Physicochemical Properties Of Taq Man PROBE

TaqMan probes are covalently modified at the 5' end with a reporter dye; commonly used dyes for this purpose are listed in Table 1. When excited by incident light, these dyes emit light at wavelengths ranging from 515 nm (FAM) to 565 nm (HEX, VIC). The first nucleotide of a TaqMan probe—the one covalently labeled with the reporter dye—can only be A, C, or T; a G residue is not allowed because this base partially quenches reporter fluorescence, whether the probe is cleaved or not.

The quencher dye can be positioned in the middle of the probe, but the 3' end is preferred because the dyes can interact more effectively due to the greater flexibility of the probe, thereby increasing FRET efficiency and lowering reporter fluorescence before cleavage. Positioning the quencher at the 3' end also ensures that the endonuclease activity of Taq can be activated at any point along the length of the probe, and still result in separation of the dyes. Tetramethyl rhodamine (TAMRA) is a commonly chosen dye because through FRET it can effectively quench the excited state energy of all the reporter dyes cited above (absorption range 500-570 nm) (Table 1). However, as it quenches the reporter, TAMRA also emits light of a different wavelength (~ 580 nm), which contributes to increasing the total fluorescence in the sample. Nonfluorescent quencher dyes such as DABCYL [4-(4'-dimethylaminophenylazo) benzoic acid] have been introduced. With an absorption range of 450 to 550 nm, DABCYL can effectively quench the fluorescence of the shorter wavelength emitting dyes (such as FAM, JOE, and TET) in the intact probe without contributing any fluorescent signal (compare the emission maxima of the reporter dyes in Table 1 with the absorption properties of the quencher dyes in Table 2). Proprietary dark quenchers with wider absorption ranges and evocative names are constantly being developed, such as Black Hole QuencherTM 1 and 2 (Biosearch Technologies), and EclipseTM dark quencher (Epoch Biosciences). Nonfluorescent quenchers are particularly useful for multiplexing (chapter on Multiplexing, EDGP), where two (or more) TaqMan probes labeled with reporters with distinct emission maxima (such as FAM and HEX) are present in the same PCR reaction to simultaneously monitor the levels of different targets in the sample.[11]

In addition to being dual fluorescent labeled at the 5' and 3' end, TaqMan probes are also modified with a phosphate group at their 3' end. This modification renders them nonextendable, preventing priming of DNA synthesis from the probe itself by Taq polymerase.

Table 1 Commonly used reporters for TaqMan probes

5' Reporter

Full name

Absorbance (nm)

Emission (nm)

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment