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´s name derives from the fact that the amplification of DNA by polymerase chain reaction (PCR) is monitored in real time. It is a quantitative method in contrast to conventional PCR, meaning that it enables the determination of exact amounts (relative or absolute) of amplified DNA in samples. Conversely, amplified DNA can only be detected after the amplification had been carried out (end-point detection) in conventional PCR.
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.
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 provides.
There are several approaches by which Cq values are obtained (Cq calling), parameters of amplification curves we should regularly check, ways of translating Cq values into absolute or relative copy numbers of gene expression, etc. Once you master these skills, qPCR becomes a really powerful technique for your research.
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 detail here. They all have one thing in common: they produce a fluorescent signal during the PCR reaction, which is quantifiably and directly proportional to the starting amount of DNA.
Figure 1 depicts a graphical representation of qPCR amplification (the first two cycles) as it happens in the PCR tube. There are different variants of qPCR signalling molecules (also called chemistries) which have slightly different ways of fluorescence labelling. Two most common principles are shown. On the left side a 5′-exonuclease variant is shown that uses FRET mechanism (fluorescence resonance energy transfer) where a fluorescence of a reporter fluorophore (R) is transferred to a quencher (Q) and is not emitted whenever the reporter and quencher molecules are in proximity (e.g. TaqMan). When the two are dislocated (when the probe is removed by 5′-exonuclease activity of TaqDNA polymerase during PCR elongation), reporter molecule freely emits the fluorescence which can then be detected. On the right side, a qPCR variant that uses an intercalating fluorophore is represented (e.g. SYBR Green). Special intercalating dyes are used that strongly increase the emission of fluorescence whenever they are intercalated in dsDNA.
Figure 2 shows amplification plot with five samples (S1 to S5). While DNA in each sample is being amplified with every cycle the fluorescence increases. In the example above, sample S1 contained the highest initial number of target DNA, resulting in the fastest increase of fluorescence. Sample S4 contained the lowest initial number of target DNA molecules while S5 did not contain any.
Good and bad sides of qPCR
Edge over conventional PCR:
- Speed: amplified DNA is being detected at the same time as the PCR reaction is taking place, so there is no need for a separate detection after, as is the case in conventional PCR (e.g. on an agarose gel)
- Throughput: qPCR is considered a high-throughput method (processing of large numbers of samples in short time), due to its compatibility 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 molecules, and it can detect down to just a few molecules of initial DNA. When compare to PCR, as little as 1/1000 the amount can be used.
- Range of quantification: broad quantification can be performed over several orders of magnitude (up to 107-fold dynamic range).
- Reproducibility: generally regarded as highly reproducible.
Disadvantages of qPCR:
- Cost of equipment: due to the optical components required for sensitive fluorescence detection the qPCR cyclers are five to ten times more expensive than conventional PCR thermal cyclers
- Cost of chemicals and consumables: qPCR is a very sensitive method therefore, precise composition and high quality of the reaction mixtures is extremely important. This is 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.
- Loading times: loading qPCR samples into plates is usually a much more precise and tedious process when compared to conventional PCR, mainly due to the higher number of reagents and samples being used and the method´s extreme sensitivity. However, loading times can be greatly reduced if a pipetting aid like PlatR is used.
- 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 reactions are sometimes inhibited by these substances, also called inhibitors of PCR reaction (DNA polymerase is susceptible to certain compounds that inhibit its activity). This can complicate the quantification process.
- Sensitivity to errors: qPCR is an extremely sensitive method and as such very prone to errors. This means that even the slightest mistakes can have a 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 (e.g. no template control, buffer control) need to be included when performing the assay to assure quality control checks in every run.
- Data analysis: data analysis and interpretation of results is more complicated than in conventional PCR, granted the results are more informative.
Where is qPCR being used?
Due to its powerful advantages, qPCR has a wide range of applications. The method had been around long enough so that the research community proved its reliability and robustness. Similarly manufacturers of qPCR cyclers developed reliable platforms and that providers of liquid handling automation devices developed qPCR-compatible automation solutions (e.g. robots).
The most evident is the use of qPCR in molecular diagnostics, where it is slowly replacing conventional methods. It is used to detect, identify and quantify microorganisms that cause diseases (bacteria, viruses and fungi). With qPCR manual labour is reduced and along that concern over contamination and erroneous results. It also allows for large amounts of samples to be processed in less time (up to 384 or even 1536 reactions per run) and has thus proven to be an irreplacable 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. Consequently, conventional microbiological techniques are sometimes still required alongside it.
Figure 3 shows a Rupestris stem pitting associated virus (RSPaV) as visualised by transmission electron microscope, one of conventional detection techniques that is being replaced 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 application is represented by 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 results with qPCR. DNA microarrays are a very powerful method on its own, but they are less sensitive and still require validation.
Conventional PCR basics
Thermostable DNA polymerase on one side
All this amplification cycles sound very sophisticated but are actually carried out automatically by a combination of temperature changes and thermostable DNA polymerase which replicates 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 the amplicon. By carefully designing of both primers (known sequence of nucleotides) we can instruct the DNA polymerase exactly which part of the DNA it should amplify. 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 on the other
In the beginning cycles the DNA is heated up to 95°C so it is denatured and single strands of DNA (ssDNA) are obtained from dsDNA. In other words, the DNA becomes exposed to the DNA polymerase. But the DNA polymerase requires a double stranded DNA where it can start adding nucleotides to the template DNA strand. Here is where a set of primers come 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 most target sequences on the template DNA even if many copies of it 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 until it runs out of the template, filling all the ‘gaps’. This is referred to as one cycle of PCR which results in double the amount of target DNA, compared to the beginning of the cycle.
The process is then repeated up to 30 – 45 times again (30 – 45 cycles), each time increasing the target DNA amount by roughly 2-fold. The only thing that is modified during the cycles is temperature, everything else is carried out by itself. By increasing and decreasing the temperature we control the amplification of the DNA (see Image 5). Sounds a lot more simple now, right? Cycling of the temperature is the reason why the PCR machines are also referred to as thermal cyclers or PCR cyclers.
Figure 5 shows a more detailed graphical representation of the PCR amplification (the first two cycles) reaction as it unfolds in the reaction tube.
Visualisation of results
In conventional PCR, we can see the results of amplification only after the complete PCR process is completed. Amplified products need to be visualised with an additional method such as an agarose gel electrophoresis or polyacrylamide gel electrophoresis (PAGE) with intercalating fluorescent dyes (e.g. ethidium bromide; see Figure 6).
Figure 6 shows a picture (negative) of an agarose gel after electrophoresis, stained with ethidium bromide and visualized 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. M ore than one PCR product was amplified in each sample (observe distinct bands of different lengths).