“A technique used to quantify the nucleic acid (DNA/RNA) present in a sample, during the PCR reaction is known as a real-time PCR or quantitative (q)PCR”
In other words, we can say,
“A molecular biology technique used to monitor the amplification of the target DNA/RNA sequence is referred to as real-time PCR or quantitative PCR.”
The PCR is the cyclic reaction based on the rapid change in temperature during each step. During PCR, our gene of interest is amplified as well as quantified.
Read these two articles to know more:
The real-time PCR assay or the quantitative PCR is something different than the conventional PCR. We will cover all the information regarding the real-time PCR in the present article. The present article is huge, gigantic and with a lot of sub-topic on real-time PCR. So take a cup of coffee, notebook, pen and read the article till the end.
The article is filled with graphics and easy to understand explanation on rtPCR. Let’s start the topic,
|PCR||Polymerase chain reaction|
|rt PCR||Real-time PCR|
|RT- PCR||Reverse transcriptase PCR|
|FRET||Fluorescent resonance energy transport|
|Taq Man||Taq DNA polymerase (Pac) Man|
Overview of Real-time PCR:
Amplification is the prime goal of any PCR reaction. Using dNTPs, primers and PCR reaction buffer, the Taq DNA polymerase amplifies our DNA in vitro. Read more on in vivo DNA synthesis: General process of DNA replication
In the traditional PCR method after the amplification, the PCR products or the amplicon are run on the agarose gel or PAGE to detect the presence or absence of DNA amplification. But in the real-time PCR, the amplification during each PCR cycle is monitored in a real-time manner, instead.
A camera or detector detects each amplicon produced during the amplification of template DNA.
Usually, the chemistry behind real-time monitoring relies on the use of fluorescent dye. As the amplification progressed, the detector detects the amount of fluorescence emitted. This is the basic and global principle behind all the types of real-time PCR.
However, some platform uses labeled probes instead of dNTPs.
Why real-time PCR is more advanced over the traditional conventional PCR?
The reason is simply clear, we can calculate or measure the amount of the amplification which is not possible in the conventional PCR. And this is the reason the technique is also named “quantitative PCR.”
In the conventional PCR, we have to do agarose gel electrophoresis that is also not required in case of the real-time PCR. By doing the melting curve analysis one can get results.
Furthermore, by running parallel reactions we can monitor and choose which reaction is performing well. Only, that reaction can be monitored. Therefore the chance of errors is minimum in quantitative real-time PCR.
Now, immediately a question strikes in mind,
What about the analysis of gene expression? Quantitative analysis of gene expression is not possible in conventional PCR.
By monitoring the reaction in real-time (how many templates amplified during each reaction) the amount of gene expression can be measured.
The principle of real-time PCR:
The principle of real-time PCR relies on the use of fluorescent dye. In general, the principle of the present method is stated below,
“The amount of the nucleic acid present into the sample is quantified using the fluorescent dye or using the fluorescent labeled oligos.”
Two types of chemistry are available for the real-time quantitative PCR:
- DNA binding dye (Intercalating dye-based method)
- Sequence-specific probe (Hydrolysis Probe-based detection method)
How can we decide which method is used for which types of a real-time experiment?
Or can we use both methods for all the samples?
Well, that depends on,
- What types of experiments we want to perform.
- The overall cost or the budget of our sample.
- The knowledge and expertise of the researcher and
- The tissue type of the sample.
DNA binding dye:
For a novice and inexperienced person, the DNA binding dye method is the best technique for real-time detection.
The dye has its own fluorescence. Once the dye bind to the double-stranded DNA the fluorescence emitted by the dye increases 100 to 1000 fold than the original signal.
However, the original dye fluorescence is taken as the baseline for the detection.
The method is rapid, quick, reliable and cost-effective. Also, the chance of error in the experiments are less and the reaction set up is simple and easy to use.
The result of the experiment depends on the specificity of the primers used in the PCR reaction.
Because even though, the primers are bind non-specifically, the DNA binding dye binds to the non-specific sequence and gives the fluorescent signals.
Because the dye detects the double-stranded DNA to binds, even if the dsDNA is non-specific, the dye must bind to it.
Therefore the chance of the non-specific detection is high in the SYBR green dye-based method.
The SYBR green is one of the most popular dyes used in real-time PCR.
The sensitivity of the experiment is limited. Again a question arise in mind,
Is it suitable for the determination of sensitive templates?
