Purification of oligonucleotides using denaturingpolyacrylamide gel electrophoresis Jack D. Pollard, Jr. 4/12/98 Several methods exist for the purification of oligonucleotidesfollowing chemical synthesis. The advantages of purification ondenaturing polyacrylamide gels are speed, simplicity, and highresolution. Denaturing polyacrylamide gels can resolve oligonucleotidesfrom 2 to 300 bases, depending on the percentage of polyacrylamideused (seeTable 2.12.1This method is thus useful not only for isolating chemically synthesizeddeoxyribonucleotides but also small RNAs or other single-strandedoligonucleotides. After gel setup, samples are loaded onto a urea-baseddenaturing gel, separated by electrophoresis, and finally recoveredfrom the crushed gel slice. Materials 10x and 1x TBE buffer, pH 8 Prepare the sample 1. Follow the appropriate deprotection protocol toprepare the sample for electrophoresis. Be sure to lyophilize the sample to dryness. Thesamples will generally appear as a an off-white powder followingdeprotection and lyophilization. If a yellowish liquid or crustypellet remains, rather than an off-white powder, resuspend thepellet in 0.5 ml distilled water and add 1/10 volume 3.0 M sodiumacetate pH 5.2. Add 3 volumes of absolute ethanol to precipitateby chilling to -80 °C for approximately 20 minutes. Centrifugefor 10 minutes at 16000xg and 4°C. Decant and save the supernatant.Wash the pellet in 70 percent ethanol Lyophilize the pellet todryness. Prepare the gel 2. Assemble the gel casting apparatus. Gel spacer and casting systems have been developedto avoid leakage. Those which avoid sealing the gel with tapeare best, and recently, gel casting boots that lack bottom spacershave become available (GibcoBRL). Greasing the side /bottom spacersor pouring an agarose plug for the gel is not necessary if somecare is taken to ensure that the bottom of the plate assemblyis completely sealed. Clean the gel plates thoroughly by washingthem with warm soapy water followed by an ethanol:water rinse.However, if the plates are particularly dirty or if the completeremoval of any residual nucleic acids is required, the platesmay be soaked in an 0.1 M NaOH for 30 minutes prior to washing.If the gel is particularly thin (<1 mm), silanizing one orboth of the plates facilitates post-electrophoretic separationof the gel from the plate. 3. Prepare the gel solution (see Table 2.12.1 forappropriate acrylamide concentrations for resolving single strandedDNAs). For a denaturing acrylamide gel of 20 cm x 16 cm x 1.6mm, 60 ml of gel solution is sufficient, and it can be made bymixing the following: 25.2 g urea (final concentration of 7 M) 6 ml 10x TBE buffer desired amount of 40 % acrylamide/ 2 % bisacrylamideneeded for resolution water to a final total volume of 60 ml Pick a concentration of acrylamide that will allowthe single stranded nucleic acid to migrate approximately one-halfto three-fourths the way through the gel when the loading dyehas reached the bottom of the gel. This allows for good separationof non- and full-length products. Use a flask that has a wide mouth and a spoutfor pouring. Caution: Always wear gloves, safety glasses, anda surgical mask when working with acrylamide powder since it isa neurotoxin. Commercially prepared polyacrylamide solutions(National Diagnostics) are available and highly recommended sincethey have long shelf lives and do not involve massing the neurotoxicacrylamide powder. 4. Heat the mixture by immersing the flask in a 60°C hot water bath or under running tap water to speed thedissolution of the urea and acrylamide. Once most of the ureaand acrylamide have gone into solution, vigorously agitate thesolution for approximately 20 minutes with magnetic stirring toensure complete mixing. 5. Add 40 mlof TEMED and swirl the flask to insure thorough mixing. Immediatelyadd 300 mlof 10 % APS and mix thoroughly. POLYMERIZATION HAS BEGUN SO ALLSUCCEEDING STEPS MUST BE PERFORMED PROMPTLY. Pour the acrylamidebetween the gel plates and insert the comb. Clamp the comb inplace at the top of the gel to avoid separation of the gel fromthe plates as the acrylamide polymerizes. Allow the gel to polymerizefor approximately 30 minutes. For thick gels pour the acrylamide directly fromthe mixing flask, but for thinner ones, a syringe fitted withthe needle is useful. By pouring the gel slowly with a tilt 45_relative to the bench top and starting from one corner, bubblesmay be largely avoided. Also, polymerize the gel while it is lyingflat to avoid undesirable hydrostatic pressure on the gel bottom. TEMED may be stored indefinitely at 4°C,but the ability of APS to efficiently initiate the free radicalinduced acrylamide polymerization diminishes greatly over time.Make a new stock every month and store at 4°C. Caution: Be sure to wear safety glasses whilepouring the gel since splashing of the neurotoxic, unpolymerizedacrylamide is common.. Run the gel 6. After polymerization is complete, remove the comband any bottom spacers from the gel. Wash the gel plates freeof spilled acrylamide and be sure that the spacers are properlyseated and clean. 