TABLE OF CONTENTS
INTRODUCTION .......................................................................... 2
PRODUCT DESIGN ...................................................................... 5
PROCEDURE-DMT-ON
Principles of Poly-Pak Purification .................................................... 7
Purification of Oligonucleotides (DMT-ON) ................................... 7
Large Scale Purification of Oligonucleotides (DMT-ON) .............. 10
Purification of Dye-Labelled Oligonucleotides (DYE-ON) ........... 11
PROCEDURE - DESALTING
Desalting of Oligonucleotides ......................................................... 14
APPLICATIONS
Purification of Unmodified Oligonucleotides .................................. 15
Purification of Amino-Modified Oligonucleotides ......................... 16
Purification of Oligophosphorothioates ........................................... 17
Purification Strategy - Thiol-Modifier C6 S-S ............................... 18
Purification of Biotinylated Oligonucleotides .................................. 20
Purification of Dye-Labelled Oligonucleotides (DYE-ON) ........... 21
SOLUTION PREPARATION ...................................................... 23
1
COMMON QUESTIONS ............................................................ 24
ORDERING INFORMATION .................................................... 28
INTRODUCTION
PURIFICATION TECHNIQUES
Advances in the chemistry of synthesis now allow rapid, facile and
efficient production of oligonucleotides. However, purification can
be time-consuming and occasionally ineffective. In some
applications, there is no need for purified product. When
purification is necessary, two traditional techniques are generally
used: polyacrylamide gel electrophoresis (PAGE) and high
performance liquid chromatography (HPLC). While the choice of
technique may depend simply on previous experience or access to
equipment, there are some basic advantages to both techniques.
PAGE
PAGE and its cousin capillary gel electrophoresis (CE) are probably
most often used for the analysis of synthetic oligonucleotides. With
UV shadowing, PAGE allows purification of small quantities of
synthetic DNA and is appropriate for sequences of any length. It is
especially useful for purification of longer sequences which exhibit
secondary structure. However, there are several drawbacks to using
PAGE for purification: it is only amenable to small-scale purification;
product recovery is usually quite low; and it is time-consuming, with
significant time required to cast and run the gel, as well as the time
required to extract and desalt the product. In addition, it may be
necessary to enrich and desalt the crude product prior to purification.
CE is an excellent analytical tool but not yet a ubiquitous purification
technique.
2
HPLC
One of the most attractive techniques for oligonucleotide purification
is reverse phase (RP) HPLC. By purifying with the dimethoxytrityl
(DMT) group still attached at the 5'-terminus of the synthetic
oligonucleotide, failure sequences which contain no DMT groups are
weakly bound to the column and easily separated from the product
which is more strongly retained and eluted later. In general, a set of
components with the DMT group still attached is revealed.
Normally, the product accounts for greater than 95% of these
INTRODUCTION
components. The buffers used in this RP HPLC technique are
volatile so the need for dialysis or desalting prior to lyophilization is
eliminated. After removal of the DMT group with aqueous acid, the
product is obtained rapidly and in good yield. RP HPLC can also
be used for the purification of detritylated oligonucleotides. The
technique retains the advantage of rapid recovery of the purified
product simply by lyophilization of the volatile solute. The two
processes can be combined on a pH stable RP packing1 to include
elution of non-DMT-containing failure sequences from the HPLC
column, followed by detritylation of the DMT-ON oligos with
aqueous trifluoroacetic acid, and finally chromatographic purification
of the product oligonucleotides2. RP HPLC purifications can be
easily scaled up to large preparative columns.
Several HPLC column manufacturers have introduced ion exchange
HPLC columns specifically for the analysis and purification of
synthetic oligonucleotides. These columns, like capillary
electrophoresis, are capable3 of superb resolution of components in
the product mixture and, consequently, provide insights into the
synthesis process. Ion-exchange columns have lower capacity for
oligonucleotides than RP columns. However, they are more efficient
for separating full-length oligonucleotides from n-1 failure sequences.
Ion exchange HPLC is often used as a second purification when
highly-purified oligonucleotides are needed.
Cartridges and Columns 3
RP cartridges containing C18 silica gel have been popular4 for some
1. M. Germann, R.T. Pon, and J.H. van de Sande, Anal. Biochem.,
1987, 165, 399.
2. A. Le, Rainin Dynamax Review, 1989, 3, 1.
3. W.J. Warren and G. Vella, BioTechniques, 1993, 14, 598.
4. K.M. Lo, S.S. Jones, N.R.Hackett, and H.G. Khorana, Proc. Natl.
Acad. Sci. USA, 1984, 81, 2285.