The answer is yes,
We can identify the non-specific bindings into the reaction by doing the melting curve analysis.
Melting curve analysis:
Once the amplification reaction is completed and the fluorescence signals are recorded, the template is melted for the determination of the non-specific bindings.
The template is melted using heating, the dye dissociates and the fluorescence signals are reduced.
The decreased transition of a wide range fluorescence is reported for the specific product while different heat transition recorded for different short non-specific bands.
“Larger sequences takes more time and higher temperature for melting while non-specific bands melt at a lower temperature and has different melting temperature curves.”
The data are plotted in fluorescence vs melting temperature graph below.
The fluorescence vs melting temperature graph is also called a dissociation curve and the method is called a dissociation curve analysis.
Image: The image shows the dissociation curve for specific product and primer dimers while another image shows different dissociation curves for two homozygous and a heterozygous.
SYBR green and EvaGreen are two main dye used in the quantitative real-time PCR. The experiments are used in the validation of the assays such as DNA microarray.
The TaqMan probe chemistry is widely used in the quantitative PCR, the TaqMan name is taken from the “Taq” Taq DNA polymerase (because the probe chemistry depends on the activity of Taq DNA polymerase) and “Man” from the PacMan game. Yes, the name is taken from the game. Remember what PacMan do?
Probe-based detection method:
The method used the single short sequence-specific probes which are of two types:
- Linear probe
- Molecular beacons
Linear probes are the TaqMan probe, relies on the activity of Taq DNA polymerase.
The probes are the labelled short single-stranded sequence-specific DNA molecules which are radio or fluorescent labelled.
Here the probe is labelled with the fluorescent dye called a reporter molecule, situated at the 3’ end. The other 5’ end has the quencher dye which is in the close proximity to the reporter dye and quenches the fluorescence of the reporter dye.
Now, in the probe base method, not only the probe but the Taq DNA polymerase plays an important role.
The Taq DNA polymerase used in the real-time PCR has the 5’ to 3’ exonuclease activity, which removes the probe by extending the DNA.
Once the probe is dissociated the reported molecules emitted fluorescent.
Because if the DNA (the sequence of our interest) is amplified, the reported molecule unquenched and releases the fluorescence.
See the figure below,
The main advantage of the probe-based method is that we can use multiple probes for multiple template DNA sequences.
which means we can amplify multiple templates in a single reaction efficiently.
TAMRA and Black Hole Quencher are two widely used quencher dyes. While FAM is the most popular reporter dye.
Here, one problem occurred which limits the TaqMan probe-based technique,
The same annealing temperature is not possible for both- primer as well as a probe.
So can it be a problem in the reaction? The answer is,
Yes, it is a problem, a big problem. By using lower extension temperature the problem can be resolved.
Instead of three steps, by using the two-step temperature profile the problem can be encountered.
Here, the annealing and the extension step is combined at 60°C. After the denaturation, the probe hybridization, primer hybridization and extension are done at a single temperature.
At 72°C extension, the Taq will be at its highest activity therefore, instead of removing the probe it facilitates strand displacement of a probe.
That is why the annealing and the extension in the linear probe-based real-time PCR are done at a single temperature.
We will prepare a whole article on the TaqMan probe and oligo probes and discuss all the advantages and disadvantage there.
“After each PCR cycle, more probes are hydrolysed, more fluorescence is generated and more amplicons are quantified.”
We had written two amazing articles on Taq DNA polymerase, read it here:
The molecular beacons operated on the mechanism of the thermodynamics in which a molecule remains in such a condition where the majority of its energy can be saved.
Here instead of binding non-specifically, the molecular beacon remains in a hairpin structure.
A linear probe can cause non-specific bindings which can be prevented by using a beacon.
Beacons are the hairpin loop-like structure of the oligonucleotides which has complementary sequences on both the ends.
The central loop is complementary to the target sequences. One end of the hairpin loop has the quencher dye and one end has the reporter fluorescent dye.
Here, interestingly, when the two ends of the hairpin stem are in close proximity with each other, the reporter molecule is quenched and cannot generate fluorescence.
But when it binds to the complementary sequence, the two ends of the hairpin separated with each other, the quencher is blocked, the reported dye released and emits the fluorescence.
The emission is recorded by the detector. See the figure below,
The molecular beacon probes are highly sequenced specific and are the best choice for sensitive reactions.
