Real time PCR

Real-Time PCR Technology Basics

The method of choice for nucleic acid (DNA, RNA) quantification in all areas of molecular biology is real-time PCR or quantitative PCR (qPCR).

The method is so-called because the amplification of DNA with a polymerase chain reaction (PCR) is monitored in real time (qPCR cyclers constantly scan qPCR plates). It is, in contrast to the conventional PCR, quantitative, meaning that it enables us to determine the exact concentration (relative or absolute) of the amplified DNA in the sample. Conversely, in conventional PCR we can see the result of amplification only after the PCR is completed (end-point detection).

Apart from DNA, RNA can also be used as a template (e.g. in case of gene expression studies or detection of RNA viruses). In this case the RNA needs to be reverse transcribed into DNA (also termed complementary DNA or cDNA) before it is amplified with real-time PCR. There is a term for this combined method: real-time reverse transcription PCR or qRT-PCR (sometimes RT-qPCR) for short.

How it works

PCR is a method where an enzyme (thermostable DNA polymerase, originally isolated in 1960s from bacterium Thermus aquaticus, growing in hot lakes of Yellowstone park, USA) amplifies a short specific part of the template DNA (amplicon) in cycles. In every cycle the number of short specific sections of DNA is doubled, leading to an exponential amplification of targets. More on how conventional PCR works can be found here.

In qPCR, exactly the same procedure happens but with two major differences: first the amplified DNA is fluorescently labelled (usually with cyanine based fluorescent dyes) and second, the amount of the fluorescence released during amplification is directly proportional to the amount of amplified DNA. Fluorescence is monitored during the whole PCR process (along all 30 to 45 cycles). The higher the initial number of DNA molecules in the sample, the faster the fluorescence will increase during the PCR cycles (see Images 1 and 2). In other words, if a sample contains more targets the fluorescence will be detected in earlier cycles. The cycle in which fluorescence can be detected is termed quantitation cycle (Cq for short) and is the basic result of qPCR: lower Cq values mean higher initial copy numbers of the target. This is the basic principle of quantitative approach that real-time PCR offers.

There are several approaches by which Cq values are obtained (Cq calling), which parameters of amplification curves we should regularly check, how to translate Cq values into absolute of relative copy numbers or gene expression, etc. Once you master these skills, qPCR becomes a really powerful technique.

There are several ways in which the amplified DNA is fluorescently labelled (also known as ‘qPCR chemistries’) but we are not going to discuss them in greater details here. They all have one thing in common: to enable production of the fluorescent signal during the PCR reaction that is directly proportional to the starting amount of the DNA.

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Image 1 shows a graphical representation of qPCR amplification (the first two cycles) as it is going on in the PCR tube. There are different variants of qPCR (also called chemistries) which have slightly different ways of fluorescence labelling. Image shows two most commonly used. On the left side a 5′-exonuclease variant is shown that uses FRET mechanism (fluorescence resonance energy transfer) where a fluorescence of a reporter fluorofore (R) is transferred to a quencher (Q) and not is not emitted whenever reporter and quencher are in proximity (e.q. linked to the same short oligonucleotide – a probe). When the two are dislocated (when the probe is dissolved away by 5′-exonuclease activity of TaqDNA polymerase during PCR elongation), reporter molecule freely emits the fluorescence which can that be detected. On the right side of the image a qPCR variant that uses an intercalating fluorofore is represented. Special intercalating dyes are used that strongly increase emission of fluorescence whenever they are intercalated into a dsDNA.

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Image 2 shows amplification plot showing of five samples (S1 to S5). As the target DNA in each sample is being amplified through cycles the fluoresce increases. Sample S1 contained the highest initial number of target DNA molecules, resulting in the fastest increase of fluorescence. Sample S4 contained the lowest initial number of target DNA molecules while S5 did not contain any target DNA.

Good and bad sides of qPCR

Advantages to conventional PCR:

Speed: the amplified DNA is being detected during the PCR reaction so there is no need for a separate detection after as is in the case of conventional PCR (e.g. agarose gel electrophoresis with intercalating fluorescent dyes)

Throughput: qPCR is considered a high throughput method (processing of large numbers of samples in short time), because it is compatible with liquid handling automation stations for sample preparation (DNA/RNA isolation and loading onto qPCR plates).

Sensitivity: qPCR is able to distinguish two fold differences in quantity of target DNA molecule. And it can detect down to a few copies of DNA (sometimes even one).

Lower amounts of starting material: as low as 1/1000 of the amount required for conventional PCR

Broad dynamic range of quantification: quantification can be performed over several orders of magnitude (up to 107-fold dynamic range)

Repeatability: high.

Disadvantages of qPCR:

Cost of equipment: due to the optical components for sensitive fluorescence detection the qPCR machines are 5 to 10-fold more expensive than conventional PCR thermal cyclers

Cost of chemicals and consumables: qPCR is a very sensitive method therefore the precise composition and high quality of the reaction mixtures is extremely important. This is the reason why ready-to-use reaction mixtures are usually purchased (master mix). Because of the sensitive detection method (fluorescence) a specific set of plastic-ware is required.

Inhibition of PCR reaction: due to the complex nature of biological samples, imperfect purification processes during isolation of nucleic acids may leave traces of various substances in isolated samples. PCR reaction is sometimes inhibited by these substances, therefore they are called inhibitors of PCR reaction (DNA polymerase is an enzyme and as such is susceptible to certain compounds that inhibit its activity – polymerisation of DNA). This can complicate the quantification.: due to the complex nature of biological samples, imperfect purification processes during isolation of nucleic acids, etc. PCR reaction is sometimes inhibited by so called inhibitors of PCR reaction (DNA polymerase is an enzyme and as such is susceptible to certain compounds that inhibit its activity – polymerisation of DNA). This can complicate the quantification.

