A,C,G,T mix
A260/A280 Ratio
ABI 392/394: Minor Base Bottle Change
AMA Reagent
Analysis of Short RNA
Biotin Labelling
Biotin: Absorbance
Biotin: How to Detect Presence
Bottle Pressure
Crosslinking: 4-Thio Pyrimidines
Crosslinking: Br-dU, I-dU
Crosslinking: Psoralen
deoxyInosine: Degenerate Base
Deprotection: 8-oxo-dG and AMA
Deprotection: Thio-modified Oligos
Desalting -  Gel Filtration vs Reverse Phase
Dye Quencher Plot
Ethidium BromideStaining
Extinction Coefficient of Oligos
Extinction Coefficient of Oligos
Extinction Coefficients
Extinction Coefficients: Dyes
Extinction Coefficients: Fluoresceins
Fluorescein Degradation
Glen Gel-Pak: Exclusion cutoff length
Glen-Pak DNA and RNA Cartridges
Glen-Pak vs Poly-Pak?
H-Phosphonate Solutions
MALDI Analysis
Nonaqueous Oxidation using TBHP
Oligonucleotide-Peptide Conjugates
Phosphorylation: 5'-Modified Oligos
Polymeric Support
Priming Tips
Psoralen Labelling
RNA Supports for DNA Modification
Storage of Oligonucleotides
Synthesis of Long Oligos
Thiol Modifier C6
Ultra-Fast Deprotection
Universal Support for RNA Synthesis



QUESTION: Glen Research offers a selection of 5’-Amino-Modifiers but which one is appropriate for which application?


QUESTION: Your ACGT mix is 10-1023. How is this calculated?

RESPONSE:This mix is prepared on the basis of an equimolar mix. It is therefore corrected for molecular weight but NOT for phosphoramidite reactivity. If the customer knows the ratios they need, then we will make that as a SPECIAL.

QUESTION: Does anyone regularly measure the 260/280 for their oligos and what kind of numbers do you get? (From the ABRF e-mail network)

RESPONSE:1. We used to measure 260/280 routinely, but rarely bother now for a couple of reasons. First, the A260 value for oligos depends very much on the base composition since the extinction coefficients for G and A are much higher than those for T and C. We found that we got 260/280 ratios ranging from1.4 to 2.2 even on highly purified oligos, so the ratio was not a very good measure of purity without going through the calculations for each oligo. On long DNA strands the purines and pyrimidines tend to average out, so one can expect a more stable average of 1.8.

2. We do not measure such ratios. My feeling is that they are not very useful unless the DNA is REALLY filthy. See the following reference:

Glasel, J.A. (1994);Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. BioTechniques. 18(1): p. 62-63.

3. I'll answer your question with a question. If you are working with synthetic oligos, what is the advantage of determining the 260/280 ratio?

4. We use them to satisfy customer curiosity. A customer will call when his oligo gives a 1.3 ratio. Having expected a 1.8 ratio, he assumes the oligo is bad. It depends on the base composition. We ran poly dA, dG, dC, & dT oligos and found the average 260/280 ratios: dA=2.5, dG=1.85, dC=1.15, and dT=1.4 for nonpurified oligos. So for a 20mer with 5 A's, G's, C's, & T's, the expected ratio is 1.725. If that 20 mer has A=1, G=2, C=11, T=8, then the ratio =1.5. For our nonpurified oligos, this is accurate. For the purified primers, the ratios are slightly lower and I would welcome an explanation for this. And after saying all that, a higher C composition seems to lower the ratio more than expected, so the 1.3 ratio is very likely. If the expected ratio is 2.0 and we get a 1.3, then we would not ship the oligo. I don't think that it is an indicator of purity, but it is something that an investigator will look at, for whatever reason, so we need to have an answer.

1. Lawrence Washington, lwashing@bio.indiana.edu
2. COREY.LEVENSON@roche.com
3. Russ Lehrman, rlehrman@nexagen.com
4. dna core, uc4dna@UCBEH.SAN.UC.EDU

QUESTION: What is a good procedure to load minor bases on an ABI 392/394 synthesizer with a minimum loss of amidite in the priming step?

RESPONSE:When minor base amidites are loaded on an ABI 392/394 synthesizer using the bottle change procedure ~ 400 µl of amidite solution are consumed in the prime step. To minimize this loss a manual procedure can be used to load minor bases with a minimal loss of amidite (~ 200 µl). This procedure can be used in the manual control mode on the ABI synthesizer using timed delivery of the reagents to change minor bases.