If the probe (molecular beacon) cannot find its complementary sequence, it remains in the hairpin loop form and prevents non-specific bindings.
Here, the sensitivity of the PCR is not compromised, as the probe chemistry is not relying on the Taq DNA polymerase activity.
The polymerization reaction is performed in three different steps and the Taq works at its full activity at 72°C.
In the molecular beacon chemistry, the structure of the beacon stem is very important. Designing the loop for the beacon is a crucial step for getting specific amplification.
Suppose, if the structure of the hairpin loop is too stable, it can not be separated and unquenching cannot happen.
The hybridization cannot occur and the reaction fails. Melting curve analysis is necessary to assist the function of the molecular beacons.
We had covered a dedicated article on the molecular beacon, you can read it here: Molecular Beacon: A hairpin that enhances real-time PCR specificity.
Scorpion probes are other types of a probe or we say it is a type of molecular beacons in which instead of two different probes and primer, the hairpin loop is incorporated directly at the 5′ end of the primer. The 3′ end contains the complementary sequence to our target DNA.
The scorpion probe is even more specific than the molecular beacons.
Steps and procedure of real-time PCR:
The quantification is achieved by amplifying and monitoring the DNA or RNA present into the sample. For the quantification of the gene expression, the RNA is quantified into the real-time PCR.
If DNA is present into the sample in higher quantity, amplification and quantification start at the early stage of the reaction otherwise, the amplification starts in late stage.
As like the conventional PCR, there are three main steps in real-time PCR;
Denaturation occurs at 94°C where the double-stranded DNA is denatured and two single-stranded DNA is generated. The DNA is melted.
This single-stranded DNA is the sight of the annealing for the primers in the later step of the amplification.
Annealing occurs at 55°C to 66°C in which the sequence-specific primer bind to the single-stranded DNA. Along with it, the fluorescent dye or the probe binds to the DNA sequence too.
Extension occurs at 72°C at which the Taq DNA polymerase activated highest. In this step, the Taq adds dNTPs to the growing DNA strand.
Note: if the amplicons are less, combine the extension step with the annealing step (for real-time PCR only).
Components used into the real-time PCR:
As like the conventional PCR, the real-time PCR reaction contains almost the same components except for the fluorescent dye or fluorescent labelled probe.
Let’s start with the dNTPs
dNTPs are added during the synthesis of the growing DNA strand by the Taq DNA polymerase. The dNTPs remain the same as like the conventional PCR.
For more detail read the article: The Function of dNTPs in PCR reaction
Taq DNA polymerase:
Normal Taq cannot work efficiently for the real-time PCR instead always use the hot-start Taq DNA polymerase. The hot start Taq DNA polymerase is the best choice for the quantification.
Read our article on Hotstart PCR: What is a hot-start PCR?
Magnesium ion also plays a crucial role in the amplification during the real-time PCR. However, the concentration of the Mg2+ ions is different than the conventional PCR. Use 3 to 5mM of MgCl2 in the real-time PCR.
Read the article on MgCl2: Role of MgCl2 in PCR reaction
100pg to 1microgram template DNA is sufficient for the real-time PCR. We required only 100 copies of genomic DNA/RNA fragment for the amplification and to start the reaction. The template DNA or RNA must be pure and free from any contaminants.
Read more on template DNA: What are the properties of PCR (template) DNA?
Do not worry about the primers of real-time PCR. Use ready to use primer kits. If you want to design the primers by yourself keep several points in mind,
The primer should be shorter, it can amplify only 100 to 160bp fragments, avoid longer amplicons.
It must be unique and contains 50% GC contains with the GC sequences at the end of the 3’ end.
It should be 18 to 20 nucleotide long.
The primer contains all these criteria are the best for a real-time PCR assay.
More detail read our primer design guidelines: PCR primer design guidelines.
Note: In each section, we had given the link of related articles for more detail. You can read it for understanding the topic better.
The procedure of the real-time PCR starts with the extraction. The DNA or the RNA is extracted and quantified using the ready to use kits.
Now, prepare the reaction as per the manufacturer’s protocol, care must be taken while preparing the reaction.
Set the cyclic condition of the PCR and put the samples inside the machine. After the amplification, standard curve analysis or relative quantification is performed, instead of agarose gel electrophoresis.
For quantification of different types of samples, Endpoint PCR or real-time PCR can be performed.
