplated base (usually A) to the 3´ end:
NNNNNNN…NNNNNNNA
ANNNNNNN…NNNNNNN

9. Cloning PCR-generated Fragments
Generation of compatible ends.
T/A cloning vectors

10. PCR Mutagenesis
Add restriction sites for cloning Generate deletional mutants Generate single point mutations

11. THE PROBLEM QUANTITATION OF mRNA northern blotting
ribonuclease protection assay
in situ hybridization
PCR
most sensitive
can discriminate closely related mRNAs
technically simple
but difficult to get truly quantitative results using conventional PCR
Northern blotting and RPAS are the gold standards, since no amplification is involved.
In situ hybridization is qualitative rather than quantitative.
Techniques such as Northern blotting and ribonuclease protection assays (RPAs) work very well, but require more RNA than is sometimes available. PCR methods are particularly valuable when amounts of RNA are low, since the fact that PCR involves an amplification step means that it is more sensitive. However, traditional PCR is only semi-quantitative at best, in part because of the insensitivity of ethidium bromide (however, there are more sensitive ways to detect the product) and, in part, as we shall discuss later, because of the difficulties of observing the reaction during the truly linear part of the amplification process. Various competitive PCR protocols have been designed to overcome this problem but they tend to be cumbersome.
Real-time PCR has been developed so that more accurate results can be obtained. An additional advantage of real-time PCR is the relative ease and convenience of use compared to some of these older methods (as long as one has access to a suitable real-time PCR machine).

12. Protocol RT-PCR Isolate RNA (total or polyA selected)
Use RNA to generate cDNA (reverse transcriptase)
Use cDNA from RT reaction as template for PCR amplification.

13. RT-PCR vs “Real-time” RT-PCR
RT-PCR is endpoint analysis
“Real-time” (fluorescent; quantitative) PCR is continual analysis

14. Application of “Real-time” PCR
Expression analysis (RNA)
Identification/quantification of single base pair polymorphisms (DNA)
Genotyping (DNA)
Exposure monitoring RNA)
Clinical Screening
Cancer (DNA)
Pathogen detection (DNA/RNA)

15. Considerations for Fluorescent “Real-time” RT-PCR
Primer sets
Chosing target sequence
Checking primer sets
Chosing fluorescent label
Quantification
Relative quantification
Absolute quantification
Standard curves

16. Basics of real time RT-PCR Abundance of mRNA
Central hypothesis: Changes in gene expression reflect changes in cell function
Nearly all physiological changes in animals are accompanied by changes in gene expression
Immune response
Toxic response
Pharmacological response

17. Outline Basics of PCR and real time PCR Experimental Design
Data Analysis
Application to Drug Development and Design

18. Polymerase Chain Reaction
In vitro method for the amplification of short (up to ~5000bp) pieces of DNA
Relies on a thermostable form of DNA polymerase
Thermus aquaticus

19. Polymerase Chain Reaction
Required reagents:
Template DNA
Primers*
DNA polymerase
dNTPs*
Thermocycler

20. Polymerase Chain Reaction
Proper controls
Water blank
Concentration curve
Internal standard control
Genomic DNA contaminant control

21. Internal standard
Gene of interest

22. Polymerase Chain Reaction
Advantages
Sensitive
Versatile – easy to test new genes
Primers are inexpensive
Reliable
Much more than microarrays for individual transcripts
Standardized competitor templates or standard curves
Allow comparison between expts
Internal standards
Addresses variation in tissue starting amounts or loading errors

23. Polymerase Chain Reaction
Disadvantages
Optimizations required
Annealing temperature
Number of cycles
Small order of magnitiude sensitivity for detection