INTRODUCTION
time for rapid and inexpensive purification of synthetic
oligonucleotides. The technique involves evaporating the ammonia
used for deprotection (since the silica gel-based support is not stable
to high pH), redissolving the crude mixture (with DMT group still
attached), and introducing the mixture onto the cartridge. The
purification procedure is similar to that described above for RP
HPLC columns. The DMT groups are later removed by treatment
with acetic acid prior to lyophilization to give the purified product.
Several disadvantages, however, exist with this technique. During the
step to concentrate the ammonia, a second base, for example,
triethylamine, must be added to maintain a basic solution and avoid
the loss of a small percentage of the DMT group. In addition, the
acetic acid deprotection necessitates two lyophilization steps after
purification and an optional extraction to remove dimethoxytritanol
from the product.
These disadvantages can be overcome5 by using a polymeric RP
packing in a cartridge or syringe barrel. Because these resins are
stable in the pH range 1-13, the ammonia solution, diluted with
water, can be added directly to the purification matrix. After elution
of failure sequences, the DMT group can be deprotected with
aqueous acid. The purified product can then be eluted and isolated
by lyophilization.
4
5. L.J. McBride, C. McCollum, S. Davidson, J.W. Efcavitch, A.
Andrus, and S.J. Lombardi, BioTechniques, 1988, 6, 362.
POLY-PAK鈩? CARTRIDGES
PRODUCT DESIGN
Glen Research has selected for use in Poly-Pak cartridges a highly
purified, pH-stable polymeric resin which has been optimized for the
purification of synthetic oligonucleotides. Poly-Pak cartridges exhibit
the following attractive features:
1) The ability to prepare up to 20 A260 units from a 0.2 碌mole
synthesis and 50 A260 units from a 1 碌mole synthesis. This feature is
highly significant for the production of oligonucleotides destined for
use in DNA amplification experiments.
2) Wash volumes are diminished to a level where the total eluent in
a 40 nmole or 0.2 碌mole purification is less than 15 mL. This speeds
the process dramatically in comparison to other RP cartridges.
3) Product oligonucleotides are eluted in as little as 0.5 mL of
aqueous acetonitrile, thereby minimizing the time needed for solvent
evaporation.
The original Poly-Pak cartridges are used for purification of
oligonucleotides produced on 40 nmole or 0.2 碌mole scales. Poly-
Pak II cartridges are for 1 碌mole purifications.
Female Luer fitting for attachment of
syringe reservoir.
Poly-Pak cartridges are molded from
translucent polyethylene.
Polymeric packing held in a
chromatographic bed.
5
Packing is retained by frits in a packed
chromatographic bed. Typically,
oligonucleotides are eluted in 0.5mL, Male luer for simple attachment of
thereby minimizing evaporation time. collection tubing or for attachment to
a vacuum manifold.
ANATOMY
OF A
POLY-PAK鈩? CARTRIDGE
Poly-Pak is a trademark of Glen Research Corporation
PRODUCT DESIGN
POLY-PAK鈩? BARRELS
Glen Research has selected for use in Poly-Pak barrels a highly
purified, pH-stable polymeric resin which has been optimized for the
purification of synthetic oligonucleotides. Poly-Pak barrels exhibit the
same features as cartridges but are designed specifically for use on
vacuum manifolds.
The original Poly-Pak barrels are used for purification of
oligonucleotides produced on 40 nmole or 0.2 碌mole scales. Poly-
Pak II barrels are for 1 碌mole purifications.
Poly-Pak barrels are molded from
translucent polypropylene.
Polymeric packing held in a
chromatographic bed.
6
Packing is retained by frits in a packed
chromatographic bed. Typically,
oligonucleotides are eluted in 0.5mL, Male luer for simple attachment of
thereby minimizing evaporation time. collection tubing or for attachment to
a vacuum manifold.
ANATOMY
OF A
POLY-PAK鈩? BARREL
PRINCIPLES OF POLY-PAK PURIFICATION
PROCEDURE-DMT-ON
Automated synthesis of oligonucleotides is carried out from the 3'- to
the 5'-terminus. During the synthesis process, failure sequences
(caused by incomplete monomer addition) are end-capped.
Assuming that end-capping is efficient, only the full-length sequence
should contain a dimethoxytrityl (DMT) group at the 5'-terminus on
completion of the synthesis. Since the DMT group binds strongly to
reverse phase supports, the full-length sequences can be retained in a
cartridge while the failure sequences are eluted. The DMT group is
then removed on the cartridge and the purified product is eluted.