Endpoint PCR vs Real-time PCR:
The main fundamental behind the real-time PCR is around the intensity of the fluorescence emitted whether it is emitted during the PCR or at the end of the PCR.
In the endpoint PCR, quantification is performed after the completion of the PCR reaction.
Based on the total fluorescence emitted, the amount of template is determined into the sample. The method is also called as the semi-quantitative PCR.
However, this method has one major problem.
At the later stage of the amplification the reagents available for the amplification are less (because it is consumed during the early reaction) also the amplification inhibitors are active more. Hence accurate measurement is not possible in this method.
Therefore this method is inconsistent and inaccurate.
Even if we amplified the identical sample multiple times, the result does not remain the same in all reaction.
The end-point semi-quantitative method is best for just confirming the amplicons, it is not suitable for the gene expression and viral titer measurements.
However, the method is rapid and faster than conventional PCR.
The real-time quantitative PCR is more sensitive and accurate than the endpoint PCR.
Because here, the amplification is measured in the real-time, during the reaction. After each reaction, the fluorescence is emitted and it is reported by the detector.
The signals are recorded during the exponential phase of the reaction.
The figure below is the graph of a real-time PCR reaction,
Here, the amplification is not recorded during the late phase of the reaction. The reason is the same as the endpoint PCR.
The real-time PCR method is undoubtedly more accurate and reliable than other methods.
It is used for the quantification of DNA, RNA and gene expression.
The sample source for the real-time quantification is gDNA, cDNA, RNA, Gene of interest, synthetic oligos, total RNA or plasmid DNA.
The real-time or quantitative analysis is divided into two other methods:
- Standard curve analysis
- Relative quantification
Standard curve analysis:
In the standard curve analysis method, the serially diluted sample or template is quantified against the known template.
Here the known template is serially diluted many times and quantified. The source of the information is used for the sample and unknown template which is also serially diluted and measured against the known.
In simple words, we can say that each unknown sample dilution is compared with each known standard dilution.
The method is also called as absolute quantification. The method is one of the best choices for the viral load quantification and bacterial load quantification present into the sample. Also, the absolute quantification method is rapid and more accurate.
By comparing the Ct value of both the standard and the unknown template, the linear curve graph is generated.
For each know and unknown dilution, the Ct of all is plotted on the graph and by comparing the data the initial concentration of the unknown template is determined.
However, the method of calculation contains so much maths, hence we are not discussing it but it is automatically calculated by the machine.
Another method is for those types of the template which does not have reference value. Or it is totally unknown.
Here, the calibrator is used to create the baseline for the experiment, and with respect to the baseline calibrate and the Ct value of the template sample, the amount of the expression of the gene into the unknown sample can be determined.
The figure below shows the graph of different templates,
|Ct value||The point at which the fluorescent is measurable.|
|Baseline||Point of the initial amplification where the fluorescent is nearly zero. No template zone.|
|Threshold||A threshold is a set of single which distinguish amplification signals from the background signals|
|Background signals||Signals of unamplified DNA|
|Exponential phase||The phase at which the reported amplification at its highest peak.|
Advantages of Real-time PCR:
The method is cost-effective.
The conventional PCR method is costly than the qPCR due to the use of so many other chemicals and agarose gel electrophoresis.
It is time-efficient.
Definitely, it is.
The average time consumed by the PCR reaction along with the agarose gel electrophoresis and data interpretation is approximately 4 to 4.5 hours.
Contrary, real-time qPCR gives results in ultra-fast time. The average duration of the qPCR reaction is around 30 minutes to 2 hours.
More sensitivity and specificity.
The quantitative real-time PCR method is more sensitive, specific and efficient.
Though the probes and primers are highly sequence-specific, if any non-specific bindings occurred, it is monitored immediately during the reaction. Also, the main reaction or the quantification of our template cannot be influenced by the non-specific bindings.
Fewer templates required:
The overall assay required less amount of the template material. It required 1000 folds less template DNA or RNA for the reaction to occur as compared with the conventional PCR.
Melting curve analysis:
The main advantage of the quantitative PCR is the confirmation of the analytes through the melting curve analysis.
We can measure and quantify how many amplicons are generated and how many non-specific or primer-dimers are formed during the PCR reaction by doing the melting curve analysis.
The major advantage over the other PCR technique is the quantification. It quantifies the template DNA or RNA present into the sample.
