Dual-Labeled Probe for qPCR

The optimal length of Double-Dye Oligonucleotide probes with a 3’ BHQ dark quencher is 25 bases. In case extra long Double-Dye Oligonucleotides probes are required, this BHQ1 and BHQ-2 can be coupled internally.

TAMRA has an absorbance maximum of 565 nm and an emission maximum of 580 nm. In FRET application, a common donor-acceptor pair is 6-FAM (donor) / TAMRA (acceptor), due to their good spectral overlap. As the donor, 6-FAM is excited at 492 nm and transfers this energy to TAMRA, which then emits light at 580 nm. This FRET oligo probe design are widely used to in vivo studies to monitor biochemical reactions ¹ .

TAMRA also can be used as a FRET-based quencher moiety in real-time PCR probes such as TaqMan probes² , Scorpion primers³ and Molecular Beacons ⁴ . For such probes, 6-FAM is used as the reporter moiety, and its emission at 521 nm is monitored. When 6-FAM and TAMRA are in close proximity, the former’s fluorescence at 521 nm is quenched by the latter. After sufficient spatial separation of the two dyes during the course of the assay, 6-FAM’s fluorescence is no longer quenched, and its fluorescence signal becomes observable.

TAMRA also can be used as a FRET-based quencher moiety in real-time PCR probes such as TaqMan probes² , Scorpion primers3 and Molecular Beacons⁴ . For such probes, 6-FAM is used as the reporter moiety, and its emission at 521 nm is monitored. When 6-FAM and TAMRA are in close proximity, the former’s fluorescence at 521 nm is quenched by the latter. After sufficient spatial separation of the two dyes during the course of the assay, 6-FAM’s fluorescence is no longer quenched, and its fluorescence signal becomes observable.

TAMRA also can be used to label DNA oligos for use as hybridization probes in a variety of in vivo and in vitro research or diagnostic applications, as well as for structure-function studies of DNA, RNA, and protein-oligonucleotide complexes. Oligos labeled with TAMRA at the 5’-end can be used as PCR and DNA sequencing primers to generate fluorescently-labeled PCR, sequencing or genetic analysis (AFLP or microsatellite) products.

We offer 3'-TAMRA CPG for 3' labeling and TAMRA-dT for labelling within the sequence. We also offer TAMRA NHS ester for labelling amino-modified oligonucleotides post-synthetically. All Double-Dye Oligonucleotides are single ie-HPLC or dual rp-HPLC and ie-HPLC purified. The length of Double-Dyes probes should be comprised between 15 and 35 bases. The optimal recommended length of Double-Dye Oligonucleotides with a 3’ TAMRA is 25 bases.

Carboxytetramethylrhodamine (TAMRA) is a fluorescent dye that is a derivative of rhodamine, and is used to label oligonucleotides at the 5’- or 3’-ends, or internally. TAMRA has an absorbance maximum of 565 nm and an emission maximum of 580 nm. TAMRA-modified oligonucleotides play a particularly important role in both fluorescence resonance energy transfer (FRET) and real-time PCR applications.

FRET is a distance-dependent interaction between two dye molecules in which excitation is radiationlessly transferred from one dye (the donor) to the second dye (the acceptor), due to spectral overlap. Because the efficiency of the energy transfer is extremely sensitive to the distance between the molecules (varying as the inverse sixth power of that distance) (1), FRET can be used to study biological phenomena that produce changes in molecular proximity (2). For oligonucleotides slated for use in FRET application, a common donor-acceptor pair is 6-FAM (donor) / TAMRA (acceptor), due to their good spectral overlap. As the donor, 6-FAM is excited at 492 nm and transfers this energy to TAMRA, which then emits light at 580 nm. FRET oligo probes are widely used to monitor biochemical reactions, particularly in in vivo studies (3).

Besides being used as a FRET fluorophore, TAMRA also can be used as a FRET-based quencher moiety in real-time PCR probes such as TaqMan probes (4), Scorpion primers (5) and Molecular Beacons (6). For such probes, 6-FAM is used as the reporter moiety, and its emission at 521 nm is monitored. When 6-FAM and TAMRA are in close proximity, the former’s fluorescence at 521 nm is quenched by the latter. After sufficient spatial separation of the two dyes during the course of the assay, 6-FAM’s fluorescence is no longer quenched, and its fluorescence signal becomes observable.

TAMRA also can be used to label DNA oligos for use as hybridization probes in a variety of in vivo and in vitro research or diagnostic applications, as well as for structure-function studies of DNA, RNA, and protein-oligonucleotide complexes. Oligos labeled with TAMRA at the 5’-end can be used as PCR and DNA sequencing primers to generate fluorescently-labeled PCR, sequencing or genetic analysis (AFLP or microsatelite) products.

Note that, because TAMRA is in the form of an NHS ester, the oligo first must be synthesized with an Amino C6 Linker (for the ends) or the Amino C6 version of the base phosphoramidite (for internal labeling). The TAMRA-NHS ester is then manually attached to the oligo through the amino group in a separate reaction post-synthesis.

References:

1. Didenko, V.V. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications. Biotechniques (2001), 31 : 1106-1121.
2. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. (1995), 4 : 1-6.
3. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. (2000), 28: 3752-3761
4. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.

The Eclipse quencher developed by Epoch, is a highly stable molecule and can be used safely in all common oligo deprotection schemes. The absorbance maximum for Eclipse Quencher is at 522 nm, compared to 479 nm for dabcyl. In addition, the structure of the Eclipse Quencher is substantially more electron deficient than that of dabcyl and this leads to better quenching over a wider range of dyes, especially those with emission maxima at longer wavelengths (red shifted) such as Redmond Red and Cy5. In addition, with an absorption range from 390 nm to 625 nm, the Eclipse Quencher is capable of effective performance in a wide range of colored FRET probes.

The optimal length of Double-Dye Oligonucleotide probes with a 3’ Eclipse® Dark Quencher is 25 bases.

The Instant Quencher IQ4, IQ2 are highly stable molecule and can be used safely in all common oligo deprotection schemes. IQ Quencher is capable of effective performance in a wide range of colored FRET probes from FAM (EX 495 nm) to Cy5 (Ex 650 nm).

The optimal length of Double-Dye Oligonucleotide probes with a 3’ IQ4 dark quencher is 25 bases. In case extra long Double-Dye Oligonucleotides probes are required, this IQ4 can be coupled internally

Blackberry quencher BBQ650 can be use to replace BHQ3. BBQ650 dark quencher has broad absorbance spectra ranging from 550-750 with peak absorbance at ~650 nmm. This quencher is ideal for use with Texas Red®, Cy5^TM, TAMRA, Redmond Red, DyeLight and other fluorescent dyes that emit in the red spectral range.

The optimal length of Double-Dye Oligonucleotide probes with a 3’ BBQ650 dark quencher is 25 bases. In case extra long Double-Dye Oligonucleotides probes are required, this BBQ650 can be coupled internally.