(Note: It is normally an option on the synthesizer to remove or to
retain the acid-labile DMT group at the 5'-terminus. For the
standard Poly-Pak purification procedure to operate, the synthesis
MUST be carried out DMT-ON.)
Deprotection of synthetic oligonucleotides with ammonium
hydroxide (30%; SG=0.88) removes the sequence from the support,
while removing all base-labile protecting groups. Following this
procedure, there is no need to lyophilize the ammonium hydroxide
solution since the polymeric resin is stable at high pH. However, if
the product has been lyophilized, simply reconstitute it in 0.1M
triethylamine acetate (TEAA).
PURIFICATION OF OLIGONUCLEOTIDES (DMT-ON)
7
Materials Amount Used
Poly-Pak Poly-Pak II
Cartridge 1 1
HPLC grade Acetonitrile 2mL 4mL
2.0M Triethylamine Acetate (TEAA) (pH7) 2mL 4mL
Deionized Water 4mL 8mL
Ammonium Hydroxide/Water (1:10) or (1:20) 3mL 6mL
2% Trifluoroacetic Acid (TFA)/Water 2mL 4mL
20% Acetonitrile/Water ~1mL ~2mL
PROCEDURE-DMT-ON
What's Happening
Procedure
A good steady flow rate is usually
The flow rate of solvents through
sufficient to flush the bed of
the cartridge should be regulated at
purification matrix.
a rate of ~1-2 drops per second.
Cartridge Preparation
Cartridge Preparation
1. We use 5mL disposable syringes.
1. Connect a syringe to the female
2. The acetonitrile washes organic
luer of the cartridge and have the
residues from the resin and wets it,
male luer terminate in a waste
while the TEAA acts as an ion
vessel.
pairing reagent to enhance the
2. Flush the cartridge with 2mL
binding of the oligonucleotide to the
(4mL Poly-Pak II) acetonitrile
resin.
followed by 2mL (4mL) 2M
TEAA.
Sample Preparation
Sample Preparation
3. For this procedure to be successful,
the synthesis must have been carried
3. Following synthesis, deprotect
out DMT-ON. The base labile
the DMT-ON oligonucleotide in
protecting groups must have been
ammonium hydroxide as normal.
removed with ammonium
For this procedure, there is no
hydroxide.
need to lyophilize the
4. The volume of diluted ammonium
ammonium hydroxide solution.
hydroxide applied to the cartridge
4. Add 3 parts deionized water to 1
has little or no effect on the
part of the deprotected DMT-
purification. (If the ammonium
ON oligonucleotide in the
8
hydroxide solution has been
ammonium hydroxide solution.
lyophilized, reconstitute the pellet in
Up to 10mL of sample solution
3mL of 0.1M TEAA.)
may result.
Purification Procedure
Purification Procedure
5. During the loading process, the
5. Load the sample solution onto
DMT-ON oligonucleotides tend to
the cartridge. Collect the eluted
stick to the polymeric packing
fraction and again push it
while the failure sequences do not.
through the cartridge.
6. Flush the cartridge with 3mL 6. This wash flushes the remainder of
PROCEDURE-DMT-ON
(6mL) of ammonium hydroxide the failure sequences from the
(1:20) for oligos 鈮?35mer, or cartridge.
ammonium hydroxide (1:10) for 7. Water flushes excess ammonium
oligos >35mer. hydoxide from the cartridge.
7. Flush the cartridge with 2mL 8. A faint pink/orange band may be
(4mL) of deionized water. observed in the cartridge.
8. Detritylate the support-bound 9. Water flushes excess 2% TFA. The
oligonucleotide by flushing the DMT group remains adsorbed to
cartridge with 2mL (4mL) of 2% the resin.
TFA. (10. Longer oligonucleotides may
9. Flush the cartridge with 2mL contain contaminating sequences
(4mL) deionized water. produced by depurination of dA
(10. For oligos >50mer, repeat steps sites. These shorter sequences are
6 and 7.) truncated at the 3' terminus and are
11. Elute the purified, detritylated removed to an extent in these
oligonucleotide by flushing the additional steps.)
cartridge with 20% acetonitrile. 11. Rather than count drops, it may be
Collect the eluted fractions. The convenient to elute with 0.5mL
first 4 drops of eluent can be (1mL) of 20% acetonitrile. A
discarded and the product is further 1mL (2mL) of eluent may
normally in the next 4 to 6 drops. be collected for security until the A260
12. Determine the A260 units and units are determined. An unusual
store any unused oligonucleotide sequence may elute in the 1mL
as a lyophilized solid at -20藲C. (2mL) wash.
