1. Real time Pcr Dr. Basim Ayesh Biotechnology training course
Real-Time PCR Training
Real time Pcr
Biotechnology training course
August, 2009
Dr. Basim Ayesh

2. The Evolution of PCR to Real-Time
Real-Time PCR Training
The Evolution of PCR to Real-Time
Traditional PCRs use Agarose gels for detection of amplification at the final phase or end-point of the PCR reaction
Real time PCR has advanced to allow detection before the end-point of the reaction.

3. Real-time PCR detection
Real-Time PCR Training
Real-time PCR detection
While the reaction is occurring
monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle
Real-Time chemistries allow for the detection of PCR amplification during the early phases of the reaction

4. Real-Time PCR Training

5. PCR Phases Exponential: Linear (High Variability):
Real-Time PCR Training
PCR Phases
Exponential:
Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.
Linear (High Variability):
The reaction components are being consumed, the reaction is slowing, and products are starting to degrade.
Plateau (End-Point: Gel detection for traditional methods):
The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.
Figure 3 shows three replicates of a sample. The replicates have the same starting quantity. As the PCR reaction progresses, the samples begin to amplify in a very precise manner. Amplification occurs exponentially, that is a doubling of product (amplicon) occurs every cycle. This type of amplification occurs in the exponential phase. Exponential amplification occurs because all of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon.
However, as the reaction progresses, some of the reagents are being consumed as a result of amplification. This depletion will occur at different rates for each replicate. The reactions start to slow down and the PCR product is no longer being doubled at each cycle. This linear amplification can be seen in the linear phase of the reaction. The three samples begin to diverge in their quantities during the linear phase.
Eventually the reactions begin to slow down and stop all together or plateau. Each tube or reaction will plateau at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase. The plateau phase is where traditional PCR takes its measurement, also known as end-point detection.
Figure 3 also shows that the three replicate samples, which started out at the same quantity in the beginning of the reaction, reflect different quantities at the plateau phase. Since the samples are replicates they should have identical quantities. Therefore, it will be more precise to take measurements during the exponential phase, where the replicate samples are amplifying exponentially.
The amplification phases can be viewed differently to assess the PCR phases. The figures that follow show the phases of PCR in a Logarithmic scale view and a Linear scale view (Figures 4 and 5).

6. Real-Time PCR Training

7. Problems with detection in the Plateau phase of PCR
Real-Time PCR Training
Problems with detection in the Plateau phase of PCR
The plateau effect on 96 replicates:
The 96 replicates in the exponential phase are very tight in both the linear and logarithmic views.
The reactions show a clear separation in the plateau phase; therefore, if the measurements were taken in the plateau phase, quantitation would be affected.

8. The plateau effect on a fivefold dilution series:
Real-Time PCR Training
The plateau effect on a fivefold dilution series:
The 5-fold dilution series, seems to plateau at the same place even though the exponential phase clearly shows a difference between the points along the dilution series.
This reinforces the fact that if measurements were taken at the plateau phase, the data would not truly represent the initial amounts of starting target material.
As you can see from the figure, the samples in the gel contain 10 copies and 50 copies, respectively. It is hard to differentiate between the 5-fold change on the Agarose gel. Real-Time PCR is able to detect a two-fold change (i.e. 10 Vs. 20 copies) .

9. Limitations of End-Point PCR
Real-Time PCR Training
Limitations of End-Point PCR
Poor Precision
Low sensitivity
Short dynamic range < 2 logs
Low resolution
Non - Automated
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide for staining is not very quantitative
Post PCR processing

10. Real-Time PCR Training
Real-time Principles
Three general methods for the quantitative detection:
Hydrolysis probes (TaqMan, Beacons, Scorpions).
Hybridisation probes (Light Cycler).
DNA-binding agents (SYBR Green).

