qPCR relies on fluorescence from intercalating dyes or hydrolysis probes to measure DNA amplification after each thermal cycle. The most common dye-based method is SYBR Green, and the most common probe-based method is TaqMan. In this video, we explain the difference between the two methods and explore the advantages and disadvantages of each.
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What is qPCR?
SYBR Green and TaqMan are qPCR methods. In contrast to standard PCR that monitors DNA amplification upon reaction completion, qPCR uses fluorescent signals to monitor DNA amplification as the reaction progresses. This is why qPCR is also referred to as real-time PCR, quantitative PCR or quantitative real-time PCR.
How SYBR Green works
Like standard PCR, the SYBR Green protocol consists of denaturation, annealing and extension phases. The difference being that you add some double-stranded DNA binding dye, SYBR Green I, to your master mix during qPCR setup. This fluorescent dye intercalates into double-stranded DNA sequences during the extension phase, where it shows a strong increase in fluorescent signal. Measuring this signal at the end of every thermal cycle will allow you to determine the quantity of double-stranded DNA present.
How TaqMan works
Instead of using intercalating dyes, the TaqMan assay uses TaqMan probes with a 5' fluorescent reporter dye and a 3' quencher dye. These probes are target-specific, and only bind to the DNA sequence of interest downstream of one of the primers during the annealing step. When the Taq polymerase enzyme encounters the TaqMan probe during the extension phase, it displaces and cleaves the 5' reporter dye. Once the reporter dye has been separated from the quencher dye, its measurable fluorescent signal at the end of every qPCR cycle increases significantly.
Pros and cons of SYBR Green vs TaqMan
The advantage of the SYBR Green method is that it is more affordable to buy a SYBR Green I dye than to design and purchase target-specific TaqMan probes. The downside of the SYBR Green assay, however, is that the dye binds to any double-stranded DNA sequence. This means that you could also detect fluorescence emitted from non-specific qPCR products, such as primer dimers.
Compared to the SYBR Green assay, the use of TaqMan probes is more expensive, but also offers two significant advantages. The first one is that TaqMan only measures amplification progression of the target sequence, as the probes are target specific. The second one is that you can monitor the quantity of various qPCR products in a single reaction by adding different primers and TaqMan probes with different reporter dyes to the master mix. This multiplex approach allows you to detect several fluorescent signals at the end of every thermal cycle.
Video content
[00:00] - What is qPCR
[00:31] - How qPCR works
[00:55] - How SYBR Green works
[01:32] - How TaqMan works
[02:13] - Pros and cons of SYBR Green
[02:39] - Pros and cons of TaqMan
[03:21] - More information
[03:32] - Outro
#qPCR #SYBRGreen #TaqMan #PCR
27 июл 2024
