Oligonucleotide Dye Labeling
The use of fluorescent detection system for the detection of DNA or RNA is developing
rapidly, particularly in the area of fluorescence in situ hybridization and in hybrid
detection on DNA chips. These fluorescent labelled probes have been proven to have
very sensitive detection. High sensitive detection systems using BNA probes have
increased sensitivity for distinguishing DNA base-pair mismatches and are commonly
used for SNP genotyping assays.
Bridged Nucleic Acids (BNAs) can be incorporated into dual-labeled probes. Since
BNA bases significantly increase Tm, BNA probes can be designed with shorter lengths
than standard dual labeled probes. Shorter BNA probes have provided better quenching
and a higher signal-to-noise ratio and are, therefore, more sensitive. More importantly,
these probes offer an improved ability to distinguish mutations or single nucleotide
polymorphisms. A DNA dual labeled probe typically has a ΔTm of ~5oC between
perfect-match and mismatch hybridization. A BNA probe can have a ΔTm of >15oC,
greatly increasing accuracy of allele determination in real-time PCR or other methods
that use differential hybridization to distinguish polymorphisms.
Probe design services
Bio-Synthesis offers design assistance for your single or dual-labeled DNA, LNA
probes or Molecular Beacons probes. Using sophisticated algorithms, BSI can design
dual-labeled probes and molecular beacons that will dramatically improve the success
of your assays. Primers and probes on multiple sequences are designed in a single
search run and are screened for all possible secondary structures to ensure optimal
signal strength. Tm is calculated using nearest neighbor thermodynamic theory and
highly accurate Santa Lucia values. For effective primer and probe design for your
Multiplex or SNP genotyping assays, contact your local BSI Representative for more
information.
Quality Control
All of our oligonucleotides undergo vigorous process monitoring and strict quality
control. Length and labeling are systematically controlled by PAGE and characterized
by MALDI-TOP mass spectrometry analysis. Quantity is determined and validated by
UV Absorbance at 260 nm.
You will receive a Quality Assurance Certificate that includes quantitated yield,
melting temperature, MW and µg/OD with every oligo. You will also receive an additional
label for use in your records or on an additional vial.
Purification
Some dye labels can be incorporated at the time of synthesis. However, double HPLC purification is highly recommended for labeling which require a post-synthetic labeling method where a functionalized oligo is covalently linked to a dye using appropriate cross linking chemistry. After synthesizing a modified oligo, failure sequences are removed during the first HPLC purification. After labeling, the second HPLC is performed to remove unlabeled oligonucleotides and excessive dye in order to obtain a full length labeled oligonucleotide. Dyes which are labeled post-synthetically, often result in lower yield. However, since the dye has never been exposed to acid during chemical synthesis, there is no nick on the fluorescent ring that causes reduction of fluorescent intensity.
Guaranteed Yield
Due to the chemistry of many modifications, yield will be approximately 40 - 60 %
less than a standard oligo. Please see our yield chart for details.
Delivery times
Expected shipment time for common dye modifiers is 3 - 5 working days. More exotic
modifications are usually completed within 5 - 7 working days.
Packaging
Lyophilized
Shipping Conditions
Room temperature
Storage Conditions
Fluorescent dye-labeled oligos are light sensitive material and are more fragile
than unmodified oligos. Their ability to fluoresce will decrease over time. To ensure
high quality, store the oligo dry at -20°C in small aliquots. Note also that fluorescent
dye-labeled oligos should be stored in the dark as light can slowly degrade the
fluorescent moieties.
For optimal long-term storage of fluorescent dye-labeled oligos except Cy5 and Cy3,
it is recommended that the oligos be resuspended in a slightly basic solution (i.e.,
TE at pH 8). If brought to a pH below 7, it has been shown that the oligo can begin
to degrade and may be unusable within a few weeks. Cy5 and Cy3 begin to degrade
at a pH above 7. For best results, resuspend Cy5 and Cy3 labeled oligos at pH 7,
aliquot, lyophilize, and store at -20oC. Fluorescent dye-labeled oligos
can be used up to 6 months from purchase when stored at -20oC in the
dark.
Custom Fluorescent Labeling Oligonucleotide
- More than 200 fluorescent labels
- Wide spectrum coverage using alternative dyes from Dyomics, Atto-Tec, Luminartis,
Abberior, Dylight
- All dye labeling are HPLC and/or Dual HPLC purified
- Fast, high quality and competitive price
Some dye labels can be incorporated at the time of synthesis. However, double HPLC purification is highly recommended for labeling which require a post-synthetic labeling method where a functionalized oligo is covalently linked to a dye using appropriate cross linking chemistry. After synthesizing a modified oligo, failure sequences are removed during the first HPLC purification. After labeling, the second HPLC is performed to remove unlabeled oligonucleotides and excessive dye in order to obtain a full length labeled oligonucleotide. Dyes which are labeled post-synthetically, often result in lower yield. However, since the dye has never been exposed to acid during chemical synthesis, there is no nick on the fluorescent ring that causes reduction of fluorescent intensity.