11. Real-Time PCR Training
TaqMan Chemistry
Real-time systems using fluorogenic-labeled probes that use the 5´ nuclease activity of Taq DNA polymerase.
Detecting only specific amplification products.
Eliminate post-PCR processing for the analysis of probe degradation

12. Real-Time PCR Training
A fluorogenic probe enables the detection of a specific PCR product as it accumulates during PCR.
The TaqMan® Probe is designed with a high-energy dye termed a Reporter at the 5 end, and a low-energy molecule termed a Quencher at the 3 end.
While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET) through space.
When reporter and quencher are separated the reporter fluoroscence

13. How TaqMan Sequence Detection Chemistry Works
Real-Time PCR Training
How TaqMan Sequence Detection Chemistry Works
If the target sequence is present, the probe anneals downstream from one of the primer sites
During extension the probe is cleaved by the 5´ nuclease activity of Taq DNA polymerase.
The reporter dye is separated from the quencher dye, increasing the reporter dye signal.
The probe is removed from the target strand, allowing primer extension to continue to the end of the template strand..
Additional reporter dye molecules are cleaved from their respective probes with each cycle.
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.

14. Real-Time PCR Training
The fluorescence intensity is increased proportional to the amount of amplicon produced.

15. Real-Time PCR Training
Molecular Beacons

16. Real-Time PCR Training
Scorpions

17. Real-Time PCR Training
Hybridization Probes
Fluorescence Resonance Energy Transfer (FRET) Concept
Donor fluorophore is excited by appropriate wavelength
Donor energy is transferred non-radiatively to the acceptor fluorophore
Excited acceptor emits at longer wavelength
Increase in fluorescence with each cycle
The signal depends only on hybridization

18. How the Hybridisation probes work?
Real-Time PCR Training
How the Hybridisation probes work?
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.
Hybridisation 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 hybridisation probes can anneal, resulting in higher fluorescence signals.

19. SYBR® Green I Dye Chemistry
Real-Time PCR Training
SYBR® Green I Dye Chemistry
Small molecules that bind to double-stranded DNA can be divided into two classes:
Intercalators
Minor-groove binders
There are two requirements for a DNA binding dye for real-time detection of PCR:
Increased fluorescence when bound to double-stranded DNA
No inhibition of PCR

20. How the SYBR Green I Dye Chemistry Works
Real-Time PCR Training
How the SYBR Green I Dye Chemistry Works
When SYBR Green I dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample.
During the PCR, the DNA Polymerase amplifies the target sequence, and creates the PCR products.
The SYBR Green I dye binds to each new copy of double-stranded DNA.
As the PCR progresses, more amplicons are created. The result is an increase in fluorescence intensity proportionate to the amount of PCR product produced.
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.

21. Advantages of SYBR Green I Dye
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Advantages of SYBR Green I Dye
It can be used to monitor the amplification of any double-stranded DNA sequence.
No probe is required, which reduces assay setup and running costs.
Disadvantage of SYBR Green I Dye
SYBR Green I dye chemistry may generate false positive signals
SYBR Green I dye binds to nonspecific double-stranded DNA sequences.
Needs fully optimized PCR system -no primer dimers or non-specific amplicons.

22. When not to choose SYBR Green
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When not to choose SYBR Green
Allelic discrimination assays.
Multiplex reactions.
Amplification of rare transcripts .
Low level pathogen detection .

23. Terms Used in Quantitation Analysis
Real-Time PCR Training
Terms Used in Quantitation Analysis
Amplification plot: The plot of fluorescence signal versus cycle number

24. Real-Time PCR Training
Baseline: The initial cycles of PCR, in which there is little change in fluorescence signal
Passive reference: A dye that provides an internal reference to which the reporter dye signal can be normalized during data analysis.
Rn (normalized reporter): The fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye.
Normalization is necessary to correct for fluctuations caused by changes in concentration or volume.

