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

Biotechnology training courseAugust, 2009

Dr. Basim Ayesh

Real-Time PCR Training 1

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.


Real-time PCR detectionWhile 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



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.



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.


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.


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


Real-time Principles

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


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


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


How TaqMan 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.


• The fluorescence intensity is increased proportional to the amount of amplicon produced.


Molecular BeaconsReal-Time PCR Training 15

ScorpionsReal-Time PCR Training 16

Hybridization Probes

Fluorescence Resonance Energy Transfer (FRET) ConceptDonor fluorophore is excited by appropriate wavelengthDonor energy is transferred non-radiatively to the acceptor

fluorophoreExcited acceptor emits at longer wavelength

Increase in fluorescence with each cycleThe signal depends only on hybridization


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.


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


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.


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.


When not to choose SYBR Green

Allelic discrimination assays.Multiplex reactions. Amplification of rare transcripts . Low level pathogen detection .


Terms Used in Quantitation Analysis

Amplification plot: The plot of fluorescence signal versus cycle number


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.


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-)]


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


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


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


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




By Real time PCR


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


Real-Time PCR Training

34

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


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).


Relative QuantitationStandard curve method

Using a separate standard curve for the target and endogenous control, in separate tubes.

Requires the least amount of optimization and validation.


Relative QuantitationComparative 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.


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


Melting Curve AnalysisReal-Time PCR Training 40

Multiplexing

TaqMan:different dyes for each

target (FAM, TET, VIC and JOE)

SYBR green:different melting points for

each targetExtensive optimisation is

requiredOne-step PCR cannot

be used


Multiplexing: SYBR greenReal-Time PCR Training 42

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


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.


The Effect of Master Mix Components on Ct

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.


The fluorescent emission of any molecule is dependent on environmental factors such as the pH of a solution and salt concentration.

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.


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.


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.


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) –1The efficiency of the PCR should be 90-110% (ideal slope =

3.32)The PCR efficiency is dependent on:

the assay and the master mix performancesample quality. length of the ampliconsecondary structureprimer design



Using the PCR Equation

Xn = X0(1 + E)n

Xn = PCR product after cycle n

X0 = initial copy number

E = amplification efficiencyn = cycle number


Xn

XX00

cycle number

Effect of Amplification Efficiency


Xn = X0(1+E)n

Case 1: E = 0.9 Case 2: E = 0.8Case 1: E = 0.9 Case 2: E = 0.8

XXnn = 100 (1+0.9) = 100 (1+0.9)3030 XXnn = 100 (1+0.8) = 100 (1+0.8)3030

XXnn = 2.3 x 10 = 2.3 x 101010 XXnn = 4.6 x 10 = 4.6 x 1099

ResultA difference of 0.1 in amplification

efficiencies created a five-fold difference in thefinal ratio of PCR products after 30 cycles

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)

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


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 .



Quantitation of gene expressionArray verificationQuality control and assay validationBiosafety and genetic stability testingDrug therapy efficacy / drug monitoringViral quantitation Pathogen detection DNA damage (microsatellite instability) measurement In vivo imaging of cellular processes



Mitochondrial DNA studies Methylation detection Detection of inactivation at X-chromosomeo Determination of identity at highly polymorphic HLA loci o Monitoring post transplant solid organ graft outcome o Monitoring chimerism after transplantationo Monitoring minimal residual disease after HSCT o Genotyping (Allelic discrimination) :

Trisomies and single-gene copy numbers Microdeletion genotypes HaplotypingQuantitative microsatellite analysis Prenatal diagnosis from fetal cells in maternal blood Intraoperative cancer diagnostics


Real-time PCR advantagesNot influenced by non-specific amplificationAmplification 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-foldRequirement 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 analysisMost specific, sensitive and reproducibleNot much more expensive than conventional PCR (except

equipment cost)


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