1. Transfer ~ 3.0 ml anhydrous ACN to a clean amidite bottle.

2. Remove old amidite bottle from the "5" position. Wipe line and replace with amidite bottle containing ACN.

3. Rinse bottle 5 line with ACN and Argon.

FunctionFunction TypeTime (sec)
1Block Flush5

6418 to waste10

545 to waste30

6418 to waste10

7418 to 510

10Flush to 510

4. Place new amidite bottle on position 5

a) Remove rinse bottle

b) Wipe line

c) Replace with new amidite bottle

FunctionFunction TypeTime (sec)

10Flush to 510

101Phos. Prep15

545 to waste1 (use timed delivery)

6418 to waste10

1Block flush10

5. Start synthesis. Do not use begin cycle.

QUESTION: Can the AMA reagent be made up and stored? for how long? in what kind of container?

RESPONSE:Yes. Store tightly sealed in a glass bottle in the refrigerator. The reagent does not decompose but routine opening will lose some of the volatile bases. Dispose properly after 4-6 weeks.

Candy Lee, Dartmouth

QUESTION: What is the extinction coefficient of Amino-Modifier C6 dT and C2 dT?

RESPONSE:The extinction coefficient of DNA at 260nm is around 10,000/mole or 10/µmole. The extinction coefficient of Amino-Modifier C6 dT and C2 dT is around 8.7. The synthesis of these monomers does not pass through the deoxynucleoside and this figure is estimated by comparison to dT-CE Phosphoramidite.

Note: dA=15.4, dC=7.4, dG=11.5, T=8.7

QUESTION: I'd like to know which columns and mobile phase systems are best for resolution of rna's in the 7-12 mer range, on an analytical scale. I've seen reference to a Dionex column PN 43010, 250 mm x 4 mm--but have had no luck in Dionex calling me back! I have reverse phase C4 and C18
columns if that would be adequate. This will be an assay for an in vitro system, not for purification of synthetic oligomers.

Deb McMillen, Institute of Molecular Biology, University of Oregon (From the ABRF e-mail network)


1. The ZORBAX Oligo column is a mixed-mode (reversed phase/ion exchange) column that will work in this range. Typical mobile phase is A: 20:80 Acetonitrile:20mM sodium phosphate, pH 7.0, B: 20:80 acetonitrile:20mM sodium phosphate pH 7.0 + 0.8M NaCl. Part number for the 6.2x80mm column is 820940-901. This column is available in the US from Mac-Mod Analytical at 800-441-7508. The column is silica-based and originally processed at very high temperature that should easily denature RNases, but does not undergo any RNase-free QC testing. Thus, you should wash the column with a gradient of 5-95% acetonitrile up over 20 minutes and back down in 10 minutes before using.

2. At the recent Western Biotech Conference in San Diego, Ravi Vinayak (Applied Biosystems) described an anion-exchange HPLC method for anaylsis of RNA. He used a Dionex Nucleo-PAC PA-100 column at 1 ml/min running at 50-75 degrees C. Buffer A was 10% acetonitrile in 20 mM lithium perchlorate + 20 mM sodium

acetate (pH 6.5) and buffer B was 10% acetonitrile in 600 mM lithium perchlorate + 20 mM sodium acetate. The gradient was 0-50% B over 40 minutes. Fractions containing RNA product were treated with 3-4 volumes of 1-propanol and chilled at -20 for afew hours to precipitate the nucleic acid (the lithium salts stay in solution).

3. Our experience is that ion exchangemore efficiently resolves low molecular weight oligos (including RNA) than reverse phase columns. My experience has been with Nucleopak PA-100 (Dionex) and GenPakFax (Waters).

Both work well in our hands.

4. The following gradient can be used successfully to analyze RNA oligomers on the Dionex NucleoPac columns.


NucleoPac PA-100 (Dionex) column Cartridge: 250 x 4 mm (analytical 250 x 9 mm (semi-preparative)

Gradient system 1:

Start Time (Min)%B at start time


Mobile phase :Solvent A: 10 mM NaClO4/H2O
Solvent B: 300 mM NaClO4/H2O (pH 8.0-8.5)
Flow Rate :1.0 mL/min
Gradient system 2:
Start Time (Min)%B at start time
Mobile phase:Solvent A: 20 mM LiClO4 + 20 mM NaOAc in H2O: CH3CN (9:1)
(pH 6.5 with dil. AcOH)
Solvent B:

600 mM LiClO4 + 20 mM NaOAc in H2O: CH3CN (9:1)

(pH 6.5 with dil. AcOH)
Flow Rate :

1.0 mL/min

1. Stephen W. Coates, swcoates@zorbax.com
2. COREY.LEVENSON@roche.com
3. Russ Lehrman, rlehrman@nexagen.com
4. Ravi Vinayak, vinayars@ccmail.apldbio.com

QUESTION: Do you have a biotin phosphoramidite containing a disulfide linker which can be cleaved later with DTT to release the DNA from a streptavidin support?

