Real-time PCR can be used for both qualitative and quantitative analysis; choosing the best method for your application requires a broad knowledge of this technology. This section provides an overview of real-time PCR, reverse-transcription quantitative PCR techniques, and the choice of instruments that Bio-Rad offers for these techniques. In real-time PCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle.
First, amplification reactions are set up with PCR reagents and unique or custom primers. Reactions are then run in real-time PCR instruments and the collected data is analyzed by proprietary instrument software. Real-time detection of PCR products is enabled by the inclusion of a fluorescent reporter molecule in each reaction well that yields increased fluorescence with an increasing amount of product DNA.
The fluorescence chemistries employed for this purpose include DNA-binding dyes and fluorescently labeled sequence-specific primers or probes. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence signal as amplification occurs. The measured fluorescence is proportional to the total amount of amplicon; the change in fluorescence over time is used to calculate the amount of amplicon produced in each cycle.
The main advantage of real-time PCR over PCR is that real-time PCR allows you to determine the initial number of copies of template DNA the amplification target sequence with accuracy and high sensitivity over a wide dynamic range. Real-time PCR results can either be qualitative the presence or absence of a sequence or quantitative copy number. In contrast, PCR is at best semiquantitative. Additionally, real-time qPCR data can be evaluated without gel electrophoresis, resulting in reduced bench time and increased throughput.
Finally, because real-time qPCR reactions are run and data are evaluated in a unified, closed-tube qPCR system, opportunities for contamination are reduced and the need for postamplification manipulation is eliminated in qPCR analysis. In research laboratories, qPCR assays are widely used for the quantitative measurement of gene copy number gene dosage in transformed cell lines or the presence of mutant genes. In combination with reverse-transcription PCR RT-PCR , qPCR assays can be used to precisely quantitate changes in gene expression, for example, an increase or decrease in expression in response to different environmental conditions or drug treatment, by measuring changes in cellular mRNA levels.
In this plot, the number of PCR cycles is shown on the x-axis, and the fluorescence from the amplification reaction, which is proportional to the amount of amplified product in the tube, is shown on the y-axis. The amplification plot shows two phases, an exponential phase followed by a non-exponential plateau phase. During the exponential phase, the amount of PCR product approximately doubles in each cycle. As the reaction proceeds, however, reaction components are consumed, and ultimately one or more of the components becomes limiting.
At this point, the reaction slows and enters the plateau phase cycles 28—40 in Figure 1. Figure 1. Amplification plot. Baseline-subtracted fluorescence versus number of PCR cycles. Initially, fluorescence remains at background levels, and increases in fluorescence are not detectable cycles 1—18, Figure 1 even though product accumulates exponentially. Eventually, enough amplified product accumulates to yield a detectable fluorescence signal.
The cycle number at which this occurs is called the quantification cycle, or C q. Because the C q 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 based on the known exponential function describing the reaction progress.
This is necessary for gel electrophoresis and most forms of DNA sequencing. But, it also limits how informative PCR can be. These two elements turn qPCR from a processing step in another procedure — to a measurement technique in its own right. The fluorometer detects that fluorescence in real time as the thermal cycler runs, giving readings throughout the amplification process of the PCR. In combination with appropriate standard curves and reference values, this real-time information about reaction rates and times translates into information about relative and absolute amounts of DNA present.
Gene expression, or mRNA synthesis, is a critical part of protein synthesis. Gene expression is an area of active inquiry for molecular biologists — which aids in understanding numerous biological pathways and diseases. The resulting readings provide information about the amount of otherwise difficult-to-measure mRNA present in the original, un-reverse-transcribed sample. Below, we have provided an overview of the different PCR methods and the reagents we provide at Enzo Life Sciences for your research needs.
We aim to help scientists quickly access PCR reagents to use in their next research project! The denaturation, annealing, and elongation process over a series of temperatures and times is known as one cycle of amplification. Each step of the cycle should be optimized for the template and primer set used. This cycle is repeated approximately times and the amplified product can then be analyzed.
As PCR is a highly sensitive method and very small volumes are required for single reactions, preparation of a master mix for several reactions is recommended. The master mix must be well mixed and then split by the number of reactions, ensuring that each reaction will contain the same amount of enzyme, dNTPs and primers. GC-rich sequences are more stable than sequences with lower GC content.
Furthermore, GC-rich sequences tend to form secondary structures, such as hairpin loops. As a result, GC-rich double strands are difficult to completely separate during the denaturation phase. Consequently, DNA polymerase cannot synthesize the new strand without hindrance.
A higher denaturation temperature can improve this and adjustments towards a higher annealing temperature and shorter annealing time can prevent unspecific binding of GC-rich primers. Additional reagents can improve the amplification of GC-rich sequences. DMSO, glycerol and betaine help to disrupt the secondary structures that are caused by GC interactions and thereby facilitate separation of the double strands. Therefore, the chosen extension temperature should be in this range. The enzyme can, however, also be active to a lesser degree, at lower temperatures.
At temperatures that are far below the annealing temperature, primers tend to bind non-specifically, which can lead to non-specific amplification, even if the reaction is set up on ice.
This can be prevented by using polymerase inhibitors that dissociate from the DNA polymerase only once a certain temperature is reached. The inhibitor can be an antibody that binds the polymerase and denatures at the initial denaturation temperature.
An additional step allows the detection and amplification of RNA. The efficiency of the first-strand reaction can affect the amplification process. RNA is single-stranded and very unstable, which makes it difficult to work with. This technique has many benefits due to a range of methods and chemistries available.
During each cycle, the fluorescence is measured. The disadvantages to dye-based qPCR are that only one target can be examined at a time and that the dye will bind to any ds-DNA present in the sample. In probe-based qPCR, many targets can be detected simultaneously in each sample but this requires optimization and design of a target specific probe s , used in addition to primers. There are several types of probe designs available, but the most common type is a hydrolysis probe, which incorporates the use of a fluorophore and quencher.
Fluorescence resonance energy transfer FRET prevents the emission of the fluorophore via the quencher while the probe is intact.
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