DNA PURIFICATION BY REVERSED PHASE AND ANION EXCHANGE HPLC

Transcription

1 DNA PURIFICATION BY REVERSED PHASE AND ANION EXCHANGE HPLC Hamilton Company offers three HPLC columns for the separation and purification of synthesized DNA. Two for reversed phase gradient elution chromatography: PRP -, PRP-3 and one for anion exchange gradient elution chromatography: PRP-X600. Reversed Phase HPLC Purification of DNA Hamilton polymeric reversed phase columns are copolymers of styrene and divinylbenzene. PRP- and PRP-3 columns provide a number of benefits for anyone purifying synthesized oligomers by reversed phase HPLC. These include: Excellent sample recovery, of greater than 95%. High sample capacity, 0 mg or more for a 4. x50 mm column. ph stability for purification of DNA with secondary structure at ph.7. Long life, the packing is stable to a wide range of solvents and mobile phases. Wide Application PRP- and PRP-3 reversed phase columns are your best choice for purification of synthetic DNA,RNA,and chimeric DNA-RNA oligomers. With PRP- and PRP-3 polymeric reversed phase columns you can purify (5 -monomethoxytrityl, 5 -dimethoxytrityl,, fluorescein,phosphorothioate 4, tert-butyldimethylsilyl 5,7, and N-Acetyl- -aminofluorene 3,0 ) protected and deprotected oligomers with excellent recovery. Oligomers with secondary structure, e.g.hairpin loops and guanine rich sequences, are easily purified as well. Excellent Sample Recovery Recovery of trityl-on DNA and chimeric DNA- RNA oligomers is greater than 95% 7 and recovery of oligomers containing secondary structure is greater than 90% with PRP- columns. This is possible because the polymeric (poly(styrene-divinylbenzene)) packing in PRP- and PRP-3 columns are free from acidic silanol groups. C8 columns typically recover only 50-80% of protected DNA because the silanol groups on the column irreversibly bind the protected DNA or hydrolyze the dimethoxytrityl (DMT) protecting group during purification. High Sample Capacity The sample capacity of a 4. x50 mm PRP- column was determined to be 0 to 5 mg of DMT protected DNA per run by Ikuta et al.. When compared to mg/run for a C8 column of the same size, it is readily apparent that PRP- and PRP-3 are the columns of choice for protected oligodeoxynucleotide purification. Loadings up to 500 mg have been obtained on a.5 x 50 mm column. A column s sample capacity is directly proportional to its volume. For example, a 4. x 50 mm analytical column has approximately.0 ml of volume, while a.5 x 50 mm preparative column has a volume of 90.8 ml. If you can purify 0 mg on the analytical column you will be able to purify 45 times that amount or 450 mg on the preparative column.

2 Easy Purification Scaleup Changing from an analytical column to a semiprep or preparative column is easy. Applications #, and 3 illustrate the reproducible scaleup from a 4. mm column to a 0.6 mm ID column. Application # PRP-, 4. x 50 mm Isocratic Scaleup, 4. mm ID Conditions: 0.05M Citric Acid, ph 4.0, 0.6 ml/min,ambient, UV at 54 nm. Application # PRP-,.5 x 50 mm Application #3 PRP-, 0.6 x 50 mm. Cytosine. Uracil 3. Uridine Isocratic Scaleup,.5 mm ID Conditions: 0.05M Citric Acid, ph 4.0, 7.6 ml/min,ambient, UV at 54 nm Cytosine. Uracil 3. Uridine. Cytosine. Uracil 3. Uridine Isocratic Scaleup, 0.6 mm ID Conditions: 0.05M Citric Acid, ph 4.0,60 