7. Fill the lower reservoir of the electrophoresistank with 1X TBE. Initially, place the gel into the lower tankat an angle to avoid air bubbles forming between the plates andthe gel bottom. Clamp the gel plates to the top of the electrophoresistank and fill the upper reservoir with 1X TBE so that the wellsare covered. A syringe with a bent needle may be used to removeair bubbles trapped under the gel that will disrupt the currentflow. 8. Use a DC power supply to prerun and warm the gelfor a least 30 minutes at 20-40 V/cm (constant voltage). 9. Resuspend the oligonucleotide pellet obtainedfrom step 1 in 1X urea loading buffer by heating it at 90°Cfor 5 minutes. The amount of sample that can be loaded dependson the efficiency of the synthesis reaction. At least 10 mgof material in a single band 2 cm wide is required to cast a clearUV shadow. The longer the oligonucleotide, the less full lengthproduct obtained. Use an amount of loading buffer that is consistentwith loading approximately 25 % of a 0.2 m-molsynthesis of a 20-mer oligonucleotide per 2 cm X 2 cm X 1.6 mmwell. This will give sharp bands with good resolution. Up to 4fold more may be added, but the resolution will suffer. 10. Rinse the wells thoroughly with 1XTBE solutionimmediately prior to gel loading. The 7 M urea dissolved in the gel will start todiffuse from the wells thereby creating a dense layer at the bottomof the wells that prevents sample loading and decreases resolution.Rinsing eliminates this problem. 11. Load the samples. Tracking dyes such as bromophenol blue and xylenecyanol (see Table 2.12.1 for migration data) may be added to thesamples or in empty lanes to monitor migration. 12. Electrophorese the gel at 20-40 V/cm (constantvoltage) until the tracking dyes indicate that the oligonucleotidehas migrated one-half to three-fourths the way through the gel. The speed of electrophoresis is directly proportionalto the voltage gradient across the gel. The current in the circuitand the heat generated for higher percentage gels (>15 % acrylamide)are corresponding smaller since the increased acrylamide concentrationleads to greater resistance. While some heating of the gel duringelectrophoresis is desirable since it helps to denature the sample,temperatures in excess of 65° C should be avoided. All gelsshould be monitored to make sure that they do not generate somuch heat that the plates crack. For example, while a 20 % gelcan be electrophoresed at 800 V with few problems, an 8 % gelunder the same conditions would likely generate too much heatfor the apparatus to dissipate. 13. When the oligonucleotide is sufficiently resolved,turn off the power supply and detach the plates from the electrophoresistank. Pry off the top plate. Cover the gel with plastic wrap (takingcare to avoid bubbles and folds) and invert the plate onto a TLCplate with a fluorescent indicator. Using a spatula, peel a cornerof the gel away from the plate and onto the plastic wrap. Pryoff the remaining plate and place another sheet of plastic wrapon top of the gel. Recover the oligonucleotide 14. Visualize the bands on the gel by briefly exposingthem to short-wave ( 254 nm) radiation from a handheld lamp. Thebands will appear as black shadows on a green background. Outlinethe bands using a marking pen. The desired band is generally the darkest oneon the gel (excluding material that runs at the dye front); itshould also be the slowest migrating band unless deprotectionwas incomplete. Lighter bands containing partially protected oligonucleotidesif they are present will migrate considerably above the majorfully deprotected band. If the stepwise efficiency of the synthesisis low, a smear may be seen instead of a clear band. Cut out thetop of the smear. Avoid unnecessarily long UV exposure which willdamage the nucleic acids. Unpolymerized acrylamide absorbs strongly at 211nm and may also cause shadowing that is confined to the edgesand wells of the gel. 15. Cut out the bands directly with a clean scalpelor razor blade. 16. Chop the gel slabs into fine particles by forcingthe gel through a small bore syringe to aid the diffusion of theoligonucleotide from the matrix. Place the crushed gel slab ina 15 ml spin tube capable of withstanding high temperatures. 