RESPONSE:No. However, this can be produced on the synthesizer by adding to the 5'- terminus first 5'-thiol-modifier C6 S-S (10-1936) followed by BioTEG phosphoramidite (10-1955). This should generate a biotinylated primer with a long spacer arm containing the disulfide linkage which can be cleaved later with DTT.

1. If Biotin-dT is inserted at the 5'-terminus, will it affect the function of kinase?
2. Does Biotin-dT affect hybridization?

RESPONSE:1. Biotin dT will not affect the ability of kinase to phosphorylate at the 5'-terminus.

2. The presence of Biotin-dT within an oligonucleotide does not affect hybridization relative to the same oligo containing dT(1). The biotin resides in the major groove of the formed double-stranded DNA and is readily available for binding to avidin or streptavidin.

(1) J. Telser, K.A. Cruickshank, L.E. Morrison, and T.L. Netzel, J. Am. Chem. Soc., 1989, 111, 6966-6976.

QUESTION: What absorbance does biotin have at 260nm? The HPLC trace shows absorbance at 254nm?

RESPONSE:Biotin is transparent at 260nm. The UV detector in the HPLC trace of biotin phosphoramidites is monitoring the absorption of the DMT group on the spacer arm.

QUESTION: How can I tell if the biotinylated oligonucleotide I have made really does contain biotin?

RESPONSE:A colorimetric assay for biotin can be quite effective. The color results from the reaction of biotin with p-dimethylaminocinnamaldehyde in the presence of sulfuric acid.

1. Spot 0.2 A260 units of biotinylated oligonucleotide on a silica gel TLC plate.

2. Dry the plate.]

3. Spray with a solution of 2% p-dimethylaminocinnamaldehyde (Sigma), 2% conc. sulfuric acid in ethanol.

4. Heat the plate and the presence of biotin will be indicated by the formation of a pink spot.

Since the intensity of the biotin spot is quite low, it is prudent to compare with an unlabelled oligonucleotide similarly treated.

QUESTION: What is the pressure ratings on our bottles for solvents?

RESPONSE:In general, any bottle designed for transportation by needs to be tested to 250kPa (40psi). All of our solvent bottles have been tested to 40psi. Recommended pressures for use should be 5-10psi. As with any glass bottle, a chip may weaken the glass and cause it to burst at lower pressures so precautions must be used to prevent injury when pressurising any glass bottle.

QUESTION: What are the options for chemical phosphorylation?


QUESTION: Can 4-thiodU be used for crosslinking and how is it prepared?

RESPONSE:4-thioU is efficiently activated for crosslinking by exposure to long-wavelength UV light for up to 10 minutes. Crosslinks are formed with RNA and proteins.

Oligonucleotides containing 4-thiodU (10-1051) and 4-thioT (10-1033) can be produced from the triazole modified phosphoramidites using the convertible nucleoside strategy (1,2). However, we now offer the nuclesides 4-thio-dT (10-1034), 4-thio-dU (10-1052), 2-thio-dT (10-1036), and 4-thio-U (10-3052) for direct incorporation into oligonucleotides and the convertible monomers have been discontinued. A further enhancement of this strategy used 4-thioxU as an intermediated in the preparation of thiocarbonyl crosslinkers (3).

(1) Y.Z. Xu, Q. Zheng, and P.F. Swann, J. Org. Chem., 1992, 57, 3841.
(2) The Glen Report, 1993, 6.1, 1, and references cited therein.
(3) R.S. Coleman and J.M. Siedlecki, Journal of the American Chemical Society, 1992, 114, 9229-9230.

QUESTION: What methods are available for crosslinking DNA with DNA, RNA, proteins?

RESPONSE:The most widely used method seems to be using Br-dU and, more recently, I-dU (1). The substitution of photoreactive Br and I for the 5-Me group of thymidine is attractive since their radii are similar to that of the methyl group. These modifications do not significantly affect the binding of oligonucleotides with proteins. Br-dU is irradiated at 308nm and crosslinking is typically not greater than 40%. Crosslinking of I-dU at 308nm is higher but is optimal at 325nm. Laser excitation is preferred.

After the synthesis of oligonucleotides containing Br-dU or I-dU, care must be taken to avoid loss of the halogens. Even though both modifications are quite stable to ammonium hydroxide, it is sensible to play safe and carry out the deprotection at room temperature for 24 hours. Even better, use the UltraMild monomers and deprotect with potassium carbonate in methanol at room temeperature. The oligonucleotide products should be protected from light - plastic tubes and amber vials are safe. Gel electrophoresis should be carried out protected from light.