Sensitivity to errors: qPCR is extremely sensitive methods and as such sensitive to errors. This means that even the slightest mistakes can have significant influence on the final results. The most variable and critical point is the preparation of the samples (DNA extraction and reverse transcription). That is why several control reactions need to be included along samples when performing qPCR to assure quality control checks of every qPCR run.

Data analysis: data analysis and interpretation of the results is more complicated than in conventional PCR but the results are more informative (quantitative).

Where is qPCR being used?

Due to several powerful advantages qPCR has a wide range of applications. The method has also been around long enough so that the research community proved its reliability and robustness, that manufacturers of qPCR machines developed reliable qPCR platforms and that manufacturers of liquid handling automation developed qPCR-compatible automated solutions.

The most evident is the use of qPCR in molecular diagnostics, where it is slowly displacing conventional methods. It is used to detect, identify and quantify microorganisms that cause diseases (bacteria, viruses and fungi; see Image 3). With qPCR manual labour is reduced and along that carry over contamination and erroneous results. It is also considered a high-throughput method, meaning that large amounts of samples can be processed in less time (384 or 1536 reactions per plate). qPCR has thus proven to be an excellent method in diagnostic laboratories. It has to be noted, though, that the method detects only the presence of DNA or RNA of a microorganism and does not report its viability. Therefore conventional microbiology techniques are sometimes still required alongside qPCR.

Real Time Pcr 3

Image 3 shows a Ruperstis stem pitting associated virus (RSPaV) as visualised by transmission electron microscope, one of conventional and labourious detection techniques that is being displaced by RT-qPCR (photo: NIB).

qPCR is also used to detect and quantify genetically modified organisms or to perform genotyping. The latter means that different alleles of the same gene or single nucleotide polymorphisms (SNPs) can be detected which can be used as genetic diagnostic or prognostic markers for certain diseases.

A very important field of use are gene expression studies that help us understand the biological processes in various fields of biology, microbiology, medicine and other life sciences. A very useful, almost blockbuster combination is a genome-wide gene expression screening with DNA-microarrays followed by validation of the results with qPCR. DNA microarrays are a very powerful method on its own but they are less sensitive and still require validation. qPCR is therefore a very important research technique.

Conventional PCR basics

Thermostable DNA polymerase is one key

All this amplification that sounds very sophisticated is actually done automatically by thermostable DNA polymerase, the role of which is to replicate DNA. In order to prevent the random uncontrolled replication of all or unwanted parts of DNA in the sample, a set of primers are introduced into the reaction mixture: a forward primer that marks the beginning and a reverse primer that marks the end of a section of DNA that is to be amplified. By careful design of both primers (known sequence of nucleotides) we instruct the DNA polymerase exactly which part of the DNA it should amplify. And it does so. These short sections of DNA are usually a few hundred base pairs long in conventional PCR and only a few ten base pairs in qPCR.

Temperature cycling is the other key

In the beginning of PCR DNA is heated up to 95°C so that the DNA is denatured (single strands of DNA are obtained). In other words the DNA becomes exposed to DNA polymerase. But the DNA polymerase requires a double stranded DNA to start the polymerisation on the template DNA strand. Here is where a set of primers steps in. The temperature is now lowered and the primers anneal to the complementary part of the DNA (according to A-T and G-C base pairing). Because there is an enormous excess of primers, they anneal to every specific target sequence on the template DNA even if many copies of the target are present. Now the DNA polymerase can start filling in the complementary DNA strand along the template DNA. The DNA polymerase replicates the short section of a DNA (also termed amplicon) until it runs out of DNA template. It fills all the ‘gaps’. This was one cycle of PCR and there are twice as many specific parts of the DNA at the end of it as were in the beginning.

Now the temperature is raised again so that the DNA is denatured and, again, becomes exposed to DNA polymerase. This time not only the DNA that was originally present in the sample but also the complementary strands which were synthesised in the first cycle represent the template for the DNA polymerase. During the second cycle the number of specific sections of DNA is thus doubled (see Image 4). This happens over and over again for 30 – 45 cycles. As you can see the only thing that is being changed during the cycles is temperature, everything else is done by itself (by DNA polymerase to be more specific). By increasing and decreasing the temperature we control the amplification of the DNA (see Image 5). Very simple, right? This cycling of the temperature is the reason why the PCR machines are also called thermal cyclers or PCR cyclers.

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Image 4 shows one polymerase chain reaction (PCR) cycle. Cycling of temperature is the basis of PCR.

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Image 5 shows a more detailed graphical representation of PCR amplification (the first two cycles) as it is going on in the reaction tube.

Visualisation of results

In conventional PCR we can see the result of amplification only after the PCR is completed.Amplified products need to be visualised with another method such as agarose gel electrophoresis with intercalating fluorescent dyes (e.g. ethidium bromide; see Image 6).

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Image 6 shows a picture (negative) of an agarose gel after electrophoresis stained with ethidium bromide under UV light. Columns ‘M’ contain molecular weight markers, each band representing a DNA fragment of a known length (shortest being on the bottom of the gel and longest being on the top of the gel). Samples ‘S1’ to ‘S4’ contain PCR products that were amplified during PCR. As you can see more than one PCR product was amplified in each sample (observe distinct bands of different lengths).

 





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