Realtime PCR Probes

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Reagent Ordering Form

 

Intercalating dyes (eg SYBR green or Sito9) versus realtime probe systems

Initially, intercalating dyes (SYBR green, Sito9 etc) were used to measure real-time PCR products. The primary disadvantage of this approach is that the detection includes both specific and non-specific PCR products. Intercalating dyes will bind to any DNA and will give a fluorescent signal that is the combination of specific and non-specific PCR product.

If there is non-specific product present this will lead to an overestimation of the levels of gene expression for your target gene. DNA binding probe systems get around this issue by being a target specific method for realtime PCR detecting only the specific product. This is particularly useful if you have found non specific amplification occurs with your target gene by conventional agarose gel analysis. If you have found that your PCR is highly specific and no side product is amplified you may as well use intercalating dyes to perform realtime PCR as they are much cheaper.

For intercalating dye relatime PCR you just need two primers, the intercalating dye and a cheap and efficient mastermix. We sell various intercalating dyes and mastermixes, please have a look at our shop webpage. you can order your primers through our Oligo Ordering Service.

 

The chemistry of realtime PCR probes

Both Taqman probes (Thermofisher) and LNA probes are dual-labeled probes for realtime PCR gene expression analysis. Both types utilize the 5´–3´ exonuclease activity of Taq polymerase to hydrolyze nucleotides of the target bound probe. with hydrolyzing nucleotides from the 5' end of the probe the reporter fluorochrome (R) is released which is usually attached to the first 5' nucleotide.

In the inactive state (before  5´–3´ exonuclease activity) the Reporter is quenched by the Quencher (Q) at the 3' end of the probe by a mechanism called "FRET". FRET stands for Fluorescence Resonance Energy Transfer and happens between the Reporter and Quencher as long as they are located in close proximity within one molecule. Once the Reporter is cut off the probe it is no longer quenched and will create a signal that is detected by the instrument. 

In most cases the Reporter (R) will be FAM labelled and only occasionally the Reporter is VIC labelled (house keeper or control genes or custom made probe sets). The old type Quenchers were TAMRA labelled. TAMRA has a small amount of emission of a longer wavelength. Modern Quenchers have almost no residual fluorescence and are therefore called "Blackhole" or "Zerowave", they convert the absorbed FRET energy into heat. 

Realtime Probes ChemistryRealtime Probes Chemistry method

 

During PCR, when the polymerase replicates a template onto which a probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage.

In the first ~20 cycles of the PCR there is Reporter produced and the fluorescence increases but this is not detected by the instrument because the number of molecules produced remains under the detection limit of the detector of the instrument. But as PCR is a logarithmic amplification there will be that cycle in which suddenly by doubling the number of Reporter molecules the detection limit (called cT = cycle Treshold) will be reached and the fluorescence will be detected as can be seen in the graph below.

Realtime Probe chemistry PCR pic

 

A Comparison of Taqman  and LNA Probes

Instruments

Taqman and LNA probes work on both instruments (LightCycler 480 and ABI Prism 7900HT). Yet the default settings for both instruments make it easier to work with LNA probes on the LightCycler 480 and to amplify the Taqman assays on the Quant7. However, we can help you set up the instrument for LNA or Taqman assays if you have booked the "wrong" instrument or the other instrument is heavily booked.

Ordering probes 

LNA probes or Beacon probes are ordered via Sigma or IDT

Taqman probes are ordered via Thermofisher or IDT offers a similar product called MGB probes

Mastermixes

LNA probes work best with the Roche Mastermix (sold in the facility in 5ml aliquots) and Taqman assays work best with Gene Expression Mastermix from Thermofisher (sold in the facility in 5ml aliquots). For prices, please see our shop webpage. There are a lot of competitor mastermixes on the market and they may be better priced and work sufficiently well or even better. We only sell the recommended mastermixes from Roche or Thermofisher for probe based realtime PCR setup, for Sybrgreen or related non-probe based realtime PCR we supply other mastermixes than Roche and Thermofisher. Please talk to our facility staff or see our shop webpage.

Proprietary differences

Where the Taqman probes and primers of an assay bind is not disclosed by Thermofisher. Instead the position of the target binding side and primer binding region is given with the assay design for the LNA probes.

 

Assay Design

Both systems use an online assay design webpage to select probes or assays for your target gene(s). For more information please see below.

Taqman Assays

For more information about Taqman assays including what the differences are in the assay names and quantities please see the Taqman webpage.

LNA Probes

For more information about LNA probes including assay design and assay setup please see the IDT webpage.