12. Poly-Pak purification should yield
Note: on a 0.2 碌mole scale, 10-20 A260
If the oligonucleotide is to be stored units, and Poly-Pak II on a 1 碌mole 9
for a period longer than a few scale, 40-70 A260 units, depending
months, elute the product with on the sequence.
water:acetonitrile:ammonium
hydroxide (90:10:1). The Note:
ammonium salt of the We thank Bruce Kaplan of City of
oligonucleotide is stable in storage. Hope, Duarte, CA for suggesting an
alternative elution of product to give
better stability in storage.
Note: Volumes for Poly-Pak II cartridges on a 1 碌mole scale are shown in parenthesis.
PROCEDURE-DMT-ON
LARGE SCALE PURIFICATION (DMT-ON)
The packing material used in Poly-Pak cartridges is available in bulk
and may be used for large scale purification of oligonucleotides. The
particle size distribution of the packing is 7-38 碌m and filter funnels
capable of retaining such small particles must be selected. The
following volumes and quantities are approximate and should be
optimized for the actual purification being undertaken.
Materials Amount Used per
10 碌moles of Synthesis
Poly-Pak鈩? Packing 2.5g
HPLC grade Acetonitrile 15mL
2.0M Triethylamine Acetate (TEAA) (pH7) 15mL
Deionized Water 15mL
Ammonium Hydroxide/Water (1:10) 25mL
2% Trifluoroacetic Acid (TFA)/Water 15mL
20% Acetonitrile/Water ~10mL
The following general procedure is repeated at each step. Slurry and
stir the packing with each solvent in 3 portions. Apply vacuum to the
funnel to wash and filter the packing.
1. Prepare the packing by slurrying and washing with Acetonitrile then with
2M TEAA. This is analogous to Steps 1 and 2 on Page 8.
2. Prepare the sample solution as described in Steps 3 and 4 on Page 8.
3. Slurry the sample solution with the conditioned packing and filter. Collect
the filtrate and apply it to the packing a second time. Collect and retain
10
the filtrate for analysis.
4. Slurry and wash the packing with dilute ammonium hydroxide, water, 2%
TFA, and water, in a manner analogous to steps 6-9 on Page 9. Keep each
wash solution separate and retain them until a satisfactory mass balance has
been achieved.
5. Elute the purified oligonucleotide with 20% Acetonitrile/Water.
Analysis of mass balance can be determined by UV spectroscopy but
critical solutions should also be analyzed by HPLC. These notes are
provided as guidelines but each large scale purification must be
accompanied by careful analysis.
PURIFICATION OF DYE-LABELLED OLIGONUCLEOTIDES
PROCEDURE-DYE-ON
In general terms, carry out the synthesis of oligonucleotides using 5鈥?-
fluorescent dye labels, e.g., 5'-Fluorescein (6-FAM), 5'-Hexachloro-
Fluorescein (HEX) or 5'-Tetrachloro-Fluorescein (TET), using the
standard DMT-ON synthesis cycle. (These products do not have a
DMT-group to remove). For oligonucleotides prepared with dye
amidites containing a DMT group (e.g., fluorescein and acridine) or
an MMT group (e.g., Cy-3鈩? and Cy-5鈩?) use the DMT-OFF cycle.
(Cy-3 and Cy-5 are further protected with a monomethoxytrityl
(MMT) group. The absorbance of the MMT cation (yellow) is
different from the DMT cation (orange) so absorbance-based trityl
monitors will detect it incorrectly as a low coupling. Conductivity
monitors will interpret the release more accurately.)
All of these dye-labelled oligonucleotide can be deprotected at room
temperature in concentrated ammonium hydroxide for a time
appropriate for the monomers used. A minimum of 24 hours at RT
is required for oligos containing ibu-protected dG residues. 6-FAM
labelled oligos can be heated in ammonium hydroxide as normal. For
the other dyes, monomers with protecting groups which can be
removed in 2 hours or less at 55掳C are preferred. Do not exceed this
time or temperature when using HEX or Cy-5 labelled oligos.
Remove the ammonia immediately after deprotection.
PURIFICATION OF OLIGONUCLEOTIDES (DYE-ON)
11
Materials Amount Used
Poly-Pak Poly-Pak II
Cartridge 1 1
HPLC grade Acetonitrile 2mL 4mL
2.0M Triethylamine Acetate (TEAA) (pH7) 2mL 4mL
Deionized Water 4mL 8mL
8% Acetonitrile/0.1M TEAA (pH7) 3mL 6mL
20% Acetonitrile/Water ~2mL ~4mL
Cy-3 and Cy-5 are trademarks of Biological Detection Systems, Inc.