ml/min, Ambient, UV at 54 nm. Another key to reproducible chromatography and scale-up is column re-equilibration with 0 or more column volumes of mobile phase A prior to sample injection and gradient elution. Since most analytical HPLC pumps have an upper flow rate limit of 0 ml/min,the retention time of the oligomer will increase when using a larger column without increasing the flow rate proportionally. The following table can be used to determine the expected oligomer capacity of a column and gives you an idea of the equilibration volumes recommended for gradient elution. It is best to determine a column s sample capacity using an analytical column, before deciding which preparative column to purchase. Other analytical, preparative and semipreparative column sizes are available, contact the Hamilton Company. Column Column Equilibration Size Volume Volume 4. x 50 mm 0.7 ml 7 ml 4. x 50 mm.0 ml 0 ml 4. x 50 mm 3.3 ml 33 ml 7.0 x 305 mm.7 ml 7 ml 0.0 x 50 mm.8 ml 8 ml 0.0 x 50 mm 9.6 ml 96 ml.5 x 50 mm 54.4 ml 544 ml.5 x 50 mm 9 ml 90 ml 50.8 x 50 mm 507 ml 5,070 ml 0.6 x 50 mm,07 ml 0,70 ml Semipreparative and preparative purifications (00 optical density units or greater) are best measured at 97nm. This prevents saturation of the detector. Analytical separations are best monitored at 60 nm. Long Column Life The poly(styrene-divinylbenzene) packing in PRP- and PRP-3 columns is stable. This stability prevents column degradation by any of the purification methods mentioned in this brochure, assuring you reproducible separations and long column life. The inertness of the stationary phase also contributes to the exceptional recovery (>95%) of protected and deprotected oligomers. Example Purification Protocol A common DNA purification protocol for DMT-on oligomers follows:. Load the DMT-on oligomer into the HPLC injector after it has been cleaved from the CPG support and prior to detritylation.. Using mobile phases consisting of: A) 00 mm Triethylammonium Acetate ph 7.5; B) Acetonitrile. Run a gradient of 0-30% B in 0 minutes (see Application #4). The order of elution is as follows: failure sequences, oligomer without the dimethoxytrityl (DMT), then the purified DMT-on oligomer elutes. 3. The DMT-oligomer is collected and treated with 500 µl of 80% acetic acid for 30 minutes. The acid treatment removes the acid labile DMT protecting group from the oligomer. Since DNA is not stable in acidic solutions, we recommend to neutralize the solution with careful addition of Ammonium Hydroxide solution. 4. If needed, the detritylated oligomer can be repurified on the same column using the following isocratic conditions: 9: 00 mm Triethylammonium Acetate, ph 7.5/Acetonitrile. The detritylated oligomer elutes quickly (see Application #5). 5. The detritylated oligomer is collected and desalted if desired. For additonal information,please consult the references listed at the end of the Mobile Phases section. Application #4 PRP-, 4. x 50 mm Application #5 PRP-, 4. x 50 mm. Failure Sequences. 5 -DMT 6mer DMT-on 6mer Purification Conditions: A) 00 mm Triethylammonium Acetate ph 7.5; B) Acetonitrile. Linear Gradient 0-30% B (0-8 min), 30-80% B (8-5 min)..0 ml/min, Deprotected 6mer Repurification of a Deprotected 6mer Conditions: 9: 00 mm Triethylammonium Acetate ph 7.5 : Acetonitrile..0 ml/min, Ambient, UV at 60 nm.

