Real-Time PCR

PCR was traditionally limited to end-point analysis using agarose gels Limitations of end-point PCR:
Poor precision Low sensitivity Short dynamic range Low resolution Size-based discrimination Ethidium bromide for staining does not allow for accurate quantitation Requires post-PCR processing 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 detect a two-fold change (i.e. 10 Vs. 20 copies).

 First of all we need to think about the nature of the PCR reaction in order to understand real time quantitation. The amount of DNA theoretically doubles with every cycle of PCR and this is what this shows, after each cycle the amount of DNA is twice what it was before. After one cycle of PCR, the amount of DNA is twice what it was before, so after two cycles one has 2x2 times as much, after 3 cycles - 2x2x2 times as much or 23 times as much, after 4 cycles 2x2x2x2 times as much or 24 times as much. Thus after n cycles there will be 2n times as much DNA.

CYCLE NUMBER 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

AMOUNT OF DNA 1 2 4 8 16 32 64 128 256 512 1,024 2,048 4,096 8,192 16,384 32,768 65,536 131,072 262,144 524,288 1,048,576 2,097,152 4,194,304 8,388,608 16,777,216 33,554,432 67,108,864 134,217,728 268,435,456 536,870,912 1,073,741,824 1,400,000,000 1,500,000,000 1,550,000,000 1,580,000,000

Linear
1600000000 1400000000 AMOUNT OF DNA 1200000000 1000000000 800000000 600000000 400000000 200000000 0 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER

Logaritmica
10000000000 1000000000 100000000 10000000 1000000 100000 10000 1000 100 10 1 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER

A MOU N T OF D N A

Linear

If we plot these figures in the standard fashion (top), we cannot detect the amplification in the earlier cycles because the changes do not show up on this scale. Eventually you see the last few cycles of the linear phase (pink) as they rise above baseline and then the non-linear or plateau phase (red) - in real life this starts somewhat earlier than shown here.

Logaritmica If we plot the amount of DNA on a logarithmic scale, we can see the small differences at earlier cycles - in real time PCR we use both types of graph to examine the data. Note that there is a straight line relationship between the amount of DNA and cycle number when you look on a logarithmic scale - because PCR amplification is a logarithmic reaction.

Logaritmica

Linear

Here is a real-time PCR trace for a single well on a 96-well plate, cycles are shown along the Xaxis, and arbitrary fluorescence units (actually these are fold increase over background fluorescence) are shown on the Y-axis - the results are similar to our theoretical graph (see insert)

AMOUNT OF DNA 0

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35

PCR CYCLE NUMBER

Here is the same real time PCR trace shown on a logarithmic scale - again it is similar to our theoretical curve (inset).
A MOU N T OF D N A 0

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PCR CYCLE NUMBER

 How Real-Time PCR Works

During the first cycles of a PCR reaction, the amount of amplicon doubles. The amount of amplicon after each cycle then multiplies exponentially in proportion to the starting amount of template in the sample. At some point, this doubling slows as the amount of substrate, nucleotides, and primers become used up. The DNA polymerase also becomes less active after the prolonged heating within the thermal cycler. The loss of doubling efficiency results in a plateau effect and the amount of amplicon produced with the later thermal cycles is no longer proportional to the amount of template DNA in the sample (Figure 1).

After enough cycles, all amplicons reach a certain maximum concentration, regardless of the initial concentration of template DNA.

 How Real-Time PCR Works

The key to determining the quantity of original template DNA present in a sample during amplification is to examine the initial thermal cycles before reaching the plateau region of amplification. To do this, the level of amplification is monitored continuously during the thermal cycling Initially, fluorescence remains at background levels, and increases in fluorescence are not detectable (cycles 1-18 in Figure 1) even though PCR product accumulates exponentially. At a cycle number, enough amplified product accumulates to yield a detectable fluorescent signal.

threshold cycle, or CT

The cycle number at which this occurs is called the threshold cycle, or CT.

Ct = cycle number in wich amplified product accumulates to yield a detectable fluorescent signal

Since the CT value is measured in the exponential phase when reagents are not limited, real-time qPCR can be used to reliably and accurately calculate the initial amount of template present in the reaction.

