RealTime PCR Training Real time Pcr Biotechnology training

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

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. 2

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 3

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Real-Time PCR Training 5 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.

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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. 7

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. 8

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 9

Real-Time PCR Training Real-time Principles � Three general methods for the quantitative detection: 1. Hydrolysis probes (Taq. Man, Beacons, Scorpions). 2. Hybridisation probes (Light Cycler). 3. DNA binding agents (SYBR Green). 10

Real-Time PCR Training Taq. Man 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 11

Real-Time PCR Training 12 �A fluorogenic probe enables the detection of a specific PCR product as it accumulates during PCR. � The Taq. Man® 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

Real-Time PCR Training How Taq. Man Sequence Detection Chemistry Works 1. If the target sequence is present, the probe anneals downstream from one of the primer sites 2. 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. . 3. Additional reporter dye molecules are cleaved from their respective probes with each cycle. 13

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

Real-Time PCR Training Molecular Beacons 15

Real-Time PCR Training 16 Scorpions

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 17

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. 18

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 19

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. 20

Real-Time PCR Training �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. 21

Real-Time PCR Training When not to choose SYBR Green �Allelic discrimination assays. �Multiplex reactions. �Amplification of rare transcripts. �Low level pathogen detection. 22

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

Real-Time PCR Training 24 �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.

Real-Time PCR Training 25 �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 )]

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 26

Real-Time PCR Training 27 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

Real-Time PCR Training 28 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

Real-Time PCR Training 29 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

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Real-Time PCR Training 31 20 min break

Real-Time PCR Training 32 Quantitation By Real time PCR

Real-Time PCR Training 33 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

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Real-Time PCR Training 35 � 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

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). 36

Real-Time PCR Training 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. 37

Real-Time PCR Training 38 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.

Real-Time PCR Training 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 39

Real-Time PCR Training Melting Curve Analysis 40

Real-Time PCR Training 41 Multiplexing �Taq. Man: �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 PCR Training Multiplexing: SYBR green 42

Real-Time PCR Training 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 43

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. 44

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 p. H 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. 45

Real-Time PCR Training 46 �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.

Real-Time PCR Training 47 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.

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. 48

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 90 110% (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 49

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Real-Time PCR Training 51 Using the PCR Equation Xn = X 0(1 + E)n Xn �Xn = PCR product after cycle n �X 0 = initial copy number �E = amplification efficiency �n = cycle number X 0 cycle number

Real-Time PCR Training 52 Effect of Amplification Efficiency Xn = X 0(1+E)n Case 1: E = 0. 9 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 Result A difference of 0. 1 in amplification efficiencies created a five fold difference in the final ratio of PCR products after 30 cycles

Real-Time PCR Training 53 �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)

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 54

Real-Time PCR Training 55 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.

Real-Time PCR Training 56 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

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

Real-Time PCR Training 58 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)