3 Mobile Phases A wide variety of mobile phases and sample preparation conditions can be used with PRP- and PRP-3 reversed phase columns for purification of protected and deprotected DNA oligomers. The information that follows represents a summary of the Literature References listed at the end of this brochure. Application #7 PRP-, 4. x 50 mm. Failure Sequences. 5 -DMT 30mer Application #8 PRP-, 4. x 50 mm. Failure Sequences. 5 -DMT 36mer Application # 6 PRP-3, 4.6 x 50 mm DMT-on 30mer Purification Conditions: A) 00 mm Triethylammonium Acetate ph 7.5; B) Acetonitrile. Linear Gradient 0-30% B (0-8 min), 30-80% B (8-5 min)..0 ml/min, Ambient. UV at 60 nm. DMT-on 36mer with Secondary Structure Conditions: A) 50 mm Sodium Hydroxide ph.7; B) : 50 mm Sodium Hydroxide : Acetonitrile. Linear Gradient 0% B (0- min), 0-50% B (-5 min)..0 ml/min,60 C. UV at 60 nm Short Tandem Repeat Primers Conditions: A) 00mM TEAA, 0. mm EDTA ph 7; B) 75:5 00mM TEAA, 0. mm EDTA ph 7 : Acetonitrile. Linear Gradient 5-50% B in 40 min. Temperature Gradient 80-90ºC in 6.7 min. Hold at 90 º C. ml/min. UV 60 nm. Purification of DMT-on and DMT-off DNA Buffer A) 00 mm Triethyammonium Acetate ph 7.5 Buffer B) Acetonitrile Advantages: DNA is typically purified with the DMT group attached, then deprotected and repurified with a shallower gradient. Literature Reference: #9 Purification of DMT-on DNA with Secondary Structure Buffer A) 0 mm Ethylenediammonium Acetate ph 7.6 Buffer B) :0 mm Ethylenediammonium Acetate ph 7.6 : Acetonitrile Advantages: High column capacity of 0 mg for the 4. x 50 mm size. Purify DNA with self complementary sequences. Disadvantages: Column temperature is 60 C. Literature Reference: # MOBILE PHASE # Buffer A) 50 mm Sodium Hydroxide ph.7 Buffer B) : 50 mm Sodium Hydroxide : Acetonitrile Sample Prep: None except some very stable structures must first be treated with formaldehyde. Advantages: Straight forward,easy to use method. Disadvantages: Column must be heated to 60 C. Method will not work with modified, base labile DNA. Literature Reference: # MOBILE PHASE #3 Buffer A) 0 mm Potassium Phosphate ph 7 Buffer B) 7:: V:V:V Acetonitrile : Methanol : 0 mm Potassium Phosphate ph 7 Sample Prep: Up to 0 OD of crude DNA were predissolved in 400 ul of 0 mm Potassium Phosphate ph 7. Then 00 ul of formamide is added, vortexed and heated in a water bath at 90 C for 3 minutes and injected. Advantages: These buffers and the sample preparation allow the purification of strongly interacting DNA with guaninerich or self-complementary regions without column heating or high ph. This method is also useful for purification of chemically modified or adduct containing DNA. These buffers are useful for purification of samples containing less than 5 to 0 OD s. Disadvantages: More laborious sample prep Literature Reference: #8 MOBILE PHASE #4 Buffer A) 50 mm Triethylammonium Acetate ph 7 : 5% Acetonitrile Buffer B) 50 mm Triethylammonium Acetate ph 7 : 50% Acetonitrile Sample Prep: Desalted DNA was predissolved in 0 µl of Deionized Water. Then 80 µl of formamide was added, vortexed and heated in a water bath at 90 C for 3 minutes. Then chilled in an ice bath. 400 ul of chilled 50 mm Triethylammonium acetate ph 7 was added, vortexed and injected. Advantages: These buffers and the sample preparation allow the purification of strongly interacting DNA with guanine rich or self-complementary regions without column heating or high ph. This method is also useful for purification of