PROCEDURE-DYE-ON
Procedure What鈥檚 Happening
Sample Preparation Sample Preparation
1. Dry the dye-labelled 1. The volume of sample solution
oligonucleotide and dissolve applied to the cartridge has
the residue in 1mL 0.1M little or no effect on the
Triethylammonium acetate purification.
(TEAA).
Cartridge Preparation Cartridge Preparation
2. Connect a syringe (we use 2. The acetonitrile washes organic
5mL disposable syringes) to residues from the resin and wets
the female luer of the it, while the TEAA acts as an
cartridge and have the male ion-pairing reagent to enhance
luer terminate in a waste the binding of the
vessel. oligonucleotide to the resin.
3. Flush the cartridge with 2mL
(4mL) acetonitrile followed
by 2mL (4mL) 2M TEAA.
Purification Procedure
Purification Procedure 4. During the loading process, the
4. Load the sample solution dye-labelled oligonucleotides
from step 1 onto the tend to stick to the polymeric
cartridge. Collect the eluted packing while the failure
fraction and again push it sequences do not.
through the cartridge. 5. The 8% acetonitrile wash
5. Flush the cartridge with 3mL flushes the remainder of the
12
(6mL) 8% acetonitrile in failure sequences from the
0.1M TEAA. cartridge. This step was
6. Flush the cartridge with 2mL devised by researchers at
(4mL) of deionized water. Applied Biosystems Division of
7. Elute the purified Perkin Elmer.
oligonucleotide by flushing 6. Water flushes excess of the above
the cartridge with ~1mL solution from the cartridge.
(~2mL) 20% acetonitrile. 7. It may be prudent to elute with
Collect the eluted fractions. 1mL (2mL) of 20%
Use the color of the product acetonitrile. A further 1mL
PROCEDURE-DYE-ON
to determine the extent of (2mL) of eluent may be
elution from the cartridge. collected for security until the
8. Determine the A260 units A260 units are determined.
and store any unused These unusual oligos may elute
oligonucleotide as a slowly but their color, when
lyophilized solid or in neutral present, is useful in determining
aqueous media at -20掳C. the extent of elution.
8. Poly-Pak purification should
Note: yield on a 0.2 碌mole scale, 10-
Dye-labelled oligonucleotides 20 A260 units, and Poly-Pak II
should be stored in the dark, on a 1 碌mole scale, 40-70 A260
either dry or in neutral units, depending on the
aqueous media at -20掳C. Do sequence.
not store the crude dye-
labelled oligonucleotide in
ammonia solution.
13
Note: Volumes for Poly-Pak II cartridges on a 1 碌mole scale are shown in parenthesis.
PROCEDURE-DESALTING
DESALTING OF OLIGONUCLEOTIDES
Poly-Pak cartridges may be conveniently used for desalting
oligonucleotides which have been purified by ion exchange HPLC or
by gel electrophoresis.
Materials Amount Used
Poly-Pak Poly-Pak II
Cartridge 1 1
HPLC grade Acetonitrile 2mL 4mL
2.0M Triethylamine Acetate (TEAA) (pH7) 2mL 4mL
0.1M Triethylamine Acetate (TEAA) (pH7) 2mL 4mL
50% Acetonitrile/Water ~1mL ~2mL
Procedure What's Happening
Cartridge Preparation Desalting Procedure
1. Prepare the cartridge as 2. For this procedure to be
described for DMT-ON successful, the oligonucleotide
purification. must be applied to the cartridge
in an aqueous solution or a
Desalting Procedure solution containing less than
2. Load the solution containing 5% organic solvent. The
the oligonucleotide onto the volume of the sample solution is
cartridge. not critical but it is always
3. Flush the cartridge with prudent to keep the unretained
3mL (6mL) of 0.1M TEAA. solvent until the purified
14
4. Elute the desalted product is quantified.
oligonucleotide by flushing 3. 0.1M TEAA removes the salts
the cartridge with 0.5mL from the cartridge.
(1mL) of 50% acetonitrile. 4. The product should elute totally
5. Determine the A260 units. in 0.5mL (1mL) of 50%
Store any unused acetonitrile.
oligonucleotide as a 5. Up to 70 A260 units can be
lyophilized solid at -20藲C. desalted on Poly-Pak cartridges.
Note: Volumes for Poly-Pak II cartridges on a 1 碌mole scale are shown in parenthesis.