Only Real-time is sufficient:
No post PCR processing and data processing is required in the quantitative real-time PCR. As like the conventional PCR, agarose gel electrophoresis and interpretation is not need in the qPCR.
Limitation of real-time qPCR
Although the advantages of the quantitative rt PCR are far more than the conventional PCR, still the technology has several limitations.
The instrument is itself is too costly as compared with the conventional PCR.
Also, the multiplexing is still limited in the Real-time PCR.
Kits are not available for all kind of genes and disorders. The technical and standardized protocols are limited. Furthermore, higher expertise and technical skills are required for developing a novel qPCR assay.
Applications of quantitative PCR:
From gene quantification to gene expression, the real-time qPCR is the ocean of the different applications in different fields. Apart from gene expression studies, it is further used in food industries, microbial identification and disease diagnosis.
One of the most powerful uses of any PCR techniques is in the inherited disease diagnosis.
The real-time PCR is used in the diagnosis of a single gene and multigenic disease. It is used to quantify the mutated gene in the disease patient. The quantitative real-time PCR is even used in the determination of copy number variation in different tissues for different inherited disorders.
Micro RNAs are the smaller RNA molecules of 20 to 25 nucleotides in length. It plays an important role in the gene regulation pathway. The qPCR assay used to quantify the microRNA from different tissues.
By quantifying it we can estimate the gene expression level in different tissues influenced by the microRNA.
Circulating tumour cells contains the mutant mRNA which transports to different tissues if it is malignant.
By quantifying the total mRNA against the mutant mRNA into the samples of cancer or from the cancer cell. The stage of cancer can be determined. By which the severity of the carcinoma can be estimated.
The mutant mRNA is the best biomarker for the gene expression studies for cancer.
Furthermore, real-time quantification can be helpful in measuring the recovery in cancer therapy. After each therapy, the gene expression level of the mutant oncogenic cancer genes from the affected tissues is determined.
By doing rt PCR, the success of the therapy can be estimated.
Therefore, cancer diagnosis, prognosis and monitoring to the response of therapy can be done using the real-time qPCR.
Microbial load testing:
The accurate microbial load testing from any biological sample is nearly impossible without the qPCR.
Microbial load in the fermented sample, soil sample, water sample, food and food spoilage can be accurately estimated by the real-time PCR.
Also, the estimation of the active microbial load is determined. Additionally, microbial risk assessment can also be possible by this method.
Genetically modified organisms are the organism whose genetic makeup is altered using the genetic engineering or transgenic technique specifically into plant, animal and microorganism.
Now, this is something interesting,
Inserting DNA through the vector is not sufficient to do so. For successful development of the GMOs, one has to estimate the expression or the protein formed by the inserted gene.
For that, only gene detection cannot help, the mRNA expression quantification through real-time PCR helps to detect the amount of the gene expressed into the GMO.
Further, the amount of inheritance of that inserted gene is determined as well.
Genotyping and quantification of pathogens:
Infectious diseases are the second most reason for death in the world. Conventional PCR can help in genotyping of the pathogen (it can only detect the strains and species of the pathogen). However, for estimating the severity of the infection quantification is required.
By quantifying the DNA from the infection sample, through the melting curve and dissociation curve analysis the exact number of the pathogen can be determined.
qPCR is applicable in Identification, characterisation, genotyping and quantification of an infectious pathogen.
Identification and quantification of circulating nucleic acid:
Cell-free fetal DNA and circulating micro RNA are two types of the circulating nucleic acid present into the bloodstream. Both types of nucleic acids are present in very less quantity.
Even if it is very difficult to extract it, qPCR helps in the identification and quantification of circulating nucleic acid.
Circulating mRNA is the best marker for the cancer diagnosis. Prenatal diagnosis of several inherited disorders is well characterised using the real-time PCR.
Detection of pathogenic SNPs:
Melting curve analysis helps in the identification of many pathogenic SNPs, it is the most recommended method for characterization and identification of some of the disease.
Apart from these applications, the real-time PCR is also used in the forensic studies, evolutionary studies, mutation creation, fossil studies and in other applied fields.
Real-time PCR is one the best method for quantification in recent days. For sensitive detection such as TB PCR, it facilitates great precision in less time.
The quantitative real-time PCR is an accurate, fast, sensitive, cheap and adequate method in the genomic research. The diagnostic value of real-time PCR is more than any other PCR techniques.