(1) M.C. Willis, B.J. Hicke, O.C. Uhlenbeck, T.R. Cech and T.H. Koch, Science, 1993, 262, 1255.

QUESTION: What is the sequence requirement for psoralen mediated cross-linking in double stranded DNA?

RESPONSE:Psoralens are a class of naturally occurring heterocyclic compounds which intercalates between bases in double-stranded or triple stranded DNA. Upon exposure to long wavelength light (350 nm) Psoralen forms covalent linkages to Thymidine (cyclobutane linkage). Psoralen is a bifunctional reagent and can form either a monoadduct, linking one adjacent thymidine on the same or complimentary strand, or a diadduct, linking adjacent thymidines on the same or complimentary strands. Diadducts formed between adjacent thymidines are photoreversable with short wavelength UV light (254 nm).

Type of Adduct

Optimal Sequence







350 nm




350 nm

254 nm

Note: For adducts between double-stranded DNA use Psoralen C210-1982.
For adducts between triple-stranded DNA use Psoralen C6 which has a longer linker arm 10-1983.

1. U. Pieles and U. Englisch, Nucleic Acids Res., 1989, 17, 285.

QUESTION: What is the base-pairing specificity of dI in an oligonucleotide?

RESPONSE:Deoxy-Inosine is often used as a degenerate base in an oligonucleotide probe or primer. This is possible since the structure allows it to base pair with all four bases in various wobble structures. However the base-pairing is not equivalent with each of the 4 naturally occuring bases. The overall preferential order of base-pairing is.

dI-dC > dI-dA > dI-dG = dI-dT

Martin,F.H. et.al., (1985), Nucleic Acids Res., 13, 8927-8938.
Case-Green,S. C., Southern, E.M., (1994), Nucleic Acids Res., 22, 131-136.

QUESTION: I have 8-oxo-dG in an oligo with a dye that is not compatible with deprotection in ammonium hydroxide. Can I deprotect an oligo containing 8-oxo-dG in AMA?

RESPONSE:Yes. 8-oxo-dG can be cleanly deprotected in AMA with 1 hour at 55 °C.

QUESTION: What is the best method to deprotected thio-modified oligos (10-1926)?

RESPONSE:The trityl group used to protect the thiol is not acid labile and therefore can not be removed on a DNA synthesizer using

the normal acid deprotection. Cleavage of the oligonucleotide from the support and removal of the base-protecting groups are carried out with ammonium hydroxide in the normal manner. If purification is desired, it should be done before removing the trityl group. The presence of the trityl group allows standard trityl-on reverse phase (RP) purification techniques to be used.

Final deblocking of the oligonucleotide involves cleavage of the trityl-sulfur bond. This is accomplished by oxidation with silver nitrate with the excess silver nitrate being precipitated with dithiothreitol (DTT). Excess DTT can be removed by extraction with ethyl acetate, by desalting or by ethanol precipitation.


1. Deprotect with ammonium hydroxide in the normal manner.

2. Purify the trityl containing oligonucleotide by HPLC or Poly-Pak cartridge.

3. Evaporate the product solution to dryness.

4. Suspend the product in 0.1M triethylammonium acetate (TEAA), pH6.5 at a concentration of ~100 A260 units/mL.

5. Add 0.15 volumes of 1M aqueous silver nitrate solution, mix thoroughly, and react at room temperature for 30 min.

6. Add 0.20 volumes of 1M aqueous DTT solution, mix thoroughly, and leave at room temperature for 5 minutes.

7. Centrifuge the suspension to remove the silver DTT complex. Remove the supernatant. Wash the precipitate with 1 volume of 0.1M TEAA. Centrifuge and combine the supernatant with the first volume.

8. Proceed directly to the conjugation reaction. (If desired, excess DTT can be removed by ethyl acetate extraction. The free thiol oligonucleotide must be stored under an inert atmosphere to avoid oxidative dimerization to the disulfide.)

QUESTION: Is desalting with Glen Gel-Pak cartridges preferable to desalting with Glen-Pak cartridges?

RESPONSE:In gel filtration, the mobile phase is an aqueous solution and the stationary phase is a porous resin, as in Glen Gel-Pak. The pores of the resin are sized such that they allow small molecules to enter the pores, yet exclude larger molecules from the pores. The small molecules, such as salts and hydrolyzed protecting groups, diffuse into the pores of the resin and move more slowly through the column. The larger molecules, such as DNA or proteins, are excluded from the pores and move quickly through the column. The end result is that the larger molecules elute first in the column void volume while the small molecules are still flowing through resin of the column.