25. Real-Time PCR Training
Rn+: The Rn value of a reaction containing all components, including the template
Rn-:The Rn value of an un-reacted sample. The Rn-value can be obtained from:
The early cycles of a real-time PCR run (those cycles prior to a detectable increase in fluorescence), OR
A reaction that does not contain any template
ΔRn (delta Rn): The magnitude of the signal generated by the given set of PCR conditions. [ΔRn = (Rn+) – (Rn-)]

26. Real-Time PCR Training
Threshold: The average standard deviation of Rn for the early PCR cycles, multiplied by an adjustable factor. The threshold should be set in the region associated with an exponential growth of PCR product.
Ct (threshold cycle): The fractional cycle number at which the fluorescence passes the fixed threshold

27. Reporters The classical reporter dye is 6-FAM (fluorescein)
Real-Time PCR Training
Reporters
The classical reporter dye is 6-FAM (fluorescein)
Other reporters used for multiplexing are Joe and Vic.
Some real-time machines can use red dyes as reporters

28. Quenchers The classic quencher dye has been TAMRA (rhodomine)
Real-Time PCR Training
Quenchers
The classic quencher dye has been TAMRA (rhodomine)
Newer quenchers are the dark dyes: DABYCL and the black hole quenchers (BQ)
MGB
A nonfluorescent quencher at the 3´ end - The SDS instruments can measure the reporter dye contributions more precisely because the quencher does not fluoresce.
A minor groove binder at the 3´ end - The minor groove binder increases the melting temperature (Tm) of probes, allowing the use of shorter probes.
Recommended for allelic discrimination assays

29. Internal Reference Dyes
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Internal Reference Dyes
TAMRA-quenched probes do not require a reference dye; they can use the TAMRA itself
Single probe reactions quenched by dark dyes should use an internal reference dye, classically ROX (dark red)
Multiplex reactions usually use dark quenchers and ROX

30. Real-Time PCR Training

31. Real-Time PCR Training
20 min break

32. Real-Time PCR Training
Quantitation
By Real time PCR

33. Absolute Quantitation
Real-Time PCR Training
Absolute Quantitation
Used to quantitate unknown samples by interpolating their quantity from a standard curve.
Standard: A sample of known concentration used to construct a standard curve.
From a standard curve you can extrapolate the quantity of an unknown sample.
Unknown: A sample containing an unknown quantity of template. This is the sample whose quantity you want to determine

34. Real-Time PCR Training

35. Real-Time PCR Training
The standard curve resulting from Ct value and standard concentrations should be a linear graph with a high correlation coefficient (> 0.99).
Theoretically a single copy of the target should create a CT value of 40 (if efficiency is 100%), which is the y-intercept in a standard curve experiment
CT value of 40 or more means no amplification and cannot be included in the calculations

36. Relative Quantitation
Real-Time PCR Training
Relative Quantitation
A relative quantitation assay is used to analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).
Relative quantitation might be used to measure gene expression in response to a chemical (drug).

37. Relative Quantitation Standard curve method
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Relative Quantitation Standard curve method
Using a separate standard curve for the target and endogenous control, in separate tubes.
Requires the least amount of optimization and validation.

38. Relative Quantitation Comparative CT method
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Relative Quantitation Comparative CT method
The need for a standard curve is eliminated.
The target and endogenous control are amplified in the same tube.
Increased throughput:
wells no longer need to be used for the standard curve samples.
the adverse effect of any pipetting and dilution errors made in creating the standard curve samples are eliminated.
A validation experiment must be run to show that the efficiencies of the target and endogenous control amplifications are approximately equal.
limiting primer concentrations must be identified and shown not to affect CT values.

39. Endogenous/Internal Control
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Endogenous/Internal Control
Usually an abundantly and constantly expressed housekeeping gene
Best to run a validity test for the selected endogenous control
Combination may be used

40. Melting Curve Analysis
Real-Time PCR Training
Melting Curve Analysis
Genotyping for the haemochromatosis G845A mutation using melting curve analysis of FRET hybridisation probes
AA, G845A homozygotes; GA, G845A heterozygotes; GG, or “wild-type” homozygotes. Right upper panel: Plot of red fluorescence relative to reference (F2/F1) versus temperature (T) for the three genotypes. Three different melting curves are shown for the three possible genotypes. These represent changes in fluorescence of the FRET complexes as they are heated through their melting temperature at the end of PCR amplification. Right Lower panel: -d(F2/F1)/dT versus temperature (T). The apex of the curves represents the melting point for the fluorescent complexes. The FRET probes bind to both alleles to form a fluorescent complex; however they are complementary to the A allele but mismatched to the G allele by one base. Consequently the melting temperature of the fluorescent complex is higher for the A allele than the G allele. Heterozygotes have two peaks representing both alleles.

