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| Image Source: genomecore.ucsf.edu |
Real Time PCR is a molecular biology instrument which is able to monitor the amplification of the target or template DNA at each cycle. Hence the name "Real Time PCR".. It can also be called as quantitative PCR as it is able to quantify the template DNA. It works on the basis of polymerase chain reaction in addition to that it utilizes the fluorescent dyes, fluorescent reporters or probes to monitor the amplification of template in DNA by emitting the fluorescence at each cycle.
There are two main methods for the detection of amplification of DNA in real time.
1) Non-specific fluorescent dyes such as SYBR Green Dye which binds to any double-stranded DNA present in the PCR tube.
2) Sequence-specific DNA Probes consisting of oligo nucleotides which are labelled with fluorescent reporters.
Example: TaqMan.
Principle of Real-Time PCR:
"A thermal cycler which is installed with an illuminator which will illuminate the samples present in the reaction tube with a light at a specific wavelength. When illuminated, the fluorophore bound to the double stranded DNA will emit the light at certain wavelength which is captured and recorded by the detectors placed on top. These detectors then convert the light or photon signal into electronic digital signal which is acquired on a special software. This process repeats at every annealing and extension step.. The number of cycles are usually between 25 to 40 cycles per reaction". (Note: Due to small base pairs of the templates and primers, usually extension step is omitted).
How Real Time PCR actually works? (Practically)
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| Image Source: (RSC) publishing - Royal Society of Chemistry |
The prepared samples are kept on a thermal cycler block which is capable of heating and cooling. On top of this, there are lens, filters, dichroic mirrors, objective and illumination source are installed. Illumination source commonly halogen lamp or LEDs will illuminate a light beam at a specific wavelength which will travel through filter to dichroic mirror, dichroic mirror will reflect the illuminating light, this reflected light travels through the objective to the samples. In this manner, samples are illuminated. Illuminated light then excites the fluorophores bound to the double stranded DNA which will emit the light at different wavelength. (Longer wavelength than absorbed). Fluorescent molecules will bind to only double stranded nucleic acids and emit the light at certain wavelength only when they are bound (during annealing of primers and extension steps). The emitted light is then captured by the specialized CCD Camera which will process and convert the light or photon signal to electronic signal. This electronic signal is then converted to digital signal which will acquired and recorded by the specialized software installed in computer.
Based on the signals, the software will generate the plots at each cycle. Plots such as Amplification plots, melt curve plots and others.
Based on the emitted light signal, software generates amplification plot as show below.
Baseline, is where the instrument is not capable of detecting the fluorescence emitted, as emitted fluorescence is too low in beginning of the cycles.
The thermal cycle at which the signal exceeds the fluorescence detection threshold is known as the Threshold cycle (CT) or crossing point. A standard curve of log concentration against CT can be made by making use of multiple dilutions of a known amount of standard DNA. The quantity of DNA or cDNA in an unknown sample can thus be determined from its CT value.
There are three phases of PCR amplification in Real Time PCR.
1) Exponential Phase, where doubling of the template DNA takes place exponentially at each cycle. reaction is specific and precise. Real Time PCR then measures the Ct values at this phase.
2) Linear Phase, where components of the reaction are being consumed and amplification of template slows. and at this phase, the reaction becomes variable.
3) Plateau Phase, this is the final phase of the reaction where the reaction is complete and no more doubling of template DNA.
(Most of traditional PCRs take readings or its measurements during plateau phase).
Two types of Detection Chemistries:
1) Using the intercalating dyes sucb SYBR Green which incorporates between base pairs of double stranded DNA. This is suitable when the reaction generates a specific template DNA. Because this type of dyes are capable of intercalating with any double stranded DNA segments.
2) Using the primers which are highly specific to the target DNA. Fluorescent Probes, reporters and quenchers are utilized to amplify the template DNA segment. Such as using Molecular Beacons or TaqMan probes.
Based on the signals, the software will generate the plots at each cycle. Plots such as Amplification plots, melt curve plots and others.
Based on the emitted light signal, software generates amplification plot as show below.
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| schematic of amplification plot |
Baseline, is where the instrument is not capable of detecting the fluorescence emitted, as emitted fluorescence is too low in beginning of the cycles.
The thermal cycle at which the signal exceeds the fluorescence detection threshold is known as the Threshold cycle (CT) or crossing point. A standard curve of log concentration against CT can be made by making use of multiple dilutions of a known amount of standard DNA. The quantity of DNA or cDNA in an unknown sample can thus be determined from its CT value.
There are three phases of PCR amplification in Real Time PCR.
1) Exponential Phase, where doubling of the template DNA takes place exponentially at each cycle. reaction is specific and precise. Real Time PCR then measures the Ct values at this phase.
2) Linear Phase, where components of the reaction are being consumed and amplification of template slows. and at this phase, the reaction becomes variable.
3) Plateau Phase, this is the final phase of the reaction where the reaction is complete and no more doubling of template DNA.
(Most of traditional PCRs take readings or its measurements during plateau phase).
Two types of Detection Chemistries:
1) Using the intercalating dyes sucb SYBR Green which incorporates between base pairs of double stranded DNA. This is suitable when the reaction generates a specific template DNA. Because this type of dyes are capable of intercalating with any double stranded DNA segments.
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| Image Source: nptel.ac.in |
2) Using the primers which are highly specific to the target DNA. Fluorescent Probes, reporters and quenchers are utilized to amplify the template DNA segment. Such as using Molecular Beacons or TaqMan probes.
Applications of Real Time PCR:
* Gene Expression Analysis
* Absolute DNA Quantification and miRNA quantification.
* Diagnosis of infectious diseases.
* Quantification of unknown DNA segments.
*Wide range of applications in biological and biomedical applications.
* Food and agriculture industries.
*Forensic Science for Crime Investigation and DNA Fingerprinting.
*Human Genetic Testing.
*Applied Microbiology
* Genetics Variation analysis such as SNP Genotyping, Drug Metabolism Enzyme Genotyping.
*Mutation identification and analysis.
*microRNA and non-coding RNA Analysis.
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