Reverse phase cartridges contain a hydrophobic matrix, which in the case of Glen-Pak is a polymeric support.  When an aqueous solution containing an oligonucleotide along with small organic molecules and/or salts is introduced to a reverse phase cartridge, the oligonucleotide adsorbs to the packing while smaller molecules and salts are washed through.  The desalted oligonucleotide can then be eluted from the cartridge using aqueous acetonitrile.

Both column types are ideal for desalting oligonucleotides and clean up of conjugation reactions. Gel filtration allows you to elute the oligonucleotide in the buffer of your choice, for example, the conjugation buffer for the next reaction.  Glen-Pak cartridges allow simple evaporation or lyophilization of the final solvent.  Both types have their supporters, often with biologists preferring Glen Gel-Pak and chemists preferring Glen-Pak.



QUESTION: Dye Quencher Plot

RESPONSE:Dye Quencher Plot

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.


Ribo etheno-A345 nm455 nm
Oligo-etheno-dA 270 nm410 nm
300 nm410 nm

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

QUESTION: Why can't I see my phosphorothioate oligo when I stain the gel with ethidium bromide?

RESPONSE:It appears that the sulfur of the phosphorothioate linkage can quench the fluorescence of ethidium bromide. I know researchers who could see their phosphorothioate oligo by UV shadowing but when stained with EtBr, no bands were observed, while on the same gel, normal phosphodiester oligos stained perfectly.

QUESTION: How do you calculate the extinction coefficient of an oligo?

RESPONSE:Calculate extinction coefficient of an oligo by either summing up the extinction coefficients of the individual bases times their number of occurrences. Or use a formula that takes into account nearest neighbor effects. algorithm for this calculation can be found on the web (http://www.basic.northwestern.edu/biotools/oligocalc.htmll). Just type in the sequence and the program will calculate the concentration of a 1 A260/ml solution.

To calculate the MW of the aminomodified oligo just add 179.16 to the calculated MW of the unmodified oligo.

QUESTION: How do you calculate the extinction coefficient of an oligo?

RESPONSE:Calculate extinction coefficient of an oligo by either summing up the extinction coefficients of the individual bases times their number of occurrences. Or use a formula that takes into account nearest neighbor effects. algorithm for this calculation can be found on the web (http://www.basic.northwestern.edu/biotools/oligocalc.htmll). Just type in the sequence and the program will calculate the concentration of a 1 A260/ml solution.

To calculate the MW of the aminomodified oligo just add 179.16 to the calculated MW of the unmodified oligo.

QUESTION: What are the extinction coefficients for propynyl-dC and dU? Also Halogenated dC and dU derivatives? Others?

RESPONSE:See: Table of Extinction Coefficients

B. Froehler, Gilead
M. Powell
R. Somers

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


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

Oligonucleotide Properties Calculator; http://www.basic.northwestern.edu/biotools/oligocalc.html

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%

QUESTION: What are the options for 5' and 3' fluorescein labelling?


QUESTION: Questions regarding Glen Gel-Pak

RESPONSE:-The exclusion cutoff length for oligonucleotides for the Glen Gel-Paks is 10bp. Oligos longer than 10bp should be purified effectively from small molecules such as salts, dyes, labels, etc.

-Why does it matter which cap I remove first from the column? Removal of the white cap first will cause air to enter the matrix from the bottom as you remove the red cap. This may cause channeling and prevent the column from working as designed.

-What if I accidentally remove the white cap first and the red cap is still on the column? You can carefully use a needle or sharp pin to poke a small hole in the top red cap needle to vent the column as you slowly remove the cap.

QUESTION: Can I use the Glen-Pak DNA cartridge for my DMT-ON RNA purification or do I have to use the RNA Glen-Pak cartridge?

RESPONSE:You cannot use the Glen-Pak DNA cartridge for DMT-ON RNA purification, as the resins and required protocols are different.  That said; you may use the Glen-Pak DNA cartridge to desalt a DMT-Off RNA product as described in the Desalting of Oligonucleotides section of our Glen-Pak User guide.  


QUESTION: How do Glen-Pak Cartridges compare with Poly-Pak Cartridges?


Traditional oligo purification cartridges, including Poly-Pak, have the disadvantage that they require the crude oligo solution to be loaded in two passes. This is clearly unsuitable for the use of these cartridges in high throughput situations.  These cartridges also are restricted in the length of oligos they can purify - typically 50-60mer.  Because of the loading and wash cycles, they also yield only about half of the available full length oligo.  Glen-Pak cartridges, second generation products that have exquisite affinity for the DMT group, fix all of these problems and are available in a wide variety of formats.  In fact, the only situation where Poly-Pak cartridges are superior to Glen-Pak cartridges is their ability to use the Dye-ON protocol to purify oligos with 5’-fluorescein tags, like FAM, HEX or TET.