17. Add 3 ml of TE for every 0.5 ml of gel slab.Freeze the sample for 30 minutes at -80°C or until frozensolid. Quickly thaw it in a hot water bath and let soak for 5minutes at 90°C. Elute on a rotary shaker overnight at roomtemperature. This freeze-rapid thaw approach (Chen Z., et al.,1996) greatly decreases elution timeand increases yield by allowing ice crystals to break apart theacrylamide matrix. A 20-mer oligonucleotide is typically recoveredin a 80 % yield after 3 hours of rotary shaking thereby makingthis technique comparable to electroelution. Since elution is a diffusion-controlled process,more buffer will aid in elution efficiency. Also, note that longeroligonucleotides will take longer to diffuse from the gel. Ifspeed is essential and high yields are dispensable, enough samplecan be obtained for most experiments in only a few hours of extraction.Increasing the temperature to 37°C will also speed the process.Yield may be increased upon repeated elutions. 18. Spin the tube to pellet the gel fragments anduse a syringe to remove the supernatant. Filter off any remainingacrylamide fragments by passing the suspension through an 0.2micron filter and into a fresh 15 ml spin tube. 19. Concentrate the sample by extracting againstan equal volume of n-butanol. Remove the upper butanol layer andrepeat until the lower aqueous volume is convenient for precipitation. About 1/5 volume of the aqueous layer is extractedinto the organic butanol layer for every volume of butanol used.If too much butanol is added and the water is completely extractedin the butanol, simply add more water and concentrated again. 20. Add 3.0 M sodium acetate pH 5.2 to a finalconcentration of 0.3 M and use 2 volumes of absolute ethanolto precipitate DNA and 3 volumes for RNA. Chill for 20 minutesat -20°C. Pellet the oligonucleotide by centrifuging at 12000xgfor 10 minutes. Do not attempt to precipitate small oligonucleotides("20 bases) in the presence of ammonium ions. If the samplesprove refractory to precipitation, use a 1:1 mix of ethanol:acetoneor 6 volumes of acetone for precipitation. A rinse with 95 % ethanolwill remove undesired salts. 21. Redissolve the oligonucleotide in TE buffer ifappropriate. REAGENTS AND SOLUTIONS Urea loading buffer 8 M urea add one volume of loading buffer to sample if a solutionor enough to dissolve a powder. COMMENTARY Background Information The traditional alternative to gel purification ofoligonucleotides has been high-performance liquid chromatography(HPLC). Although alkali perchlorate salts HPLC systems can achievevery high resolution of small and medium sized oligonucleotides(<60 bases), electrophoresis provides superior capacity andresolution over a greater range of sizes and is simpler to setup and operate. Separation times using HPLC may be faster (<30min) than for gels, but initialization of the system and productworkup tend to negate this advantage. Purification of oligonucleotides on low-pressurereverse-phase cartridges is technically simpler than gel electrophoresisand faster (<2 hr). However, these cartridges offer no separationof desired product from failed sequences, and if not used properly,allow contamination of the final product with low-molecular-weightcompounds that often inhibit subsequent enzymatic manipulationof oligonucleotides. For short oligonucleotides synthesized withhigh yield, very simple purification methods (e.g., gel filtrationor ethanol precipitation) are adequate for some applications suchas sequencing or PCR primers which do not require absolutely homogenousmaterial. The high resolution and high capacity of polyacrylamidegels makes them the method of choice for the purification of oligonucleotides.Urea disrupts hydrogen bonding between bases and thus allows oligonucleotidesto be resolved almost exclusively on the basis of molecular weightas opposed to secondary structure. However, it should be notedthat oligonucleotides of equivalent length but different sequencewill still migrate slightly differently. Thus, mixed sequenceswill appear as broader bands than homogeneous sequences (AppliedBiosystems, 1984). Also, RNA eletrophoresesthrough the gel more slowly than does DNA of comparable size.Finally, the compatibility of the chemistries of modified nucleotidesincorporated into the nucleic acids and acrylamide matrix shouldbe checked before PAGE purification (oligonucleotides bearingthio groups seem to undergo Micheal Addition to the acrylamidethereby rendering them irreversibly capped). Critical Parameters For most applications, the separation of oligonucleotidesfrom mononucleotides and protecting groups provides adequate purification.In those cases where separation of oligonucleotides from nearbyfailure sequences is essential, however, the most critical