Real-Time PCR
Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

 The CT of a reaction is determined mainly by the amount of template present at the start of the amplification reaction. If a large amount of template is present at the start of the reaction, relatively few amplification cycles will be required to accumulate enough product to give a fluorescent signal above background. Thus, the reaction will have a low, or early, CT. In contrast, if a small amount of template is present at the start of the reaction, more amplification cycles will be required for the fluorescent signal to rise above background. Thus, the reaction will have a high, or late, CT. This relationship forms the basis for the quantitative aspect of real-time PCR.

Terminologia
Baseline
Fase onde a intensidade de sinal de produto amplificado ainda no ultrapassa a intensidade da fluorescncia encontrada no meio.

Terminologia
Threshold
Nvel de fluorescncia onde a reao detectada durante a fase exponencial; linha de comparao entre as amostras

Terminologia
Cycle Threshold (CT)
Ciclo (ponto no tempo) onde a reao cruza o limiar de deteco (Threshold)

Terminologia
Rn
Sinal do Reporter dividido pelo sinal de referncia passiva (sinal do ROX). Normalizao empregada para corrigir erros de pipetagem

Standard PCR Real-Time PCR

Plateau phase
End Point

Linear phase

Exponential phase

Area of detection for real-time PCR

QUANTITATION
Theoretically there is a quantitative relationship between the amount of starting sample and the amount of PCR product at any given sample. Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction rather than end-point plateau. The exponential phase is the optimal point for analyzing data.

Cycle threshold Ct is related to the initial target copy number

CT

Optimizing a Real-Time Quantitative PCR Assay (qPCR) A powerful way to

determine whether your qPCR assay is optimized is to run a set of serial dilutions of template DNA

A standard curve is constructed by plotting the log of the starting quantity of template (or the dilution factor, for unknown quantities) against the CT value obtained during amplification of each dilution.

Quantificao absoluta

Necessita de uma curva padro

necessrio usar padres A relao entre o log da quantidade inicial de DNA alvo e o Ct deve ser linear S devem ser usadas quantidades iniciais da amostrade DNA dentro da zona linear

Real-Time PCR
Advantages of real-time vs. end-point PCR: Collects data in the exponential growth phase (vs end-point plateau) Increase in fluorescent signal is proportional to number of amplicons generated Increased dynamic range of detection Does not require post-PCR processing Increased sensitivity (detection down to 2-fold change) Applications: Viral quantitation Quantitation of gene expression Microarray verification Drug therapy efficacy Pathogen detection Genotyping

Real-Time PCR
Detection Assays: SYBR Green Dye
SYBR Green I binds to doublestranded DNA. The resulting DNAdye-complex absorbs blue light (max = 498 nm) and emits green light (max = 522 nm)

As DNA is amplified SYBR fluorescence increases proportionally Non-specific dye used to detect the presence or absence of an amplicon

Non-target sequence-specific detections systems are susceptible to falsepositives

Real-Time PCR
Detection Assays: Sequence-specific probes
Dual fluorophore-labeled oligonucleotide probe: e.g. TaqMan

53 exonuclease activity of DNA polymerase cleaves reporter dye from quencher and allowing fluorescence.

Specific sequences are able to be detected the real-time TaqManin improves: reaction. Specificity

Product quantification Multiplex PCR

Advanced PCR
PCR has become a central tool for DNA analysis across all disciplines of biology and biochemistry Novel enzymes and instrumentation are creating new applications for PCR Other advanced PCR methods for research and diagnostic applications:

Hot start PCR (specificity) Cycling sequencing (DNA sequencing) Site-directed mutagenesis PCR Colony PCR Multiplex-PCR Error-prone PCR (mutagenesis) StEP PCR (recombination) Emulsion PCR (cell-free cloning)

Exerccios
1 - Em que condies possvel quantificar o a quantidade de DNA-alvo inicial a partir do produto amplificado? 2 - O que representa o Ct? 3 - De que forma se relaciona o Ct com a quantidade de DNA-alvo inicial (aumenta/diminui)? 4- Porque h necessidade de obteno de uma recta padro que relaciona Ct com o log da quntidade de DNA inicial?

Nos resultados para RT-PCR abaixo: 5 - Qual o Ct para quantidades inicias de RNA de 105 pg? 6 - e de 1pg?

7 -De acordo com as figura, porque se pode afirmar que as quantificaes feitas na zona do plateau no so rigorosas? 8 -O que se observa na fluorescncia na zona do plateau quando so realizadas 96 reaces para a mesma quantidade de DNA inicial? 9 -O que se observa na fluorescncia na zona do plateau quando so efectuadas diluies em srie da quantidade de DNA inicial?