Protocol

Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

As a rule of thumb, oligonucleotides >25 nucleotides should be purified by polyacrylamide gel electrophoresis, as should oligonucleotides of any length that yield anomalous results. After electrophoresis, the oligonucleotide is eluted from the gel and concentrated by reversed-phase chromatography on Sep-Pak C₁₈ columns.

The method described here is a modification of a procedure that has been in use in Michael Smith's laboratory (University of British Columbia) for more than 20 years.

MATERIALS

n-Butanol

Acetonitrile

Use 10 ml of high-performance liquid chromatography (HPLC)-grade acetonitrile for each Sep-Pak column.

Ammonium acetate (10 M)

Use 2 ml of 10 mM ammonium acetate solution for each Sep-Pak column.

Crude preparation of synthetic oligonucleotide

Formamide loading buffer without tracking dyes

This gel-loading buffer consists of undiluted formamide without the usual tracking dyes (bromophenol blue and/or xylene cyanol FF); the dyes or contaminants in them may migrate at the same rate as the oligonucleotide and interfere with its detection by absorption of UV light (please see Step 14 of protocol). If desired, 0.2% orange G can be included in the gel-loading buffer. This dye migrates with the buffer front and does not interfere with detection of the oligonucleotide.

Formamide-tracking dye mixture

This solution is a 50:50 mixture of formamide and an aqueous solution of tracking dyes (0.05% xylene cyanol FF and 0.05% bromophenol blue). It is used as a size standard in wells adjacent to those containing the oligonucleotide preparation.

Methanol:H₂O solution

Combine 6 ml of methanol with 4 ml of filter-sterilized Milli-Q H₂O. Use 3 ml of methanol:H₂O solution for each Sep-Pak column.

Oligonucleotide elution buffer

TE (pH 8.0)

METHOD

1. In a sterile microcentrifuge tube, prepare a 10 µM solution of the crude oligonucleotide in sterile, filtered H₂O (Milli-Q or equivalent). Vortex the solution thoroughly.

The solution is often slightly cloudy because of the presence of insoluble benzamides generated during the synthesis of the oligonucleotide.
Synthetic oligonucleotides are usually supplied by the manufacturer as a lyophilized powder after removal of protecting groups used in the synthetic reactions (deprotection).

Before purifying an oligonucleotide, confirm that the deprotection reaction has been carried out. If the oligonucleotide is supplied in NH₄OH, transfer 0.5-1.0-ml aliquots to 1.5-ml microcentrifuge tubes and evaporate the NH₄OH to dryness on a centrifugal evaporator (Savant SpeedVac or its equivalent) at room temperature.

When opening a tube of crude oligonucleotide for the first time, vent the tube by opening it slowly to allow ammonia gas to escape (preferably into a chemical fume hood). This reduces the chance of spraying the oligonucleotide around the room.

2. Centrifuge the tube at maximum speed for 5 minutes at room temperature in a microcentrifuge. Transfer the supernatant to a fresh, sterile microcentrifuge tube.

3. Extract the solution three times in succession with 400 µl of n-butanol. Discard the upper (organic) phase after each extraction.

4. Evaporate the solution to dryness in a centrifugal evaporator (Savant SpeedVac or its equivalent). The tube should contain a yellowish pellet and a creamy-white powder.

5. Dissolve the pellet and powder in 200 µl of sterile filtered H₂O (Milli-Q or equivalent).

6. Estimate the amount of oligonucleotide in the preparation as follows: Add 1 µl of the solution to 1 ml of H₂O. Mix the solution well and read the OD₂₆₀. Calculate the oligonucleotide concentration.

Amounts of oligonucleotides are often described in OD units. One OD corresponds to the amount of oligonucleotide in a 1-ml volume that results in an optical density of 1 in a 1-cm path-length cuvette.

Calculate the millimolar extinction coefficient of the oligonucleotide () from the following equation:

= A(15.2) + G(12.01) + C(7.05) + T(8.4)

where A, G, C, and T are the number of times each nucleotide is represented in the sequence of the oligonucleotide. The numbers in parentheses are the molar extinction coefficients for each deoxynucleotide at pH 8.0.

For example, a 19-mer containing 5 dA residues, 4 dG residues, 4 dC residues, and 6 dT residues would have a millimolar extinction coefficient of

(5 x 15.2) + (4 x 12.01) + (4 x 7.05) + (6 x 8.4) = 202.64 mM^-1cm^-1

Calculate the concentration (c) of the undiluted solution of oligonucleotide from the following equation:

c = (OD₂₆₀)(1000)/

7. Pour a denaturing polyacrylamide gel (as described in Preparation of Denaturing Polyacrylamide Gels) of the appropriate concentration (see table). The loading slots in the gel should be approx. 1 cm in length.
Range of Resolution of Gels Containing Different Concentrations of Acrylamide

Acrylamide Size of Oligonucleotides (%) (in Bases) 20-30 2-8 15-20 8-25 13-15 15-35 10-13 35-45 8-10 45-70 6-8 70-300

8. Run the gel at constant wattage (50-70 W) for approx. 45 minutes or until the temperature of the gel reaches 45-50°C (Loading and Running DNA Sequencing Gels). Turn off the power supply and disconnect the electrodes.

Prerunning the gel in this way causes ammonium persulfate to migrate from the wells and, more importantly, warms the gel to a temperature optimal for electrophoresis of DNA.

