Hairpin shaped hybridisation probes with an internally quenched fluorophore to detect specific sequences. Hydrolysis probes for TaqMan assays with a variety of dye - quencher combinations compatible with all type of instruments.ĭonor and acceptor probes to be used in FRET (fluorescence resonance energy transfer) assays. Short probes that incorporate a minor groove binder (MGB) to be used in 5’ nuclease (TaqMan - MGB) technology for human IVD applications. Highly-specific and shorter qPCR probes with locked nucleic acids Select the qPCR Probe you need for your application, method and technology: Increase the efficiency, sensitivity and specificity of your real-time PCR results by using hybridisation probes. However, if you would like to alter the sequence to change a fluorescent quencher to an NFQ-MGB quencher, you can use our Primer Express® Software.Are you performing gene expression analysis, SNP genotyping or mutation detection via real-time PCR? If you are using a published sequence, you will want to use the same quencher, or if it is not available, then the same type of quencher. If you are interested in multiplexing, we recommend the QSY® probe. In general we prefer MGB-NFQ as a quencher, as it allows for shorter probes which are more specific. For optimal performance, the quencher's absorbance spectrum should match the reporter. A wide variety of approaches have been developed for generating a fluorescent signal, the most common of which use either hydrolysis probes (e.g., TaqMan®), or a double-stranded DNA binding dye, (e.g., SYBR® Green). Quenchers may be fluorescent (TAMRA) or nonfluorescent molecules (DABCYL, Black Hole Quencher (BHQ). Quantitative PCR (qPCR) uses real-time fluorescence to measure the quantity of DNA present at each cycle during a PCR. If you are designing from scratch, you will have the flexibility to alter the sequence to meet Tm requirements as needed. Quenching molecules are typically placed at the 3' end of single molecule probes such as Dual-Labeled Probes, Molecular Beacons and Scorpions Probes. The choice of quencher will depend on whether you are designing a TaqMan probe from scratch or you already have a sequence (a probe sequence from a publication, for example). If you plan to multiplex, using a nonfluorescent quencher is usually best. They can be nonfluorescent or weakly fluorescent they can increase the Tm of the TaqMan probe or have no effect on the Tm. When choosing a quencher, keep in mind the basic types of quenchers. You must keep this in mind if you plan to multiplex with such a probe, as the TAMRA™ quencher will take up a dye option on your instrument. Similar to MGB, because the quencher does not fluoresce, the real-time instruments can measure the reporter dye contributions more precisely.Ī TAMRA™ quencher is a weakly fluorescent quencher. Mismatches between a probe and target reduce the efficiency of probe hybridization in a measurable way, which is especially important in SNP genotyping assays.Ī QSY® quencher is also a nonfluorescent quencher and is available with a variety of dyes to support multiplexing. In general, the TaqMan® MGB probes exhibit great differences in Tm values between matched and mismatched probes, which provides more accurate allelic discrimination and a more sensitive real-time assay. Note: A TAMRA™ quencher does not affect the Tm of the probe. The minor groove binder increases the melting temperature (Tm) of probes, allowing the use of shorter probes. Because the quencher does not fluoresce, the real-time PCR instruments can measure the reporter dye contributions more precisely.ģ) A minor groove binder at the 3’ end. TaqMan® MGB probes have the following features:ġ) A fluorescent reporter at the 5’ end (typically FAM™ or VIC® dye)Ģ) A nonfluorescent quencher at the 3’ end.
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