PURIFICATION OF UNMODIFIED OLIGONUCLEOTIDES
APPLICATIONS
The ability of Poly-Pak cartridges to produce high quality
oligonucleotides is demonstrated in the purification of a 21-mer.
Figure 1 contains the RP HPLC trace of the crude DMT-ON
oligonucleotide after deprotection. In Figure 2, the deprotected 21-
mer has been purified on a Poly-Pak cartridge and isolated in 4 drops
of aqueous acetonitrile. The analysis in Figure 2 is carried out using
capillary gel electrophoresis (CE).
15
Capillary: Beckman eCAP ssDNA gel
Time 0.1M TEAA(%) Acetonitrile(%)
Length: 30cm
Field: 11.1kV
0 97 3
Temp.: 30掳C
5 97 3
Flow Rate: 1mL/min.
20 75 25
Detector: UV at 254nm
30 75 25
Buffer: Tris-borate-urea
Column: Spherisorb ODS-2(150X4.6mm)
Figure 2: DMT-OFF Capillary
Flow Rate: 1mL/min.
Electropherogram of Poly-Pak
Detector: UV at 254nm
purified oligonucleotide.
Figure 1: DMT-ON Chromatography by
RP HPLC .
APPLICATIONS
PURIFICATION OF AMINO-MODIFIED OLIGONUCLEOTIDES
Amino-modification is an important technique for the preparation of
labelled oligonucleotides for use in DNA sequencing, amplification,
and in the production of diagnostic probes. A popular method for
attachment of an amino group to a synthetic oligonucleotide is the
use of a 5'-amino-modifier containing the monomethoxytrityl
(MMT) protecting group. These amino-modifiers are specifically
designed to take advantage of RP purification techniques, including
cartridge purification.
The Poly-Pak procedure for purification of amino-modified
oligonucleotides is the same as that described beginning on Page 8.
Two minor exceptions are worthy of note:
1. When the amino-modifier contains the thermally labile MMT
protecting group, carry out the deprotection at 40藲C for 24 hours.
2. During the detritylation
(Step 8, Page 9), pass 2%
TFA through the Poly-Pak
three times and leave to react
for 2 minutes each time. A
yellow color may become
visible in the cartridge.
The chromatogram in Figure 3
demonstrates the efficiency of
this technique.
3'-Amino-Modifier CPG has
16
gained popularity for the direct
synthesis of oligonucleotides
containing an amino group at
the 3'-terminus. Similarly,
Amino-Modifier-dT is used to
introduce amino groups at
specific points in
oligonucleotides. When Figure 3: RP HPLC of an amino-modified
oligonucleotide purified as
synthesized DMT-ON, these described. Column , flow rate
3鈥?-amino-oligonucleotides can and detector are as described
be purified on a Poly-Pak in Figure 1, Page 15.
cartridge using the procedure
described on Page 8.
PURIFICATION OF OLIGOPHOSPHOROTHIOATES
APPLICATIONS
Oligophosphorothioates (S-Oligos) have shown potential as
therapeutic agents due to the nuclease resistance of the
internucleotide linkages. These modified oligos can be produced
using a variety of techniques by phosphoramidite chemistry and also
conveniently by H-phosphonate chemistry. Whatever the synthetic
route, the product S-Oligos can be quickly purified on Poly-Pak
cartridges. The multiplicity of the DMT-ON product (Figure 4) is
caused by diastereomers at the phosphorothioate linkage adjacent to
the DMT group. The product analyzed by RP HPLC is shown in
Figure 5.
Oligophosphorothioates can be further analyzed by PAGE, CE, or
ion exchange HPLC following the conditions described1.
17
Figure 5: RP HPLC of a DMT-OFF
Figure 4: RP HPLC of a DMT-ON
oligophosphorothioate,
oligophosphorothioate, using
following the conditions shown
the conditions shown in Figure
in Figure 1, Page 15.
1, Page 15 but increasing the
acetonitrile content to 40% in
the same time.
1. B.J. Bergot and W. Egan, J. Chromatography, 1992, 599, 35.
APPLICATIONS
PURIFICATION STRATEGY
The purification of modified oligonucleotides using RP cartridges
can be very effective as long as the strategy has been well conceived.
The development of Poly-Pak cartridge purifications should be
carried out in conjunction with RP HPLC. With a properly
developed strategy, the method should be analogous to an affinity
purification rather than a true chromatographic technique. In other
words, you must find conditions where the desired product sticks to
the cartridge while all impurities can be washed off and then change
the conditions to elute the purified product.