24. AFLP Amplified Fragment Length Polymorphism

27. Principles of Real-Time Quantitative PCR Techniques
SYBR Green I technique: SYBR Green I fluorescence is enormously increased upon binding to double-stranded DNA. During the extension phase, more and more SYBR Green I will bind to the PCR product, resulting in an increased fluorescence. Consequently, during each subsequent PCR cycle more fluorescence signal will be detected.
Hydrolysis probe technique: The hydrolysis probe is conjugated with a quencher fluorochrome, which absorbs the fluorescence of the reporter fluorochrome as long as the probe is intact. However, upon amplification of the target sequence, the hydrolysis probe is displaced and subsequently hydrolyzed by the Taq polymerase. This results in the separation of the reporter and quencher fluorochrome and consequently the fluorescence of the reporter fluorochrome becomes detectable. During each consecutive PCR cycle this fluorescence will further increase because of the progressive and exponential accumulation of free reporter fluorochromes.
Hybridization probes technique: In this technique one probe is labelled with a donor fluorochrome at the 3’ end and a second –adjacent- probe is labelled with an acceptor fluorochrome. When the two fluorochromes are in close vicinity (1–5 nucleotides apart), the emitted light of the donor fluorochrome will excite the acceptor fluorochrome (FRET). This results in the emission of fluorescence, which subsequently can be detected during the annealing phase and first part of the extension phase of the PCR reaction. After each subsequent PCR cycle more hybridization probes can anneal, resulting in higher fluorescence signals.

28. What do mRNA levels tell us?
DNAmRNAprotein
Reflect level of gene expression
Information about cell response
Protein production (not always)

29. quantitative mRNA/DNA analysis
Direct
-Northern blotting
-In situ hybridization
PCR amplification
-Regular RT-PCR
-Real time PCR
(Microarrays)
There two general ways to analyse mRNA. Either directly like in in situ hybridiszation or throug PCR magnification. Which include regular PCR and real time PCR.
nöfn

30. Why isn´t this good enough?
This slide shows the conventional PCR based testing formats. mRNA extraction, DNA amplification and then analysis by either by gel electrophorezis which can possable be followed by a Northern blot and Enzyme linked hybridisation which is not as common.
Why isn´t this good enough?

31. What’s Wrong With Agarose Gels?
* Low sensitivity
* Low resolution
* Non-automated
* Size-based discrimination only
* Results are not expressed as numbers
 based on personal evaluation
Ethidium bromide staining is not very quantitative
End point analysis
huglægt??
ABI: Real-Time PCR vs Traditional PCR (www)

32. Different concentrations give similar endpoint results!
Endpoint analysis
exponential increase
Different concentrations give similar endpoint results!

33. Real-time Principles
based on the detection and quantitation of
a fluorescent reporter
In stead of measuring the endpoint we focus on the first significant increase in the amount of PCR product.
The time of the increase correlates inversely to the initial amount of DNA template

34. Polymerization Q R R = Reporter Q = Quencher 3 Probe Forward Primer5
Reverse
R = Reporter
Q = Quencher

35. For Real Time PCR we need a a specific probe with a fluorescent reporter.Q R

36. When in close contact with the reporter,
the quencer absobes its emission.

37. Strand Displacement 3 Q 5 R

38. Cleavage 3 Q R 5

39. Polymerization Completed3 Q R 5

40. Van der Velden. Leukemia 2003 (www)

41. * nonspecific binding is a disadvantage
SYBR Green
(double-stranded DNA binding dye)
* emits a strong fluorescent signal upon binding to double-stranded DNA
* nonspecific binding is a disadvantage
* requires extensive optimisation
longer amplicons create a stronger signal
It´s cheap

42. SYBR® Green I Chemistry
Polymerization
Forward
Primer 5' 3' 5' 5' 3' 5'
Reverse
Primer
Polymerization completed 5' 3' 5' 5' 3' 5'

43. Real-time PCR advantages
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* requirement of 1000-fold less RNA than conventional assays
(3 picogram = one genome equivalent)
* most specific, sensitive and reproducible

44. Housekeeping gene
Knowing the amount of mRNA in one sample from one specific gene does not tell us alot
You don´t know the total amount of mRNA in your sample
You also dont know how much the mRNA level has changed compared to other mRNA levels
Example:
mRNA levels increase 2x after induction
It is possable that all genexpression in the cell has increased
We have to compare the expression of our gene to another gene which expression is normally constant, a housekeeping gene

45. Real-time PCR advantages
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional assays
(3 picogram = one genome equivalent)
* detection is capable down to a 2-fold change
* confirmation of specific amplification by melting curve analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR
(except equipment cost)

46. Real-time PCR disadvantages
* setting up requires high technical skill and support
* high equipment cost
* * *
* intra- and inter-assay variation
* RNA lability
* DNA contamination (in mRNA analysis)

