The need for dye-labeled oligonucleotides is skyrocketed lately. The main areas of application are:
- Mutation analysis
- Multiplex PCR
- Quantitative PCR
- In situ hybridization (FISH)
- Structure Elucidation
- Single Molecule Spectroscopy
- Enzyme kinetics
- Diagnostic formats (LightCycler ™, TaqMan ™ and Molecular Beacons)
Quality of fluorescently labeled oligonucleotides
A measure of the quality of labeled oligonucleotides represents the emitted fluorescence, which should reach certain values depending on the dye, the amount of the oligonucleotide used and the environment. While an excess of fluorescence suggesting that free or non-covalently bound dye is present, is a lack of fluorescence due to incorrect sequence selection (please no dG in the immediate vicinity of the dye), poor quality of components or due to incorrect production conditions in the synthesis causes and purification. Here unsuitable cleavage reagents, incomplete removal of unlabeled nucleic acid sequences, poor separation of impurities such as salts and derivatives protecting group and false conditions are particularly mentioned in the finishing.
The Fa. PURIMEX can draw on more than 30 years experience. We use to manufacture our labeled oligonucleotide only blocks a renowned manufacturer. Whenever permitted by the requirements we use the amino linker / NHS ester variant for the introduction of labeling, because if the quality of the modules is good, runs fast and virtually quantitatively.Furthermore, it is a very gentle process and allows, depending on the dye, an efficient separation of the unlabeled sequences and impurities.
Methods for introducing dyes / haptens
The introduction of a dye / Haptenes in an oligonucleotide is carried out using the following methods, their advantages and disadvantages are shown in detail:
Installation via amidite
The dye / the hapten is incorporated during the synthesis at the 5 'end or within the sequence (internal, dye / hapten dT amidite) by coupling a amidite. After cleavage of the oligonucleotide from the support and removal of the protecting groups the crude product is purified by HPLC using duplicate.
- Almost quantitative reaction
- Überschüße can easily be removed
- Minimal time requirement
- Expensive Blocks
- Few compounds available (s. Below)
- Significant staff overhead by cleaning synthesis machinery
- Impairment of fluorescence by exposure to drastic reagents
We recommend the use of amidites only with double markings. In individual marks should be used for reasons of quality NHS ester blocks.
The incorporation of the dye / hapten is performed at the 3'-end by the use of a polymeric support to which the derivative is already bound. After cleavage of the oligonucleotide from the support and removal of the protecting groups the crude product is purified by HPLC using duplicate.
- Minimal time requirement
- Expensive raw materials
- Few blocks available (s. Below)
- Modification is exposed at each chain extension acid and base.
- Difficult purification, as well as truncated sequences carry the non-polar residue.
We recommend the use of CPG-blocks only with double markings. In individual marks should be used for reasons of quality NHS ester blocks.
When oligosynthesis a corresponding amino linker at the 5'-end, within the sequence or at the 3 'end is installed. The amino-oligo is purified by HPLC twice (TritylOn / troff). Subsequently, a dye / hapten-NHS-ester is specifically coupled and another HPLC separated from unlabeled oligonucleotide and excess dye / hapten. There aminolinker that allow the functional group at the 5'-end (C6 amino linker), within the sequence (internal) at each dA, dG, dC, T (aminolinker-dA / dG / dC / T) or Ru, or at the 3'-end (amino-linker CPG) to position.
- Many also diastereopure dyes available.
- High flexibility, dye can be introduced at any point of the oligos.
- High quality of products by triple HPLC and gentle conditions.
- Low proportion of unlabeled sequences
- Little shorter, labeled oligonucleotides
- Time consuming by prolonged labeling reaction and triple HPLC purification
- with heavily intercalating compounds such as TAMRA, the complete removal of excess dye only by PAGE-purification is possible
We recommend that for reasons of quality individual marks always perform with the NHS-ester method via amino linker.
When oligosynthesis a corresponding thiol linker is incorporated at the 5'-end or the 3'-end. The thiol-oligo is purified by HPLC twice or in combination with amino linker by triple HPLC (TritylOn / troff). Subsequently, a dye / hapten-maleimide is specifically coupled and another HPLC separates not labeled oligonucleotide and excess dye / hapten.
