Oligo Fluorescent Modifications

The application of fluorescent-labeled oligonucleotides is based on the principle of nucleic acid hybridization. The target DNA or RNA is analyzed by various hybridization reactions with the target nucleic acid molecule, followed by the associated fluorescent probe detection technique. BOC RNA offers a wide range of oligonucleotides for single, double and even multiple labeling using classical fluorescent dyes or newly developed alternatives.

What is Fluorescent

When ultraviolet or visible light hits certain substances, these substances emit light of varying wavelengths and intensities, and this light disappears quickly when the ultraviolet or visible light stops hitting them, and this light is called fluorescence.

The wavelength of fluorescence is somewhat longer than the wavelength of absorbed UV light. In simple terms, the process of fluorescence generation is that when a substance absorbs incident light, the energy of the photon is transferred to the substance molecule, the molecule is excited, and the molecule in the excited state is unstable, and can return to the ground state through the decay process of the radiation transition, decay The process is accompanied by the emission of photons, i.e. fluorescence. Some substances that can produce fluorescence can be applied to biological stains, which we call fluorescent dyes (fluorescent pigments) or fluorescent probes.

Fluorescent Groups

• Fluorescein labeling

There are various methods of oligonucleotide labeling of fluorescein-based dyes, and the diverse choice of labeling is related to the degree of chlorination of the aromatic ring - which determines the fluorescence emission of the dye. 6-FAM CE Phosphoramidite, 6-HEX CE Phosphoramidite and 6-TET-CE Phosphoramidite, derived from the monoisomeric 6-carboxyfluorescein, can all be used for effective labeling of the 5' end of oligonucleotides. There are also two phosphoramidite monomers that can be used for oligonucleotide fluorescein labeling: 6-Fluorescein-CE Phosphoramidite and Fluorescein-CE Phosphoramidite, both of which contain the same fluorescent dye as 6-FAM CE Phosphoramidite, but with different linkage backbones. Internal addition of fluorescein to the sequence can be achieved by replacing any suitable dT residue with Fluorescein-dT-CE Phosphoramidite.

Fluorescein labeling of the 3' end of the oligonucleotide can be achieved using four different vectors. 3'-Fluorescein CPG based on substituted fluorescein 5-isomers and 3'-(6-Fluorescein) CPG prepared from 6-FAM are commonly used for this purpose. In addition, there are 3'-(6-FAM) CPG and Fluorescein-dT CPG also derived from 6-carboxyfluorescein; among them, 3'-(6-FAM) CPG can effectively block the extension of polymerase to the 3' end and exonuclease activity.

• Cyanine dyes

Cyanine dyes as fluorescently labeled oligonucleotides are mainly used in the preparation of probes in molecular diagnostics, such as real-time PCR, fluorescence in situ hybridization (FISH), and DNA detection based on surface-enhanced resonance Raman spectroscopy (SERRS). Their emission spectra can be tuned by changing the length of polymethine chains, and their solubility in organic or aqueous solvents can be changed by substituents on the aromatic rings.

The most commonly used phosphoramidite dyes are Cyanine-3 (Cy3 MMTr CE Phosphoramidite) and Cyanine-5 (Cy5 MMTr CE Phosphoramidite). These dyes are usually attached to the 5' end of the oligonucleotide during synthesis, but can also be easily incorporated in the middle of the sequence. The hydroxyl protecting group MMT is removed in the same way as the DMTr protecting group. Mid-sequence incorporation is uncommon due to the lack of heterocyclic bases in the structure, and lacks the ability to participate in base pairing—making the duplex formed unstable. For 3'-modifications, the equivalent 3'-modified 1000 Å CPG vectors, 3'-Cyanine-3 (3'-Cyanine-3 CPG) and 3'-Cyanine-5 (3'-Cyanine-5 CPG ).

• ROX dye marker

ROX fluorescent dye (carboxy-X-rhodamine) is commonly used as a passive reference dye to provide a stable baseline for fluorescent reporter dyes (i.e., SYBR Green I or TaqMan probes in multiplex qPCR or real-time PCR).

We Provide the Following Degenerate Bases

Advantages

• BOC RNA has extensive experience in oligonucleotide synthesis and modification, and can provide customers with a comprehensive range of fluorescent dyes and quenching groups.
• Relying on our strong analytical, separation and synthesis platform, we are good at developing all kinds of products with poor stability and customization of unique products.
• Based on perfect quality management system, we ensure stable and reliable product quality with small batch-to-batch variation.
• We are able to communicate with customers in a timely manner and provide "customer-centric" services. Please contact us for more details.