chemically modified or adduct-containing DNA. These buffers are also useful for larger scale purifications (higher than 5 to 0 OD s). Disadvantages: More laborious sample prep Literature Reference: #8 Purification of DMT-on DNA for NMR Buffer A) 0 mm Potassium Phosphate ph 7.0 Buffer B) 7:: V:V:V Acetonitrile : Methanol : 0 mm Potassium Phosphate ph 7.0 Advantages: This method avoids the use of amines which can cause difficulty with NMR analysis. It also eliminates the need for the usual ion exchange purification step. Literature Reference: #3 3

4 Purification of DMT-on DNA with Secondary Structure and On-Column DMT Deprotection Buffer A) 5% Acetonitrile in 00 mm Tetraethylammonium Hydroxide Buffer B) 5% Acetonitrile in 00 mm Triethylammonium Bicarbonate ph 7.0 Buffer C) 0.5% Trifluoroacetic Acid Buffer D) Acetonitrile Advantages: Purify large amounts of DNA for NMR, x-ray crystallography and antisense studies. The alkaline denaturing conditions can purify 0 µmole scale synthesis. Detritylation on the HPLC column and load the sample in ammonium hydroxide. Good purity as determined by CE and NMR. Literature Reference: #6 Purification of Phosphorothioate modified DNA Buffer A) 00 mm Potassium Phosphate with mm Tetrabutylammonium Phosphate, ph 7.0 Buffer B) Acetonitrile Sample Prep: Plasma and urine samples were prepared with phenol extraction procedure. Advantages: Sharper peaks than silica-based column under identical conditions Literature Reference: #4 Purification of -(acetylamino) fluorene Modified DNA Buffer A) 0 mm Potassium Phosphate ph 7.0 Buffer B) 7:: V:V:V Acetonitrile : Methanol : 0 mm Potassium Phosphate ph 7.0 Advantages: This method avoids the use of amines which can cause difficulty with NMR analysis. It also eliminates the need for the usual ion exchange purification step. Literature Reference: #0 Purification of Fluorescein Modified DNA A) 0 mm Sodium Acetate ph 6.7 B) Acetonitrile (see application #9) Application #9 PRP-, 4. x 50 mm 0 6. Failure Sequences. Fluorescein mer Fluorescein Modified mer Conditions: A) 0 mm Sodium Acetate ph 6.7; B) Acetonitrile,. Linear Gradient 0-50% B (0-0 min)..0 ml/min, Ambient, UV at 60 nm. Purification of RNA and DNA/RNA Chimers Buffer A) 0 mm Tetrabutylammonium acetate, mm Tetrabutylammonium phosphate ph 7.5 Buffer B) 8: (V:V) 0 mm Tetrabutylammonium acetate, mm Tetrabutylammonium phosphate ph 7.5, Acetonitrile : Water. Column Temperature: 60 C Advantages: Long column lifetime. Very good recoveries of the sample (95%). PRP- columns are stable to the tetra-nbutylammonium fluoride used in the sample preparation. Silica-based columns are not. The level of purification possible with PRP- is comparable to that which might be achieved by preparative gel electrophoresis but PRP- is able to purify much larger samples more rapidly. Now prepare relatively large amounts of high purity chimeric ribozymes without PAGE purification. Disadvantages: Column must be heated to 60ºC Literature Reference: #7 Purification of RNA Buffer A) 00 mm Trimethylammonium acetate ph 7.0 Buffer B) Acetonitrile Column Temperature: 60 C Advantages: PRP- columns are more stable to continous heating than silica-based C8 columns. Superior recovery of RNA versus other columns. A volatile buffer eliminates the need for desalting. The Trimethylammonium salt is considerably easier to remove by lyophilization than the Triethylammonium salt. Disadvantages: Column must be heated to 60ºC Literature Reference: #5 4