47. Principles of Real-Time Quantitative PCR Techniques
SYBR Green I technique: SYBR Green I fluorescence is enormously increased upon binding to double-stranded DNA. During the extension phase, more and more SYBR Green I will bind to the PCR product, resulting in an increased fluorescence. Consequently, during each subsequent PCR cycle more fluorescence signal will be detected.
Hydrolysis probe technique: The hydrolysis probe is conjugated with a quencher fluorochrome, which absorbs the fluorescence of the reporter fluorochrome as long as the probe is intact. However, upon amplification of the target sequence, the hydrolysis probe is displaced and subsequently hydrolyzed by the Taq polymerase. This results in the separation of the reporter and quencher fluorochrome and consequently the fluorescence of the reporter fluorochrome becomes detectable. During each consecutive PCR cycle this fluorescence will further increase because of the progressive and exponential accumulation of free reporter fluorochromes.
Hybridization probes technique: In this technique one probe is labelled with a donor fluorochrome at the 3’ end and a second –adjacent- probe is labelled with an acceptor fluorochrome. When the two fluorochromes are in close vicinity (1–5 nucleotides apart), the emitted light of the donor fluorochrome will excite the acceptor fluorochrome (FRET). This results in the emission of fluorescence, which subsequently can be detected during the annealing phase and first part of the extension phase of the PCR reaction. After each subsequent PCR cycle more hybridization probes can anneal, resulting in higher fluorescence signals.

48. Schematic diagram comparing three different fluorescence-monitoring systems for DNA amplification. System A uses dsDNA-specific dyes (F) such as SYBR"Green I, which increase in fluorescence when bound to accumulating amplification product. System B uses dual-labelled probes and depends on the 5'-exonuclease activity of the polymerase to separate donor (D) and acceptor (A) by hydrolysis. Donor fluorescence is increased by removing acceptor quenching. System C depends on the independent hybridization of adjacent donor (D) and acceptor (A) probes. Their approximation increases resonance energy transfer from the donor to the acceptor. Other symbols are "hv" for excitation light and "x" for a 3'-phosphate.

49. TaqMan Probes FRET = Förster/fluorescence resonance energy transfer &
DNA Polymerase 5' exonuclease activity
* Tm value 100 C higher than primers
* runs of identical nucleotides (no consecutive Gs)
* G+C content 30-80%
* more Cs than Gs
* no G at the 5' end

50. FRET = Förster/fluorescence resonance energy transfer
ABI: Real-Time PCR vs Traditional PCR (www)

51. DNA Polymerase 5' Exonuclease Activity
Mocellin et al. Trends Mol Med 2003 (www)

52. The TaqMan 5’ Exonuclease Assay
In addition to two conventional PCR primers, P1 and P2, which are specific for the target sequence, a third primer, P3, is designed to bind specifically to a site on the target sequence downstream of the P1 binding site. P3 is labelled with two fluorophores, a reporter dye (R) is attached at the 5 end, and a quencher dye (D), which has a different emission wavelength to the reporter dye, is attached at its 3 end. Because its 3 end is blocked, primer P3 cannot by itself prime any new DNA synthesis. During the PCR reaction, Taq DNA polymerase synthesizes a new DNA strand primed by P1 and as the enzyme approaches P3, its exonuclease activity processively degrades the P3 primer from its 5 end. The end result is that the nascent DNA strand extends beyond the P3 binding site and the reporter and quencher dyes are no longer bound to the same molecule. As the reporter dye is no longer in close proximity to the quencher, the resulting increase in reporter emission intensity is easily detected.
Human Molecular Genetics 2. NCBI Books (www)

53. TaqMan Primers * equal Tm (58-600 C) * 15-30 bases in length
* G+C content 30-80%
* no runs of four or more Gs (any nucleotide)
* no more than two G+C at the 3’ end
* no G at the 5' end
* amplicon size bp (max 400)
* span exon-exon junctions in cDNA

54. SYBR Green
(double-stranded DNA binding dye)
* emits a strong fluorescent signal upon binding to double-stranded DNA
* nonspecific binding is a disadvantage
* requires extensive optimization
* requires melting point curve determination
* longer amplicons create a stronger signal
* may be multiplexed when coupled with melting curve analysis

55. Fluoresces when bound
to dsDNA

56. SYBR Green
(1) At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis. (2) After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation. (3) During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls.

