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Oligo Synthesis

Oligo Synthesis : CEPs

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Etheno-dA-CE Phosphoramidite

Etheno-dA-CE Phosphoramidite

Glen Research

Catalogue No.DescriptionPack SizePriceQty
10-1006-02Etheno-dA-CE Phosphoramidite 0.25g £300.00 Quantity Add to Order
10-1006-90Etheno-dA-CE Phosphoramidite 100µmoles £114.00 Quantity Add to Order

Description

Etheno-dA-CE Phosphoramidite

STructure

Catalog Number: 10-1006-xx

Description: Etheno-dA-CE Phosphoramidite

5'-Dimethoxytrityl-etheno-deoxyAdenosine3'-[(2-cyanoethyl)-
(N,N-diisopropyl)]-phosphoramidite
Formula: C42H48N7O6P M.W.: 777.86 F.W.: 337.23
Diluent: Anhydrous Acetonitrile
Coupling: No changes needed from standard method recommended by synthesizer manufacturer. The use of UltraMILD monomers are preferred. (Catalog Numbers: dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx). To avoid any exchange of the iPr-Pac group on the dG with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx).
Deprotection: If UltraMILD reagents were used, deprotect in 0.05M Potassium Carbonate in Methanol for 4 hours at Room Temperature OR for 2 hours at hours in 30% Ammonium Hydroxide. If standard bases were used, deprotection in Ammonium Hydroxide at Room Temperature for 24-36 hours will give acceptable yields.
Storage: Refrigerated storage, maximum of 2-8°C, dry
Stability in Solution: 1-2days

FLUORESCENT NUCLEOSIDES

Etheno-dA is a fluorescent nucleoside which is especially useful in observing the transition between DNA structural types. It is quite base labile and should be deprotected with ammonium hydroxide at room temperature for 24 hours. Alternatively, UltraMild chemistry can be used. 2-Aminopurine and AP-dC (G-Clamp) are also useful fluorescent nucleosides.

Pyrrolo-dC is a fluorescent deoxycytidine analog that is an ideal probe of DNA structure and dynamics. It base-pairs as a normal dC nucleotide. An oligo fully substituted with pyrrolo-dC has the same Tm as the control dC oligo with the same specificity for dG. Its small size does not perturb the structure of the DNA helix and it is well tolerated by a number of DNA and RNA polymerases. It is highly fluorescent and its excitation and emission are well to the red of most fluorescent nucleotide analogs, which eliminates or reduces background fluorescence from proteins. Pyrrolo-dCTP has potential uses in biological assay development.

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Protocols

Material Safety Data Sheet

Glen Report 6.1: ETHENO-DA AND ETHENO-A* - FLUORESCENT MONOMERS

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References

QUESTION: What are the relative extinction coefficients of 5'-Fluorescein, Hex and Tet etc.. at 260 nm and their Lambda max?

RESPONSE:Please see http://www.glenresearch.com/Technical/Extinctions.html

REFERENCE(S):
Oligonucleotide Properties Calculator; http://www.basic.northwestern.edu/biotools/oligocalc.html

QUESTION: What are the relative extinction coefficients of various dyes?

RESPONSE:Please see http://www.glenres.com/Technical/Extinctions.html#dyes

QUESTION: Does AMA or methylamine cause any degradation to fluorescein or fluorescein-type dyes such as FAM or FITC?

RESPONSE:Response: While AMA (Ammonium hydroxide/40% Methylamine 1:1 v/v) is considered compatible with fluorescein, the use of methylamine when deprotecting a Fluorescein-labeled oligo does lead to a small amount of degradation, which is characterized by a the appearance of a late-eluting peak by RP HPLC that shows no visible fluorescein absorbance. With standard deprotection conditions (AMA 10 minutes at 65 C) the amount of degradation is approximately 5%. The impurity is not detected with AMA at RT for 2 hours.

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Notes

FREQUENTLY ASKED TECHNICAL QUESTION

QUESTION: Can Etheno-dA be substituted for dA in a sequencing or PCR primer . What is the best method for cleavage/deprotection of oligos containing Etheno-dA. What are the excitation/emmision wavelengths for oligos containing etheno-dA.?

RESPONSE:Due to the structure of the 1, N6 etheno structure etheno dA will not base-pair with T or dU. For this reason oligonucleotides with etheno-dA in the middle or 3'-end will not act as PCR or sequencing primers. However if located at or near the 5''-end etheno-dA can be incorporated one or more times and still act as an effective sequencing or PCR primer (1).

Etheno-dA is somewhat labile to NH4OH so it is best to use base labile protecting groups such as Pac dA, isopropyl-Pac-dG and Ac-dC and either deprotect with K2CO3:MeOH, 4 hr. at RT. or NH4OH 4 hr. at RT.. If using standard protecting groups use mild deprotection conditions (0.4 M Methanolic NaOH; MeOH:H2O, 4:1) 17 hr. at room temperature.


 Excitation Emission
Ribo etheno-A 345 nm 455 nm
Oligo-etheno-dA 270 nm 410 nm

300 nm 410 nm

REFERENCE(S):
1. Srivastava, S.C., et.al., (1994), Nucleic Acids Research, 22, 1296-1304.

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Applications & Benefits

EXTINCTION DATA

Item Nucleoside λMax-1 Emax-1 λMax-2 Emax-2 E260


(nm) (ml/µmole) nm (ml/µmole) (ml/µmole)
10-1006 Etheno-dA 295 3.4 274 5.9 4.7

 

DILUTION/COUPLING DATA

The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.

ABI 392/394
Cat.No. Pack
Size		 Grams/
Pack		 0.1M Dil.
(mL)		 LV40 LV200 40nm 0.2µm 1µm 10µm
Approximate Number of Additions
10-1006-02 0.25grams .25grams 3.21 93.67 56.2 35.13 25.55 18.73 4.68
10-1006-90 100µmoles .078grams 1 20 12 7.5 5.45 4 1
Expedite
Cat.No. Pack
Size		 Grams/
Pack		 Dilution
(mL)		 Molarity 50nm 0.2µm 1µm 15µm
Approximate Number of Additions
10-1006-02 0.25grams .25grams 4.8 .07 89.6 56 40.73 5.6
10-1006-90 100µmoles .078grams 1.5 .07 23.6 14.75 10.73 1.48
Beckman
Cat.No. Pack
Size		 Grams/
Pack		 Dilution
(mL)		 Molarity 30nm 200nm 1000nm

Approximate Number of Additions
10-1006-02 0.25grams .25grams 4.8 .07 91.2 57 41.45

10-1006-90 100µmoles .078grams 1.5 .07 25.2 15.75 11.45

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Related products

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