5 Reversed Phase Column Ordering Information PRP-, 00Å Reversed Phase HPLC Columns I.D. x Length 3µm 5 µm 7 µm 0 µm. x 50 mm I.D. x Length 5 µm 7 µm 0 µm -0 µm 4.6 x 50 mm* x 00 mm x 50 mm x 50 mm* x 50 mm x 50 mm x 00 mm x 50 mm x 50 mm x 50 mm* x 00 mm* x 50 mm* x 00 mm x 305 mm x 50 mm x 00 mm x 50 mm x 50 mm x 50 mm x 50 mm x 50 mm x 50 mm 7955 * PEEK hardware PRP- Analytical Guard Columns For Steel Columns* For PEEK Columns** Starter Kits ( holder, cartridges) Replacement Cartridges (5/pk) * Analytical guard columns for steel columns are.3 x 0 mm ** Analytical guard columns for PEEK columns are 3.0 x 8.0 mm PRP- Semiprep/Preparative Guard Column For Steel Columns*** Starter Kit 79 ( holder, cartridge) Replacement 79 Cartridges (/pk) *** Semiprep/preparative guard columns for steel columns are 4.6 x 0 mm PRP-3, 300Å Reversed Phase HPLC Columns I.D. x Length 3 µm 0 µm -0 µm. x 50 mm x 00 mm x 50 mm x 50 mm x 00 mm x 50 mm x 50 mm x 50 mm* x 50 mm* x 50 mm* x 305 mm x 50 mm x 50 mm x 50 mm * PEEK Hardware PRP-3 Analytical Guard Columns For Steel Columns* For PEEK Columns** Starter Kits ( holder, cartridges) Replacement Cartridges (5/pk) * Analytical guard columns for steel columns are.3 x 0 mm PRP-3 Semiprep/Preparative Guard Column Starter Kit 793 ( holder, cartridge) Replacement 794 Cartridges (/pk) *** Semiprep/preparative guard columns for steel columns are 4.6 x 0 mm. For Steel Columns*** 5

6 Anion Exchange HPLC Purification of DNA Anion Exchange Purification of DNA Hamilton PRP-X600 anion exchange HPLC columns separate DNA oligomers according to charge. Controlled porosity provides excellent separation of n- oligomers from the full length product. See application # for the separation of a DMT-on antisense, phosphorothioate 8mer from the failure sequences. PRP-X600 columns work best when heated to 85 C. Application #0 PRP-X600, 4.6 x 50 mm Application # PRP-X600, 4.6 x 50 mm. DMT-on phosphorothioate antisense 8mer 6 Sample Capacity The 50 mm long 4.6 mm I.D. columns have a sample capacity of approximately mg. 50 x 4.6 mm long columns have a sample capacity of about 3 mg. Compatible Mobile Phases PRP-X600 weak base anion exchange columns are polymeric, stable from ph to and compatible with organic solvents. Changing Mobile Phases and Equilibration After Gradient Separation Equilibration of the PRP-X600 column requires approximately 0 column volumes. Following each gradient analysis, reequilibrate the column with Buffer A to ensure reproducible sample retention pbr3 DNA Fragment Conditions: A) 0 mm TRIS, mm EDTA ph 9.0; B) N Sodium Chloride in 0 mm TRIS, mm EDTA. Linear Gradient % B (0-5 min), % B (5-45 min)..0 ml/min, Ambient. UV at 60 nm. PRP-X600 Anion Exchange HPLC Columns I.D. x Length 7 µm 4.6 x 50 mm* x 00 mm* x 50 mm* x 50 mm* x 50 mm 7949 * PEEK Hardware PRP-X600 Guard Columns Starter Kits ( holder, cartridges) 7936 Replacement Cartridges (5/pk) DMT-on Phosphorothioate Antisense 8mer Conditions: A) 85:5 00 mm TRIS ph 8.0 : Acetonitrile; B) 85:5 00 mm TRIS ph 4.0,.5 M Lithium Chloride :Acetonitrile. Linear Gradient 0-00% B in 0 min..0 ml/min. 85 C. UV at 60 nm For PEEK Columns

