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What Is Real-Time PCR
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A Real-Time Polymerase Chain Reaction is based on a conventional polymerase chain reaction laboratory technique that used in molecular biology for real-time monitoring of the amplification of a targeted DNA molecule. It is a well established method for detection, quantification, and typing of different microbiological agents in the area of clinical, veterinary diagnostic and food safety, basic research, forensic, medicines and more.
Real-Time PCR can be used for quantitatively (Quantative Real-Time PCR, qPCR) also known as Quantitaive Real-Time PCR (qPCR). qPCR is a powerful technique allows exponential amplification of DNA sequences.
Principles Of Real-Time PCR
There are many techniques that are used for the monitor the progress of PCR reaction. All techniques have common things that DNA amplification to generate fluorescence which can be detected by a camera used during each PCR cycle.
“The principle of Real-Time PCR relies on the fluorescent dye. Nucleic acid is present in the targeted DNA or sample which is quantified by using the fluorescent dye or fluorescent labeled-oligos. When a dye or prbe binds to the targeted DNA then it releases flurochrome which then fluresces to be detected by the detector. The detector recieves a signal in the form positive template amplification.”
Amplification:
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Denaturation: In the denaturation high temperature is used to break the hydrogen bond of targetd DNA and targeted DNA is converted into single strand from double starnd DNA. Usually 95 C temperature is used in the denaturation. It can be increased if GC content is high.
Annealing: After denaturation mixture is cooled from 50-70 C. For this an appropriate temperature is used. It is based on the calculated melting temperature (Tm).
Extension: At 70-72 C DNA polymerase helps to primer for extension steps. In this step primer extension occurs at rate of upto 100 bases per second.
Detection:
Detection is based on the fluorescence technology. In the Real-Time PCR is subjected to tungsten or halogen source that lead to fluorescence the marker which is added into the sample. The signal is amplified with amplification of copy number of sample DNA.
There are two types of chemistry available in Real-Time quantitative PCR for detection:
- DNA Binding Dye: DNA binding dye has its own fluorescence. Once the dye binds to the double-stranded DNA then dye emits the fluorescence and it increases 100-1000 folds than the original signal. The emitted signal is detected by an detector and sent to the computer. It converts into the digital signal that is displayed into the computer screen. The original dye is taken as the base line for the detection.
- Probe-Based detection method: In the Probe-based detection technology linear probes are utilized:
Linear Probe:
Taqman probe is Linear Probe and it relies on the activity of Taq DNA polymerase. Probes are structurally made up of short single- stranded sequences specific DNA molecule that are fluorescent labelled. It is a hydrolysis probe that carries a reporter dye, often fluorescein (FAM), attached to 3′ end of the oligonucleotide and a tetramethylrhodamine quencher (TMRA) at the 5′ end.
In the probe base method probe as well as Taq DNA polymerase plays important role real-time PCR. Taq DNA polymerase has exonuclease activity. It removes probe and extend DNA. As the Taq DNA polymerase initiates to generate DNA strand it causes degradation of the probe by 5′ end nuclease activity. If the DNA molecule is amplified, the reporter molecule is unquenched and fluorescence is generated.
With each cycle number of signaling molecule increases, causing an increase in fluorescence that is positively related to target amplification.
Advantages:
- It helps to determine which reaction worked well and which reaction has failed.
- Reaction efficiency can be calculated accurately.
- There is no need to run PCR sample on gel electrophoresis after the reaction because melting curve analysis serves this purpose.
- Real-Time PCR data can be used to perform true quantitative anlysis of gene expression.
- Faser than normal PCR.
- Complexity such as sample quantification is reduced.
Application of Real-Time PCR
- Cancer Reasearch
- Drug Research
- Gene Expression Analysis
- Viral Quantification
- Food Testing
- GMO food
- Gene copy number
- Animal and plant breeding
- Disease diagnosis and management
Disclaimer: All the content of this article is for information purposes only.
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