Principles and Applications of real-time PCR
What is real-time PCR?
Real-time PCR (Real-time PCR) also known as quantitative PCR (qPCR) is a variant of the standard polymerase chain reaction in which amplification and simultaneous quantification of target DNA is done on the same PCR machine, using a commercially available thermal cycler for fluorescence detection. Fluorescent dyes specifically label the DNA of interest, and the amount of fluorescence produced is proportional to the amount of DNA present.
While various real-time PCR models are available, all models share common features: a standard thermal cycler platform plus an excitation source, a fluorescence detection camera, and a computer and software for data processing. Various fluorescent dyes can be used in qPCR depending on the excitation sources and detection filters available.
What are the characteristics of real-time PCR?
Amplification and product detection are done in one reaction vessel without any openings. This greatly reduces the chance of sample cross-contamination with amplification products.
• Compared to conventional PCR analysis, real-time PCR instruments not only measure amplification products (amplicons), but also quantify the number of products, thereby determining the copy number of the target in the original specimen.
• Compared to traditional PCR-based detection methods, the time required to complete real-time PCR detection is greatly reduced because the use of fluorescent probes eliminates the time required for post-PCR detection of amplified products. Additionally, some systems are capable of rapid thermal cycling, testing product within 20 to 30 minutes, depending on the instrument design.
What is the principle of real-time PCR?
Real-time PCR is done in the same way as traditional PCR methods (denaturation of double-stranded DNA, primer annealing and extension). However, its detection process differentiates real-time PCR from traditional PCR analysis. In real-time PCR assays, the accumulation of amplicons is monitored as they are generated using fluorescent dye-labeled primers. These labels produce changes in fluorescent signal that can be measured by the instrument upon direct interaction with or hybridization to the amplicon. This signal is related to the number of amplification products in each cycle and increases with the number of specific amplicons.
Currently, a range of fluorescent chemistries are used for amplicon detection; the more commonly used chemistries can be divided into two categories: (1) nonspecific binding of fluorescent dyes (such as SYBER Green I) to double-stranded DNA (2) fluorescent probes specific binding to the target.
Detection method of real-time PCR
Many real-time PCR methods have been described, but two of them are the most popular.
As the PCR product is generated, more dye-labeled probes find the target region to add, ultimately resulting in the release of the reporter molecule during subsequent amplification. The release of the reporter dye is reflected as an increase in the intensity of the fluorescent signal proportional to the number of synthesized amplicons.
Whether using a SYBR green probe or a TaqMan probe, the relationship between signal intensity and template quantity in a real-time PCR reaction provides a reliable means for quantifying nucleic acids and detecting the presence or absence of specific gene sequences.
What are the applications of real-time PCR?
Real-time PCR can calculate the starting template concentration and is therefore a common analytical tool for assessing DNA copy number, viral load, SNP detection and allelic discrimination. Prior to reverse transcription PCR, qPCR was a powerful tool for detecting mRNA expression and the gold standard for microarray gene expression data validation.
What are the advantages of Real-time PCR?
Significant advantages of real-time PCR include
• Ability to measure DNA concentration over a wide range,
• Its high sensitivity,
• Its ability to process multiple samples simultaneously and provide instant information.
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