7 Bulk Resin Hamilton HPLC supports for the separation and purification of DNA are available as bulk resin or prepacked into analytical,semi-preparative and preparative columns. Chromatographic capacity and efficiency tests are conducted on both bulk and packed column resins to ensure product integrity and reproducibility. Samples from different production lots can be purchased for method/process validation. For ease of ordering,specify the quantity of resin needed in grams. Quantities from one gram to tens of kilograms are available. One gram of resin is roughly equivalent to.5 cc s of volume. Visit the Hamilton Company web site at for technical specifications. Bulk Resin Ordering Information Support Type of Support Material Capacity Pore Size 3 µm 5 µm 7 µm 0 µm -0 µm µm µm PRP- PSDVB* N/A 00Å Reversed Phase PRP-3 PSDVB* N/A 300Å Reversed Phase PRP-X600 Polydimethyla-.6 Superficially Anion Exchange midopropylme- meq/gm porous thacrylamide * PSDVB is poly(styrene-divinylbenzene). Bulk resin is sold by the gram. Literature References. Ikuta, S.; Chattopadhyaya, R.; Dickerson, R.: Analytical Chemistry, 984, 56, 53.. Germann, M.W.; Pon, R.T.; van de Sande, J.H.: Analytical Biochemistry, 987, 56, Huang, G.; Krugh, T.R.: Analytical Biochemistry, 990, 90,. 4. Biegelow, J.C.; Chrin, L.R.; Mathews, L.A.; McCormack, J.J.: J Chrom., 990, 533, Khare, D.; Orban, J.: Nucleic Acids Research, 99, 0, Hobbs, F.W.; Yarem, J.A.: BioTechniques, 993, 4, Swiderski, P.M.; Bertrand, E.L.; Kaplan, B.E.: Analytical Biochemistry, 994, 6, Arghavani, M.B.; Romano, L.J.: Analytical Biochemistry, 995, 3, Reddy, D.M.; Iden, C.R.: American Laboratory, 995, 5, Zhou, Y.; Romano, L.: Biochemistry, 993, 3,

8 SPE Cartridges, Barrels and 96-well plates Minutes SPE Purified Oligomer Electropherogram of a 7mer purified with a Syringe Cartridge Use Hamilton SPE cartridges, barrels, and 96-well plates for rapid and easy purification of synthesized DNA. Excellent Purity - PRP- cartridges provide purity comparable to that obtained with reversed phase HPLC. Depending on your needs, you have the choice of a purified protected oligomer or a purified deprotected oligomer when you use PRP- cartridges. See Figure for an electropherogram of a 7mer purified with a PRP- cartridge. Higher Sample Capacity - One PRP- cartridge will purify 0 OD A60 units from a 0. µmole synthesis. Apply your sample once, at the rate of - drops per second for optimal binding.prp- cartridges do not require multiple applications. Low Backpressure - Cartridges have very low backpressure, so loading your sample is easier. Tight controls on particle size ensure consistent batch to batch performance. Cartridges are designed for a single use, but may be used again for purification of the same oligonucleotide. Cartridges can be used with a syringe or vacuum manifold to load, wash, and elute your samples. Barrels are designed for use with a vacuum manifold. SPE Ordering Information PRP- Syringe Cartridges Part # Description Bed Volume Application Syringe Cartridge 50/pk 50 mg 0. µmole Syringe Cartridge 0/pk 330 mg µmole Syringe Cartridge 0/pk 600 mg.0 µmole PRP- Barrels (for use on vacuum manifolds) Part # Description Bed Volume Application 7934 Barrel, ml,50/pk 00 mg 0. µmole Barrel, ml,50/pk 50 mg 0. µmole Barrel,6 ml,0/pk gm.5 µmole Barrel,0 ml,0/pk gm.5 µmole Barrel,60 ml,/pk 0 gm 5.0 µmole 96 Well Plates Part # Description Bed Volume Application Well Plate 50 mg/well 0. µmole Electropherogram courtesy of Lolita Bland, University of Virginia PRP is a registered trademark of Hamilton Company R Hamilton Company 4970 Energy Way Reno, Nevada 8950 USA Toll-Free: Telephone: Fax: hplc@hamiltoncompany.com Hamilton Bonaduz AG Via Crusch 8 CH -740 Bonaduz/Switzerland Telephone: +4-(0) Fax: +4-(0) hplc@hamilton.ch Web site: 04/03 MB

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