57. When to Choose SYBR Green
* Assays that do not require specificity of probe based assays. Detection of 1000s of molecules
* General screening of transcripts prior to moving to probe based assays
* When the PCR system is fully optimized -no primer dimers or non-specific amplicons, e.g. from genomic DNA

58. When Not to Choose SYBR Green
* Allelic discrimination assays (not an absolute one)
* Multiplex reactions (not an absolute one)
* Amplification of rare transcripts
* Low level pathogen detection

59. Real-Time Principles
Three general methods for the quantitative detection:
1. Hydrolysis probes
(TaqMan, Beacons, Scorpions)
2. Hybridization probes
(Light Cycler)
3. DNA-binding agents
(SYBR Green)

60. Mocellin et al. Trends Mol Med 2003 (www)
Molecular Beacons
Mocellin et al. Trends Mol Med 2003 (www)

61. Real-Time Principles
Three general methods for the quantitative detection:
1. Hydrolysis probes
(TaqMan, Beacons, Scorpions)
2. Hybridization probes
(Light Cycler)
3. DNA-binding agents
(SYBR Green)

62. Scorpions
Bustin SA. J Mol Endocrinol 2002 (www)

63. Scorpions
Bustin SA. J Mol Endocrinol 2002 (www)

64. * it is the parameter used for quantitation
Threshold Cycle
* threshold cycle or the CT value is the cycle at which a significant increase in DRn is first detected
* it is the parameter used for quantitation
* CT value of 40 or more means no amplification and cannot be included in the calculations

65. What is CT?
The Amplification Plot contains valuable information for the quantitative measurement of DNA or RNA. The Threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. The threshold line is set in the exponential phase of the amplification for the most accurate reading. The cycle at which the sample reaches this level is called the Cycle Threshold, CT. These two values are very important for data analysis using the 5’ nuclease assay.

67. Albumin (ALB) gene dosage by real-time PCR
Laurendeau et al. Clin Chem 1999 (www)

68. DRn
* Rn+ is the Rn value of a reaction containing all components (the sample of interest); Rn- is the Rn value detected in NTC (baseline value)
* DRn is the difference between Rn+ and Rn-. It is an indicator of the magnitude of the signal generated by the PCR
* DRn is plotted against cycle numbers to produce the amplification curves and gives the CT value

69. What is DRn? +Rn Rn Rn -Rn CT cycle number Sample 10 20 30 4010 20 30 40
cycle number Rn CT
Threshold
Rn
Sample
No Template
Control
-Rn
+Rn

70. What is DRn?
(www)

71. Endogenous/Internal Control (Normalization)
* usually an abundantly and constantly expressed housekeeping gene
* most commonly used ones are the least reliable ones
* best to run a validity test for the selected endogenous control
* combination may/should be used

72. Endogenous Control Selection
Sabek et al. Transplantation 2002 (www)

73. Approximation vs Pfaffl method (Efficiency Determination)
The slope of the log-linear phase is a reflection of the amplification efficiency
The efficiency of the reaction can be calculated by the following equation: Eff=10(-1/slope) –1. The efficiency of the PCR should be % (ideal slope = ­3.3)
A number of variables can affect the efficiency of the PCR. These factors can include length of the amplicon, secondary structure, and primer design, to name a few
Approximation vs Pfaffl method
(Efficiency Determination)

75. Using the PCR Equation Xn X0 Xn = X0(1 + E)n
Xn = PCR product after cycle n
X0 = initial copy number
E = amplification efficiency
n = cycle number
E IS MAXIMALLY 1.0, BUT USUALLY AROUND 0.9. X0
cycle number

76. Effect of Amplification Efficiency
Xn = X0(1+E)n
Case 1: E = Case 2: E = 0.8
Xn = 100 (1+0.9)30
Xn = 100 (1+0.8)30
Xn = 2.3 x 1010
Xn = 4.6 x 109
CALCULATE EFFICIENCIES FROM THE SLOPE OF YOUR DOSE RESPONSE CURVES
Result
A difference of 0.1 in amplification
efficiencies created a five-fold difference in the
final ratio of PCR products after 30 cycles