41. Multiplexing TaqMan: SYBR green: Extensive optimisation is required
Real-Time PCR Training
Multiplexing
TaqMan:
different dyes for each target (FAM, TET, VIC and JOE)
SYBR green:
different melting points for each target
Extensive optimisation is required
One-step PCR cannot be used
Real-time detection of four different retroviral DNAs in a multiplex format. Four assays were carried out in sealed tubes, each initiated with 100,000 molecules of a different retroviral DNA. Each reaction contained four sets of PCR primers specific for unique HIV-1, HIV-2, HTLV-I, and HTLV-II nucleotide sequences and four molecular beacons, each specific for one of the four amplicons and labelled with a differently coloured fluorophore. Fluorescence from the fluorescein-labelled molecular beacon (HIV-1-specific) is plotted in red, fluorescence from the tetrachlorofluorescein-labelled molecular beacon (HIV-2-specific) is plotted in green, fluorescence from the tetramethylrhodamine-labelled molecular beacon (HTLV-I-specific) is plotted in blue, and fluorescence from the rhodamine-labelled molecular beacon (HTLV-II-specific) is plotted in brown. The slight HTLV-I signal seen in the assay initiated with HTLV-II DNA is an artefact that resulted from a portion of the rhodamine fluorescence being interpreted by the spectrofluorometric thermal cycler as tetramethylrhodamine fluorescence.

42. Multiplexing: SYBR green
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Multiplexing: SYBR green

43. One-Step or Two-Step PCR
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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

44. Real-Time PCR Training
Understanding CT
CT (threshold cycle) is the intersection between an amplification curve and a threshold line.
It is a relative measure of the concentration of target in the PCR reaction.
The CT values from PCR reactions run under different conditions or with different reagents cannot be compared directly.
A: Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. In other words, Rn is the reporter signal normalized to the fluorescence signal of ROX™. In this view, Rn is graphed versus cycle. B: ΔRn is Rn minus the baseline, graphed here versus the cycle of PCR. C: Amplification plot shows the Log (ΔRn) graphed versus cycle.

45. The Effect of Master Mix Components on Ct
Real-Time PCR Training
The Effect of Master Mix Components on Ct
The fluorescent emission of any molecule is dependent on environmental factors such as the pH of a solution and salt concentration.
The fluorescence intensity is higher in Master Mix A even though the target, probe and ROX™ concentrations are the same in both cases.
The resulting ΔRn value will, therefore, vary accordingly.
Raw fluorescence data obtained with one assay and two master mixes with the same ROX™ level. The difference in signal is due to the master mix composition. Reaction was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System with a VIC® MGB probe. The X axis shows the emission wavelength of the fluorophore and the Y axis shows the intensity of the emission.

46. Real-Time PCR Training
Note that the baseline fluorescence signals, in a template-independent factor, are different for the two master mixes.
Variations in CT value do not reflect the overall performance of the reaction system.
Master mixes with equivalent sensitivity may have different absolute CT values.
Master Mix A and Master Mix B were used to amplify RNase P in equal amounts of human gDNA using the Applied Biosystems 7500 Real-Time PCR System. Figure 3A shows the Rn versus cycle number and the baselines for both reactions. Figure 3B shows the Log (∆Rn) versus cycle number. The threshold (green) is set at the same level for both master mixes. The CT value of Master Mix B (CTB) is earlier than that of Master Mix A (CTA) for identical concentrations of target, reflecting the lower baseline of Master Mix B.

47. Effect of ROX™ Passive Reference Dye on Ct
Real-Time PCR Training
Effect of ROX™ Passive Reference Dye on Ct
The Rn value is calculated as the ratio of FAM™ fluorescence divided by the ROX fluorescence.
Therefore, a lower amount of ROX would produce a higher Rn value assuming FAM fluorescence signal stays the same.
This would lead to an increase in baseline Rn and subsequently a smaller ∆Rn as well as a different CT value.