parametersto be considered are the percentage of acrylamide and the amountof sample loaded. If maximum resolution is desired, then only50 to 100 mgof material should be loaded per 2 cm x 2 cm x 1.6 mm well. Thepercentage of acrylamide that will give optimal resolution isgiven in Table 2.12.1and can be determined empirically by running a small portion ofthe starting material on trial gels and staining with ethidiumbromide. By running long (20- to 30-cm) gels, oligonucleotidesof up to 100 bases can be cleanly separated from n-1 and n+1 products.If an oligonucleotide contains extensive self-complementary sequencesor polyguanosine tracts, it may not be completely denatured in7 M urea, and thus, cannot be cleanly separated from failed synthesisproducts. To overcome this difficulty, samples can be electrophoresedon gels containing 20 M formamide instead of urea (Franket al., 1981). Troubleshooting All of the problems that apply to nondenaturing PAGEare relevant here.However, most failures in purification will occur because theinitial synthesis reaction has been inefficient. In almost allcases, it is better to resynthesize a poor yielding oligonucleotidethan to attempt to isolate a small amount of full-length productfrom a starting material seriously contaminated with failure sequences.If the oligonucleotide cannot be resynthesized, relatively smallamounts of product can be visualized by autoradiography providingthe starting material is end-labeled with polynucleotide kinaseand [g-32P]ATP(the starting material should not contain residual ammonium, whichinhibits the enzyme) (reference kinasing unit). It is convenientto end label a small amount of starting material with radioactiveATP; the remainder should be phosphorylated using cold ATP sothat it will not migrate differently from the labeled tracer.Smaller amounts of starting material should be loaded on thinner(approximately 0.75- to 1.0-mm) gels in narrower lanes (approximately1.0cm). Anticipated Results In general, the yield of purified oligonucleotidesfrom denaturing PAGE decreases as the percentage of acrylamideincreases. With crushed gel slices, an average yield of 50 percentmay be expected. Greater recoveries can be obtained by increasingthe volume of elution solution added to the gel slice or by doingserial elutions from the same gel slice. Methods employing moreactive transfers (e.g., electroelution:Smith, 1980; Vorndam and Kerschner, 1986) may givemore efficient recoveries. Also, samples (especially large syntheticRNAs) which prove particularly refractory to elution with aqueousbuffers may be eluted easily with 6 volumes of formamide (>5h at room temperature) followed by a brief elution with an aqueousbuffer (approximately 1 h). Isoamyl-alcohol may be used to concentratethe formamide-aqueous buffer extracts to a convenient precipitationvolume (Urbach, J., personal communication). Time Considerations It is usually most convenient to set up and run thegel on one day, elute the oligonucleotide overnight, then phenolextract and ethanol precipitate the sample the following day.However, a deprotected oligonucleotide can be ready for molecularbiology applications in as little as 6 hr: set up and polymerizationof gel, 1 hr; running gel, 2 hr; fragment elution, 2 hr; productrecovery, 1 hr. Maniatis, T., Jeffrey, A., and deSande, H.V. 1975.Chain length determination of small double-and single-strandedDNA molecules by polyacrylamide gel electrophoresis. Biochemistry14:3787-3794. Smith, H.O. 1980. Recovery of DNA from gels. Meth.Enzymol. 65:371-379. Vorndam, A.V. and Kerschner, J. 1986. Purificationof small oligonucleotides by polyacrylamide gel electrophoresisand transfer to diethylaminoethyl paper. Anal. Biochem. 152:221-225. Chen Z. and Ruffner D.E. 1996. Modified crush-and-soakmethod for recovering
38% acrylamide/2% bisacrylamide
TEMED (N,N,N',N'-tetramethylethylenediamine)
Urea
10% ammonium persulfate (in water < 1 monthold, store at 4° C)
urea loading buffer
3 M sodium acetate, pH 5.2
TE buffer
Thin-layer chromatography (TLC) plate with fluorescent indicator(e.g., Silica Gel F-254 or IB-F)
Glass plates, spacers, and combs for pouring gels
Acrylamide gel electrophoresis apparatus
DC power supply
0.2 micron filter (Gelman Sciences)
Additional reagents and equipment for phenol extraction and ethanolprecipitation.
20 mM EDTA
5 mM TRIS pH 7.5
0.5 % dye by mass either xylene cyanol, bromophenolblue, or both.
Frank, R., Muller, D., and Wolff, C. 1981. Identificationand suppression of secondary structures formed from deoxyoligonucleotidesduring electrophoresis in denaturing polyacrylamide gels. Nucl.Acids Res. 9:4967-4979.
oligodeoxynucleotides from polyacrylamide gel. Biotechniques.21:820-822.