9. Without delay, load approx. 2 OD₂₆₀ units of oligonucleotide (in a volume of 10 µl or less for maximum resolution) onto one or more slots of the gel as follows:

Add an equal volume of formamide loading buffer lacking dyes to the oligonucleotide solution. Mix the reagents well by vortexing, and then heat the mixture to 55°C for 5 minutes to disrupt secondary structure.

Flush out the urea from the wells with 1x TBE.

Load the heated oligomer into the slots. Load 5 µl of formamide-tracking dye mixture into an unused slot.

For further details on loading polyacrylamide gels, please see Loading and Running DNA Sequencing Gels.

10. Run the gel at 1500 V until the oligonucleotide has migrated approximately two thirds of the length of the gel.

The position of the oligonucleotide may be estimated from the positions of the tracking dyes as detailed in the table. Note that a synthetic oligonucleotide carrying a hydroxyl residue at its 5' terminus migrates more slowly through a denaturing polyacrylamide gel than does a phosphorylated oligonucleotide of equivalent length. Furthermore, the electrophoretic mobility of an oligonucleotide is dependent on its base composition and sequence. Thus, there may not be an exact correspondence between the predicted and observed positions of the oligonucleotide in the polyacrylamide gel.

Approximate lengths of Oligonucleotides Comigrating with Tracking Dyes

Polyacrylamide Xylene Cyanol FF Bromophenol Blue (%) 20 22 6 15 30 9-10 12 40 approx. 15

11. Lay the gel mold flat on plastic-backed protective bench paper with the smaller (notched) plate uppermost. Allow the gel to cool to <37°C before proceeding.

12. Remove any remaining pieces of electrical tape. Use a spacer or a plate-separating tool to slowly and gently pry apart the plates of the mold. The gel should remain attached to the longer (nonsiliconized) glass plate.

If the gel adheres to both plates, replace the partially dislodged, smaller or notched plate back on the gel, invert the plates, and try again.

13. Place a piece of Saran Wrap on the gel, turn the glass plate over, and transfer the gel to the Saran Wrap. Place a piece of Parafilm or a fluorescent thin-layer chromatographic plate under the gel where the oligonucleotide is predicted to be.

14. Use a hand-held UV lamp to examine the gel by illumination from above at 260 nm.

The DNA in the gel absorbs the UV radiation and appears as dark blue bands against a uniform fluorescent background contributed by the Parafilm or chromatographic plate. If the DNA is difficult to visualize, take the gel into a darkened room and illuminate it with the hand-held UV lamp.

15. Recover the desired oligonucleotide, which should be the slowest-migrating band (i.e., closest to the top of the gel), by excising each DNA band with a sharp, clean scalpel or razor blade. Avoid taking UV-absorbing material smaller in length than the desired oligonucleotide.

16. Transfer the gel slices to three or four microcentrifuge tubes. Add 1 ml of oligonucleotide elution buffer to each tube. Crush the slices with a disposable pipette tip, using a circular motion and pressing the fragments of gel against the sides of the tubes. Seal the tubes well. Incubate the tubes for 12 hours at 37°C in a shaker incubator.

17. Centrifuge the tubes at maximum speed for 5 minutes at room temperature in a microcentrifuge. Pool the supernatants, transfer them to a 5-cc disposable syringe, and pass them through a Millex HV filter. Collect the effluent in a 15-ml polypropylene tube.

18. Prepare a Sep-Pak C₁₈ reversed-phase column as follows:

Attach the barrel of a disposable 10-cc polypropylene syringe to the longer end of a Sep-Pak C₁₈ classic column.

Add 10 ml of acetonitrile to the barrel and slowly push it through the column with the plunger of the syringe.

Remove the syringe from the Sep-Pak column and then take the plunger out of the barrel. This prevents air being pulled back into the column. Reattach the barrel to the column.

Add 10 ml of sterile filtered H₂O (Milli-Q or equivalent) to the barrel and slowly push it through the column with the plunger. Repeat Step iii.

Add 2 ml of 10 mM ammonium acetate to the barrel and push it slowly through the column. Again remove the syringe, remove the barrel, and reattach the barrel to the column. The column is now ready for use.

19. Add the solution containing the gel-purified oligonucleotide (from Step 17) to the barrel and slowly push it through the column with the plunger. Collect the effluent in a sterile 50-ml polypropylene tube. Repeat Step 18c.

20. Add 10 ml of H₂O to the barrel and push it slowly through the column with the plunger. Repeat this wash step twice more.

21. Elute the bound oligonucleotide from the Sep-Pak column with three aliquots of 1 ml of methanol:H₂O solution. Repeat Step 18c after each elution. Collect each effluent in a separate microcentrifuge tube. Read the OD₂₆₀ of the solution in each of the three microcentrifuge tubes, using the methanol:H₂O solution as a blank. More than 90% of the oligonucleotide applied to the column should elute in the first fraction.

22. Evaporate the solution containing the oligonucleotide to dryness in a centrifugal evaporator.

23. Dissolve the oligonucleotide in a total volume of 200 µl of H₂O or TE (pH 8.0).

24. Transfer 5 µl of the solution to a cuvette containing 995 µl of H₂O. Mix the contents of the cuvette, and read the OD₂₆₀ of the diluted sample. Calculate the amount of oligonucleotide present in the total solution (Step 23) as described in Step 6 of this protocol.

REFERENCES

1. Atkinson, T. and Smith, M. 1984. Solid-phase synthesis of oligodeoxyribonucleotides by the phosphite-triester method. Oligonucleotide synthesis: A practical approach 2006: 35–82.