- High quality of products by triple HPLC and gentle conditions
- Low proportion of non-labeled sequences
- Little shorter, labeled oligonucleotides
Disadvantages as compared to the NHS-ester method
- Only the company ATTO-TEC and Dyomix provide your complete range of products as maleimides. Other manufacturers have only a few standard colors available.
- Less flexibility, since there are no components, with the thiol-function, and thus of the dye within the sequence can be positioned.
- Poorer yield in the labeling reaction, resulting in a dye-dependent on the used, more or less results in poorer quality.
Because of these drawbacks we are maleimides only with double marks when no amidite is available as a 1 component or a free amine function is needed, for example for immobilization and the dye is to stand at the other end. The selectivity of maleimide to react with thiol groups, is in the presence of a free Aminolinkers not one hundred percent, but not the desired compounds can often be well separated
Doubly labeled oligonucleotides are required for diagnostic formats such as TaqMan ™ and molecular beacons and FRET for structural studies via. For the selective labeling of an oligonucleotide with different dyes, we offer 3 methods:
- Both fluorophores are introduced as amidite or CPG blocks.
- The fluorophore is introduced as an amidite / CPG-derivative and the other via aminolinker / dye NHS ester.
- The first fluorophore is introduced via thiol / maleimide and the second via aminolinker / NHS ester. This option requires a fourfold HPLC purification and the yield is only about half as large as compared with the first two variants.
Dye / hapten amidites, mountable at the 5'-end
Biotin, BHQ 1, BHQ 2, Cy3 ™, Cy3.5 ™, Cy5 ™, Cy5.5 ™, Dabcyl, DNP-TEG, Dy 780, Eclipse ™ Quencher, fluorescein, HEX, Redmond Red ™, TET and Yakima Yellow ™
Dye / hapten dT amidites, mountable within the sequence
Biotin-dT, BHQ 1-dT, BHQ 2-dT dabcyl dT and fluorescein-dT
Dye / hapten-CPG, mountable at the 3'-end
Biotin, BHQ 1, BHQ 2, BHQ 3, Dabcyl, DNP-TEG, Eclipse ™ Quencher, fluorescein, Redmond Red ™ and Yakima Yellow ™
Dye / hapten-NHS-ester, universally mountable
All Alexa Fluor ® derivatives, AMCA, all ATTO derivatives, biotin, all BODIPY ® derivatives all Cy ™ derivatives Dabcyl, dansyl, digoxigenin, DMACA, DNP-TEG all Dyomics derivatives, fluorescein, HEX, HYCO, Marina Blue ®, MECO, Naphtofluorescein, all Oregon Green ® derivatives, Pacific Blue ™, pyrene, all QSY ® derivatives, Rhodamine Green ™, Rhodamine Red ™, ROX, R6G, SECCA, TAMRA, TET, Texas Red ®
Dye / hapten-maleimides, mountable at the 5 'or 3' end
Many Alexa Dervate, all ATTO and Dyomix derivatives, biotin, BODIPY ® 499/508, BODIPY ® 577/618, BODIPY ® TMR, fluorescein, Oregon Green ® 488, Pacific Blue / Orange ™, pyrene, QSY ® 7/9, Rhodamine Red ™, TAMRA 5/6, Texas Red ®
The delivery volumes of dye-labeled products can not be guaranteed as they depend heavily on the quality of the reactive dye. As a rule, but 50 nmol / 200 nmol / 1000 nmol 4 nmol / 20 nmol / 80 nmol be achieved with the standards.In fourfold HPLC purified compounds (double labeling with maleimides and NHS-esters) are delivered in accordance with 3.5 nmol, 17.5 nmol and 70 nmol.
For additional PAGE purification are available 50/200/1000 nmol 02/10/50 nmol in the standards.
The price includes only the dye / hapten derivative. Base cost of couplings, amino linker and purification will be charged extra.
Highlights of PURIMEX dye labeled oligonucleotides
- 100% full-length with 3x HPLC / PAGE purified oligos
- Introduction of the dye at almost all positions possible
- Little sequences unlabeled
- Wide range of dyes
- Hardly contain free dye
- High intensity of the probes
- Low Background
In the following tables you will find the wide range of dyes and haptens offered by PURIMEX ordered by absorption wavelength, or in alphabetical order. Important information on the individual products, such as fluorescent properties, structure, mounting designs, applications and references can be found online under http://www.purimex.com