In developing a Poly-Pak purification, first check the RP HPLC of
the product mixture. The desired product should elute from a RP
HPLC gradient with a retention difference of at least 7% and
preferably 10% acetonitrile.
18
Figure 6: RP HPLC of a crude oligo- Figure 7: RP HPLC of the DMT-on oligo-
nucleotide modified at the 5' nucleotide shown in Figure 6
terminus with thiol-modifier C6 modified at the 5' terminus with
S-S following the conditions thiol-modifier C6 S-S and
shown in Figure 1, Page 15. following Poly-Pak purification.
To illustrate purification strategy using Poly-Pak cartridges, the
APPLICATIONS
purification of oligos modified using 5'-Thiol-Modifier C6 S-S
is described. This is a 3 step process. Step 1 purifies the DMT-ON
oligonucleotide with the disufide group intact. Step 2 cleaves the
disulfide linkage. Step 3 purifies and desalts the 5'-thiol
oligonucleotide. Figures 6, 7 and 8 illustrate the progress of the
purification and desalting steps.
Step 1
Purify the DMT-ON oligonucleotide as described on Pages 8 and 9
with the exception that the detritylation steps (8 and 9) are omitted.
The purified DMT-ON oligonucleotide is eluted with 1 mL of 20%
acetonitrile and there is no need to evaporate the solvent.
Step 2
Add the eluate from the cartridge
to an equal volume of a solution
of dithiothreitol (DTT) (0.2M
DTT in 0.1M phosphate buffer,
pH=8.3-8.5). Leave at RT for
0.5h to cleave the disulfide.
Step 3
Add 2 volumes of water to the
incubation solution from Step 2.
Desalt the oligonucleotide on a
Poly-Pak cartridge, as described 19
on Page 14, but eluting with 20%
acetonitrile. In this way, the
DMT containing C6 thiol will
remain bound to the cartridge
while the oligonucleotide
containing the 5'-thiol elutes
from the cartridge. The two
minor impurities shown are non- Figure 8: RP HPLC of the thiol-modified
oligonucleotide shown in Figure
nucleosidic and do not affect 7, desalted using a Poly-Pak
thiol-specific conjugation. cartridge.
APPLICATIONS
PURIFICATION OF BIOTINYLATED OLIGONUCLEOTIDES
In the years since we introduced our range of Amino-Modifiers, it
has become clear that their most common use is as precursors for the
introduction of biotin by reaction with the appropriate biotin NHS
ester. While these methods have proved to be relatively successful,
the only satisfactory means of purifying biotinylated oligonucleotides
produced in this manner is by RP HPLC. The process is time-
consuming and suffers from the possibility of cross-contamination
which is potentially troublesome in PCR applications.
CE phosphoramidites based on a branched hydrocarbon backbone
terminating in a DMT group and with a linkage to biotin extending
away from the branching site have now been introduced. These
products can be added in multiple steps at the 3' or 5' terminus, can
be inserted at any position in the sequence, or can be added once at
the 5' terminus for subsequent RP
purification.
The chromatogram shown in
Figure 9 demonstrates the use
of a biotin phosphoramidite to
label the 5' terminus of an
oligonucleotide. The
biotinylated oligonucleotide was
simply purified by the DMT-
ON technique on a Poly-Pak
20
cartridge, using the procedure
described on Page 8. This
product would certainly be of
sufficient purity for use as a
biotinylated PCR primer or
probe.
Figure 9: CE of a Poly-Pak purified
biotinylated oligonucleotide,
following the conditions shown
in Figure 2, Page 15.
PURIFICATION OF DYE-LABELLED OLIGONUCLEOTIDES
APPLICATIONS
Most oligonucleotides dye-labelled at the 5鈥? terminus can be purified
on Poly-Pak cartridges in a manner analogous to the DMT-ON
procedure. The purification is possible when the fluorescent group is
sufficiently lipophilic. In these cases, the dye-labelled
oligonucleotides bind sufficiently to the packing to allow the failure
sequences to be washed away preferentially. Oligonucleotides
labelled at the 5鈥? terminus with fluorescent dyes, poly-aromatic
hydrocarbons and steroids have been purified in this way on Poly-Pak
cartridges. Biotin is not appropriate for this procedure and must be
purified DMT-ON, as described on Page 20.
Figures 10 and 11 show oligonucleotides labelled at the 5鈥?-terminus
with 5鈥?-fluorescein (6-FAM) and Cy-3 CE phosphoramidites and
purified using the procedure described on Page 11.
21
Figure 11: RP HPLC of a Cy-3 labelled
Figure 10: RP HPLC of a FAM labelled
oligonucleotide, following the
oligonucleotide, using the
conditions shown in Figure 1,
conditions shown in Figure 1,
Page 15.