QUESTION: What are the concentrations of the solutions used on an ABI for H-phosphonate chemistry?

RESPONSE:1. Adamantantanecarbonyl chloride - 2% w/v in 5%pyridine/acetonitrile.

2. Isopropyl phosphite - 1% in acetonitrile/pyridine (50:50)

3. Oxidation: Following synthesis

Step 1: React support with 4% Iodine in THF/Pyridine/Water ( 8:1:1) ~2mL for 4minutes

Step 2: Remove solution from support

Step 3: React support with mixture of 4% Iodine in THF/Pyridine/Water ( 8:1:1) (~1mL) and TEA/H2O/THF (8:1:1) (~1mL) for 3 minutes

Step 4: Flush column with acetonitrile and blow dry support

QUESTION: Why does MALDI analysis of my oligos that contain one or more Fluorescein-dTs give an incorrect mass even though they give only a single, fluorescent band on a PAGE gel?

RESPONSE:It's an artifact of the MALDI analysis. The lasers used to ablate the MALDI matrix are generally between 308 and 355 nm, with the most frequently used laser line at 337 nm. When a laser hits a strong absorbance band of a molecule, often photochemistry starts to occur - which often leads to cleavage reactions. The Fluorescein-dT has strong UV absorbance in this region which appears to lead to a 135 m/z fragment is being blown off the molecule in a rather consistent manner. For instance, your oligos D1 and D2, which have 5 and 3 Flu-dTs respectively, the observed mass difference is -676 Da and -406 Da, which nicely fits the loss of a 135 mw fragment: 5 x 135 (675) and 3 x 135 (405). We haven't seen any papers that identify this 135 m/z fragment - but it's clear to me that that is what's occurring. Changing matrix matrix used may help. The matrix 2,4,6-trihydroxyacetophenone has been used successfully to analyze a fluorescein-labeled oligos[1], though the safest bet is to use Electrospray MS rather than MALDI for mass spec analysis.

1 Pieles et al., Nucleic Acids Res., Vol 21 (14) 3191-3196 (1993)

QUESTION: What is a possible alterternative oxidizing solution to I2 ?

RESPONSE:In situations where you want to avoid the use of iodine containing oxidizing solutions, such as when using 7-deaza-dG, or when you want to use a non aqueous oxidizing solution t-butyl hydroperoxide (TBHP) has been shown to work both for DNA (1) and RNA (2).


For DNA small scale: 1.1 M TBHP in Methylene Chloride, 0.8 min oxidation (1)

For large scale RNA synthesis: 37ml TBHP solution + 63ml Methylene Chloride (approx. 2M), 6 min oxidation (2)

Mix reagents fresh prior to use, oxidizing solution only stable for a few days on synthesizer.

1. Hayakawa, Y., et al., JACS, 1990, 112, 1691.
2. Sproat, B., et al., Nucleosides & Nucleotides, 1995, 14, 255.

QUESTION: What is the best method to make peptide-oligonucleotide conjugates?

RESPONSE:It would seem that the best method to make peptide-oligo conjugates would be to use Fmoc chemistry and synthesize the peptide off an oligo synthesized on amino-CPG. However, deprotection of peptides synthesized using Fmoc chemistry requires 50% TFA and t-boc synthesized peptides require HF both of which would severely damage if not completely hydrolyze the oligo.

The best and most straight foward method is to use a heterobifunctional crosslinking reagent to link a synthetic peptide, containing an N-terminal lysine, to a 5'-Thiol modified oligo or conversely a 5'-amino modified oligo to a cysteine containing peptide . A good crosslinking reagent is N-Maleimido-6-aminocaproyl- (2'-nitro,4'-sulfonic acid)-phenyl ester . Na + (mal-sac-HNSA) from Bachem Bioscience (cat. # Q-1615). Reaction of this crosslinker with an amino group releases the dianion phenolate, 1-hydroxy-2-nitro -4-benzene sulfonic acid a yellow chromophore. The chromophore allows both quantitation of the coupling reaction as well as act as an aid in monitoring the seperation of "activated peptide" from free crosslinking reagent using gel filtration.

Method A: Couple Peptide Amine To Oligo Thiol (Note peptide MW must be > 5,000 to be excluded from desalting column). This method best for oligo-enzyme conjugation.

Step 1: Synthesize a peptide with an N-terminal, or internal, lysine (The epsilon amino group is more reactive than an alpha amino group).

Step 2: Synthesize an oligonucleotide with a 5' Thiol group.

Step 3: React peptide with excess mal-sac-HNSA (pH 7.5 Sodium phosphate)

Step 4: Seperation of peptide-mal-sac conjugate from free crosslinker and buffer exchange (pH 6.0 Sodium phosphate) using a gel filtration column (Glen Gel-Pak™ or eq.). Note peptide must be large enough to seperate from the free linker which can be visualized as a yellow band. Do not collect yellow band with peptide.

