There are many things you need to understand about quantitative polymerase chain reaction or qPCR before proceeding to any scientific research relating to DNA. The qPCR is similar to PCR, but it goes further to allow you to quantify the amount of target DNA in your sample. Two options are involved with qPCR: either you are using fluorescent probes or intercalating dye to detect your target.
Suppose you are new but have a project in genotyping, allelic discrimination, gene expression analysis, single nucleotide polymorphism detection, or pathogen detection. Before proceeding, you must understand QPCR Probes and how it works. Luckily, many labs and companies have experts who can help you move with your research and finding without sweat. They will provide you with high-quality RT-qPCR and qPCR master mixes suitable for the above projects. This article will explore everything you need about probe-based qPCR and its difference with PCR.
What Is Probe-Based qPCR
For accurate and Specific results, you must use probe-based qPCR, which uses fluorescently labeled sequence-specific DNA. When using probes, you must design the probes and primers, which might be time-consuming and more expensive than the dye-based qPCR option. However, it allows multiplexing, which helps recover the lost time back.
The Difference Between PCR and qPCR
It’s critical to note that both qPCR and PCR are polymerase chain reactions used to detect and amplify specific samples of DNA. However, one of the significant differences is that qPCR is a real-time method while PCR is not. It means that you can witness and monitor the amplification of your target DNA using qPCR in real time as it is happening.
Another difference between these methods is that PCR doesn’t require fluorescent probes or dyes, while qPCR does. These probes are crucial to qPCR since they allow you to quantify the amount of DNA present in your sample. Additionally, qPCR is used to quantify and detect RNA viruses, while PCR is often used to amplify DNA for sequencing or other downstream application.
How Probe-Based qPCR works
When considering the probe-based qPCR, you need a quenching element on one side and probes containing fluorescent reporter dye on the other. The quenching part helps prevent fluorescence by absorbing all the light emitted by the reporter. It’s crucial to take time before locating your probe and primers when designing them to avoid significant mistakes. Remember that the probes should always be placed between the primers in the middle of the sequence. The PCR will identify and bind the complementary target downstream of the primer. Further, the quencher and the fluorescent reporter dye will be separated by Taq DNA polymerase that cleaves the probe. This process allows the fluorescent signal to signal to be released, which the qPCR machine measures and quantitates the amount of DNA in the sample.
The benefit of using probe-based qPCR is you can be sure the signal comes from the intended target. The signal will only be released when the probe and primers bind together. For precise and accurate measurement, you can contact a reputable company with an expert who can guide you on interpreting results and what to expect.
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