Educational Information
What is the Principle of Real-time PCR?
Real-time PCR, also known as quantitative PCR (qPCR), is a molecular biology technique used to amplify and simultaneously quantify a targeted DNA molecule. The principle of real-time PCR involves several key components and steps:
1. DNA Amplification: Like traditional PCR, real-time PCR involves the amplification of a specific DNA sequence through repeated cycles of denaturation, annealing, and extension. This process exponentially increases the amount of the target DNA.
2. Fluorescent Detection: The key difference between traditional PCR and real-time PCR is the incorporation of a fluorescent dye or probe that allows for the detection of the DNA during the amplification process. There are two main types of detection methods:
– Intercalating Dyes: Dyes such as SYBR Green bind to double-stranded DNA. As the DNA is amplified, more dye binds to the increasing amount of double-stranded DNA, leading to an increase in fluorescence.
– Probe-based Systems: Specific probes (e.g., TaqMan probes) can be designed to hybridize to the target sequence. These probes are labeled with a fluorescent reporter and a quencher. When the probe is intact, the quencher suppresses the fluorescence. During amplification, the probe is cleaved by the DNA polymerase, leading to an increase in fluorescence that is proportional to the amount of target DNA.
3. Real-time Monitoring: The fluorescence emitted during the PCR cycles is measured in real-time, typically at the end of each cycle. The increase in fluorescence corresponds to the amount of PCR product generated, allowing for the quantification of the initial amount of target DNA in the sample.
4. Quantification: The data collected during the amplification can be analyzed to determine the starting quantity of the target DNA. This is usually done by comparing the cycle threshold (Ct) values (the number of cycles required for the fluorescence to exceed a threshold level) to a standard curve generated from known quantities of the target DNA.
5. Applications: Real-time PCR is widely used in various applications, including gene expression analysis, detection of pathogens, genotyping, and genetic variation studies due to its sensitivity, specificity, and ability to provide quantitative data.
In summary, real-time PCR combines the amplification of DNA with real-time monitoring of fluorescence, allowing for both the detection and quantification of specific DNA sequences in a sample.