Step 5: Activate thiol modified oligo, desalt and buffer exchange (pH 6 Sodium phosphate) on Glen Gel-Pak™ column.

Step 6: React acitvated peptide with Thiol modified oligo.

Step 7: Purify Peptide-Oligo conjugate by ion exchange chromatography on Nucleogen DEAE-500-10 or eq. Elution order: free peptide, peptide-oligo, free oligo.

Method B: Couple Oligo Amine To Peptide Cysteine (Note oligos > 15mers are excluded from desalting column). Use above procedure switching oligo for peptide.

Step 1: Synthesize a peptide with an N-terminal, or internal, cysteine

Step 2: Synthesize an oligonucleotide with a 5' amino modifier.

Step 3: Purify oligo Trityl-on by RP HPLC or cartridge.

Step 4: React oligo with excess mal-sac-HNSA (pH 7.5 Sodium phosphate)

Step 5: Seperation of oligo-mal-sac conjugate from free crosslinker and buffer exchange (pH 6 Sodium phosphate) using a gel filtration column (Glen Gel-Pak™ or eq.). Note oligo must be large enough to seperate from the free linker which can be visualized as a yellow band. Do not collect yellow band with oligo.

Step 6: Dissolve peptide in pH 6.0 Sodium phosphate buffer and react with activated oligo.

Step 7: Purify Peptide-Oligo conjugate by ion exchange chromatography on Nucleogen DEAE-500-10 or eq. Elution order: free peptide, peptide-oligo, free oligo.

QUESTION: Can oligonucleotides modified at the 5'-terminus with, for example, biotin be phosphorylated with kinase?

RESPONSE:Modification reagents based on a 1,2-diol, e.g., BioTEG, DNP phosphoramidites, or a 1,3-diol, e.g., fluorescein, biotin phosphoramidites, can be added several times at the 5'-terminus since they contain an alcohol group capable of further addition with phosphoramidites. Can this alcohol also be a substrate for T4 polynucleotide kinase for 32P labelling of these modified oligonucleotides? Surprisingly, the answer is yes. Teoule and coworkers have shown(1) that oligos labelled at the 5'-terminus are substrates for kinase. Interestingly, the oligos modified with reagents based on 1,2-diols are labelled to 50%, indicating that only one diastereomer is labelled, while those modified with 1,3-diol reagents are labelled to 100%.

(1) M.L. Fontanel, H. Bazin, and R. Teoule, Analytical Biochemistry, 1993, 214, 338-340.

QUESTION: What are the physical properties of the various black hole quenchers?


Quencher lmax (nm)E260 (L/mol.cm)Emax (L/mol.cm)
BHQ-0 4937,70034,000
BHQ-2 5798,00038,000
BHQ-367213,000 42,700

Glen Report 17.1

QUESTION: What is the Polymeric support used for our Oligo Affinity Support PS?

RESPONSE:The polymeric resin we use for our Oligo Affinity Support is Toyopearl AF-Carboxy 650M from TosoHaas. This is a macroporous polymethacrylate support with carboxylic acid function attached to the support through a linker. Amine containing ligands can be attached to the carboxylic acid via carbodiimide coupling. There are a number of other versions of the resin with other reactive groups that can be used for other coupling chemistries. Some of the physical characteristics of the resin are:

Particle size:40-90 µm
Pore size:1000 Å
Exclusion limit:1,000,000 +- 30% Daltons

QUESTION: What is the procedure for changing minor base bottles on a synthesizer?

RESPONSE:ABI 392/394: see ABIPriming.html

Expedite: see ExpeditePriming.html

See also: USAGE.html

QUESTION: Which should I use - psoralen C2 or psoralen C6?

RESPONSE:Psoralen C2 is designed to crosslink to a T residue adjacent to the 3'-terminus of the opposite strand of double stranded DNA. Psoralen C6 is intended to fulfill the same purpose but, with the longer spacer, crosslinks to the triple strand of triplex DNA.

Max excitation=330nm, observed at max 395nm

QUESTION: Are any changes required from standard DNA synthesis and deprotection?

RESPONSE:These supports (20-3303, 20-3315, 20-3324, 20-3330) can be used for oligodeoxynucleotide synthesis when a ribose functionality is desired at the 3'-terminus. Following periodate oxidation of the 1,2-diol, labelling or carrier molecules containing amino groups can be conjugated to the 3'-terminus. An example (1) is the use of poly-lysine as a carrier molecule.