48. Real-Time PCR Training
Low ROX concentration can result in increased standard deviation of the CT value (in case of multiple wells).
The greater the standard deviation, the lower the confidence in distinguishing between small differences in target concentration.
Master mixes containing 3 different concentrations of ROX™ were used to amplify the TGF beta assay on the Applied Biosystems 7900HT Fast Real-Time PCR System using the 96-well block. Figure 4A shows the CT value and Figure 4B shows the standard deviation with variable ROX concentrations. Decreasing ROX concentration gives an earlier CT but increases the standard deviation.

49. Effect of Efficiency of a PCR Reaction on Ct
Real-Time PCR Training
Effect of Efficiency of a PCR Reaction on Ct
The slope of a standard curve 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 = ­3.32)
The PCR efficiency is dependent on:
the assay and the master mix performance
sample quality.
length of the amplicon
secondary structure
primer design

50. Real-Time PCR Training

51. Using the PCR Equation Xn = X0(1 + E)n Xn X0
Real-Time PCR Training
Using the PCR Equation
Xn = X0(1 + E)n
Xn = PCR product after cycle n
X0 = initial copy number
E = amplification efficiency
n = cycle number Xn
E IS MAXIMALLY 1.0, BUT USUALLY AROUND 0.9. X0
cycle number 51

52. Effect of Amplification Efficiency
Real-Time PCR Training
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 52

53. The efficiency of a PCR reaction can affect CT.
Real-Time PCR Training
The efficiency of a PCR reaction can affect CT.
A dilution series amplified under low efficiency conditions could yield a standard curve with a different slope than one amplified under high efficiency conditions.
Blue: efficiency =100% (slope is -3.3)
Green efficiency =78% (slope is -4)
The blue standard curve has an efficiency of 100% (slope is -3.3). The green standard curve has an efficiency of 78% (slope is -4). Amplification of the Y quantity gives an earlier CT with low efficiency condition (green) compared to the high efficiency condition (blue). With a lower quantity (X) there is an inversion and the low efficiency condition (green) gives a later CT compared to the high efficiency condition (blue).

54. Real-Time PCR Training
Two samples amplified under low and high efficiency conditions show different CT values for the same target concentration.
Although the high efficiency condition (the blue curve ) gives a later CT at high concentration, it gives better sensitivity at low target concentration

55. Real-Time PCR Applications
Real-Time PCR Training
Real-Time PCR Applications
Real-Time PCR can be applied to traditional PCR applications as well as new applications that would have been less effective with traditional PCR.
With the ability to collect data in the exponential growth phase, the power of PCR has been expanded into applications .

56. Real-Time PCR Applications
Real-Time PCR Training
Real-Time PCR Applications
Quantitation of gene expression
Array verification
Quality control and assay validation
Biosafety and genetic stability testing
Drug therapy efficacy / drug monitoring
Viral quantitation
Pathogen detection
DNA damage (microsatellite instability) measurement
In vivo imaging of cellular processes

57. Real-Time PCR Applications
Real-Time PCR Training
Real-Time PCR Applications
Mitochondrial DNA studies
Methylation detection
Detection of inactivation at X-chromosome
Determination of identity at highly polymorphic HLA loci
Monitoring post transplant solid organ graft outcome
Monitoring chimerism after transplantation
Monitoring minimal residual disease after HSCT
Genotyping (Allelic discrimination) :
Trisomies and single-gene copy numbers
Microdeletion genotypes
Haplotyping
Quantitative microsatellite analysis
Prenatal diagnosis from fetal cells in maternal blood
Intraoperative cancer diagnostics

58. Real-time PCR advantages
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Real-time PCR advantages
Not influenced by non-specific amplification
Amplification can be monitored in 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 point analysis
Most specific, sensitive and reproducible
Not much more expensive than conventional PCR (except equipment cost)