77. Determination of real-time PCR efficiencies of reference gene (Gst), target gene 1 (TyrA) and target gene 2 (PyrB). CP cycles versus cDNA (reverse transcribed total RNA) concentration input were plotted to calculate the slope (mean ± SD; n = 3). The corresponding real-time PCR efficiencies were calculated according to the equation: E = 10[–1/slope]

78. If the CT values for each of the dilutions are plotted against concentrations, the result should be a linear graph with a high correlation coefficient (> 0.99). The slope of this graph is also a measure of efficiency, and can be readily used to calculate efficiency - this is done by most software (iCycler, for example).

79. Nigel Walker, NIEHS (www)

80. Nigel Walker, NIEHS (www)

81. Assay Validation
* Test primer pairs in all combinations with the probe with
a known template (plasmid clone, sDNA, RNA)
* Use standard assay conditions: nM primers;
100 nM probe, 3 mM MgCl2
* Choose the primer pair that gives the highest DRn
and the lowest CT
* Make a dilution of a template, either sDNA, sRNA or total
RNA for a standard curve
* Correlation coefficient of the standard curve > 0.99?
* If the slope of the standard curve of the best primer
pair is around -3.5 increase the MgCl2 to 5 mM
* If the slope is higher than -3.6, change primers
* An ideal assay will have a slope of -3.3

82. One-Step or Two-Step PCR
* one-step real-time RT-PCR performs reverse transcription and PCR in a single buffer system and in one tube
* in two-step RT-PCR, these two steps are performed separately in different tubes

83. The really big deal Isolation of DNA polymerase from Thermus
aquaticus (Mullis and Faloona 1987)
• T. aquaticus is a hot springs bacterium
• Therefore, has thermally stable DNA
polymerase (e.g. not killed by heat)

84. Importance of T. aquaticus
Extension at 72 C: Most polymerases are
active at 45 C or below (e.g. normal
body/environmental temps)-temps below
normal annealing temps-results in nonspecific
binding of primers
2. From Bacterium that lives in near boiling
water: Most polymerase inactivated by high
temperatures-initially had to add new
polymerase each cycle

85. Colony PCR
CPSC265 Class 8

86. Cloning
Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule.
These cells are clones, hence the name
This used to be the only way to amplify DNA. It is still by far the most accurate.

88. Plasmid vectors – circular, autonomous bacterial DNA

89. The vector is made with a “T” overhang

90. Taq polymerase leaves an “A” overhang
Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR.
When Taq synthesizes a new strand, it always puts an extra “A” at the end
This can be useful, but note: other polymerases do not do this, they leave “blunt” ends. Only Taq polymerase leaves ‘A’ overhangs. ‘Blunt’ end vectors do not work with Taq, we need a ‘T’ overhang.

92. DNA ligase Repairs gaps in the sugar-phosphate backbone of DNA
Creates phosphodiester bonds
Does not do anything with the bases

93. Transformation of bacteria
Two main methods for transformation
Chemical / Heat Shock
As done in last practical, this method gets DNA into the cell by making them porous using CaCl2 and a 42 C heat treatment
Electroporation
Makes cells porous using high-voltage electricity

94. Imperfect science
Most of the plasmid / insert combinations will not ligate
Most of the bacteria will not be transformed
We only need one molecule to get into one bacterium to make one colony.

95. PCR from clones
Often clones will religate containing any old DNA (eg primer dimers)..
The DNA can go in in either orientation
We can use the PCR to tell which colonies have the insert we want, and which orientation it is in.

96. Some population genetics software
Microsatellite toolkit: Excel plug-in for creating Arlequin, FSTAT and Genepop files. Microchecker: Estimate null allele frequency. Adjust allele frequencies. Arlequin: HW equilibrium, Linkage Disequilibrium, Fst, exact test of differentiation, Amova, Mantel test FSTAT: Allelic richness, Fst per locus (to check contribution of each locus to observed pattern of differentiation) Structure, BAPS: Population structuring, population assignment. Migrate: Estimates of effective population size and migration rates Bottleneck: Check for very recent population bottlenecks