These supports can be used for RNA synthesis, and our other RNA supports (now all containing 2'-acetyl protecting groups, which are completely base-labile) can also be used for DNA modification.

(1a) M. Lemaitre, B. Bayard, and B. Lebleu, Proc. Natl. Acad. Sci. USA, 1987, 84, 648-652.
(1b) M. Lemaitre, C. Bisbal, B. Bayard, and B. Lebleu, Nucleosides & Nucleotides, 1987, 6, 311-315.

QUESTION: What is the stability of deprotected oligonucleotides in NH4OH?

RESPONSE:Q. Is it OK to store deprotected oligos in NH4OH untill ready to use.

A. It's OK to store the oligo in NH4OH at -20°C especially if the DMT group has been retained for further purification. For DMT-OFF oligos its better If they are lyophilized and stored at -20°C. This is the most stable form for the oligo.

Q. What is the best way to store a crude oligo if no further purification is to be used.

A. For crude DMT off oligos it is better to do a quick EtOH ppt. before use. This will remove protecting groups and any other salts which might be present. After EtOH ppt. the oligo can again either be stored lyoplilized at -20°C or you can determine its concentration and store in solution at -20°C. A good buffer for storage is 10 mM Tris containing 1 mM EDTA to chelate any divalent cations which could activate any DNase which might be present.

QUESTION: How do the 1000Å and 2000Å supports compare in the synthesis of long oligos? Do you recommend any changes to the cycle for long oligos?

RESPONSE:In one comparison several years ago, a customer compared 1000Å, 2000Å and a low-loaded 500Å CPG for the synthesis of 200 and 400mers. Only in the case of the 2000Å CPG could the 200mer product be seen on a gel although the other supports did make product since it could be amplified by PCR. The 400mers could not be seen but could be amplified by PCR.

For the synthesis of long oligos, we recommend increasing the coupling wait step from 15 seconds to at least 30 seconds. I believe this is important later in the synthesis. Also, we recommend the use of DMAP in the Cap B solution or, if methylimidazole has to be used, increasing the capping wait step to 45 seconds. With incomplete capping, you are going to be making oligos contaminated with deletion mutations. I know DMAP has been accused of causing base modification of dG sites leading to fluorescent bands on gels but it is still the most effective capping activator. I also would recommend, if possible, an extended capping of the support (20 minutes) before the DMT is removed in the first cycle. The 1000Å and especially the 2000Å supports are very fragile and can be damaged in transit. This capping step deactivates any fresh surfaces caused by fractures. TWIST columns are assembled with 20µM frits to retain any fines coming from these friable supports.

J.S. Eadie and D.S. Davidson, Nucleic Acids Res., 1987, 15, 8333.

QUESTION: What is the "T" in the structure of thiol-modifier C6?

RESPONSE:"T" is short for trityl or triphenylmethyl. This group is not significantly acid labile and requires an oxidative cleavage with silver nitrate to remove it. It is also susceptible to oxidative cleavage with the iodine oxidizer and, for maximum yield, the iodine concentration in that oxidizer should be 0.02M, which is now virtually standard.

QUESTION: How does this work? What would happen if I used the AMA reagent with Bz protected dC?

RESPONSE:dC when protected as an amide linkage is susceptible to transamination or displacement of the amide with the deprotecting amine. Normally, the deprotecting amine is ammonia and displacement leads to the same product as hydrolysis. However, if methylamine is used for the deprotection step with a benzoyl protected dC, approximately 10% of the product is from the displacement reaction to give N-methyl-dC residues, while the remaining 90% is the desired product formed by hydrolysis. A more labile protecting group on dC is isobutyryl which is also commercially available. This group is displaced by methylamine to about 5% - an improvement but still a mutation. Ac-dC, on the other hand, is hydrolyzed virtually instantaneously by the AMA reagent and none of the slower displacement reaction gets a chance to occur.

M.P. Reddy, Beckman Instruments, personal communication.

QUESTION: Which universal support is best for RNA synthesis?

RESPONSE:Glen UnySupport is popular for RNA synthesis and deprotection of the oligo along with dephosphorylation of the universal linker at the 3’ terminus is carried out using Ammonium Hydroxide:MethylAmine (AMA) 1:1 for 1 hour at 65°C.  Our research has shown that these conditions do cause a small amount of degradation of the RNA oligo but our customers clearly are able to tolerate this low level of degradation.


The optimal universal support for RNA synthesis is our Universal Support III.  When using this support, the cleavage and dephosphorylation step is carried out using 2M anhydrous ammonia in methanol for 30 minutes at room temperature.  You can evaporate that solution to dryness and then add your favorite reagent for RNA deprotection.  Or simply add AMA to the solution and deprotect the oligonucleotide for 10 minutes at 65°C.