Page 15.
(This page is left intentionally blank.)
22
SOLUTION PREPARATION
SOLUTION PREPARATION
The following solutions may be prepared by the user. Always wear
gloves and protective eye wear while handling corrosive organic
liquids. Read and take heed of the information in Material Safety
Data Sheets supplied by the manufacturers.
1. 2M Triethylamine Acetate
Ingredients:
Glacial Acetic Acid 120mL
HPLC Grade Water 605mL
Triethylamine 275mL
Total Volume 1L
Add the glacial acetic acid to the water. With vigorous stirring,
slowly add the triethylamine. (Note that triethylamine must be of the
highest quality and must be colorless.) The triethylamine will form
an upper layer and slowly will be absorbed into the aqueous acetic
acid. The mixture will become quite warm. When the final solution
has cooled, measure the pH and adjust, if necessary, to 7.0 to 7.5 with
glacial acetic acid or triethylamine.
2. Ammonium Hydroxide/Water (1:10)
23
Add one volume Ammonium Hydroxide (SG 0.88) to ten volumes of
HPLC Grade Water.
3. 2% TFA/Water
Add two volumes of trifluoroacetic acid to 98 volumes of HPLC
Grade Water.
COMMON QUESTIONS
WHY IS MY YIELD NOT HIGHER?
This is probably the most commonly asked question and usually
follows a purification which yields a lower amount of product than
expected. First of all, Poly-Pak cartridges have a finite capacity for
oligonucleotides and this is approximately equivalent to 0.3-0.4
碌moles. A 0.2 碌mole synthesis should be completely purified by a
single cartridge, whereas a 1 碌mole synthesis can be purified using a
Poly-Pak II cartridge or barrel. Assuming a high quality 0.2 碌mole
DNA synthesis, a 20mer should yield about 30 A260 units of crude
and purification should provide at least 10 A260 units of product. The
remainder of the UV absorbing material is in the ammonium
hydroxide loading solution which contains virtually no DMT-ON
product and the diluted ammonium hydroxide wash which contains a
small amount of failure sequences as well as some DMT-ON
product.
If the yield is substantially lower, it is necessary to examine the DNA
synthesis to determine if that was problematical. If the DMT yields
were low, the amount of available DMT-ON product will be
substantially lowered, as will the number of crude A260 units. Even if
DMT yields seem to have been satisfactory, other synthesis factors
can contribute to a low yield. The only way to obtain a satisfactory
answer is to measure the A260 units in the crude product and to
analyze this by RP HPLC.
WHAT PURITY CAN I EXPECT FROM A POLY-PAK?
24
Poly-Pak cartridges and barrels are not very intelligent. They really
do not understand which of the oligonucleotides offered up to them
is the desired product. They are only capable of separating DMT-
ON oligonucleotides from non-DMT containing failure sequences
since the polymeric packing has a high affinity for the lipophilic
DMT group. Although a stepwise gradient of increasing organic
content may be successful in some separations, the Poly-Pak
purification strategy is better understood if looked on as an affinity-
type purification. If the product quality is poor after Poly-Pak
purification, all of the components formerly had a DMT group on
COMMON QUESTIONS
them.
How can that happen? Unfortunately, we do not understand this
fully. However, we do know that a well used and maintained
synthesizer tends to produce high quality products after Poly-Pak
purification while an infrequently used synthesizer with older
reagents installed tends to produce poorer quality oligonucleotides
even after Poly-Pak purification. Several mechanisms may be
implicated in the formation of DMT-ON oligonucleotides which are
not the desired length: Incomplete capping leads to sequences with
point deletions; Incomplete oxidation also leads to sequences with
point deletions; Depurination of dA sites leads to chain scission on
deprotection and oligonucleotides truncated at the 3' terminus;
Incompletely capped CPG gives rise to maverick sequences also
truncated at the 3' terminus; and Unreversed chain branching at dG
sites leads to higher molecular weight products.
Without evading the question any longer, a good synthesizer will
yield oligos after Poly-Pak purification which are >95% pure by CE
or ion-exchange HPLC and which give clean and accurate results in
their intended usage. A stressed synthesizer will yield a product
which is upgraded in quality but which is only around 90% pure.
CAN POLY-PAK CARTRIDGES BE REUSED?
The answer to this question is a qualified yes. We would not 25
recommend their reuse for the purification of PCR primers even
though non-specific binding is very low. However, they may certainly
be reused for the purification of further amounts of the same
oligonucleotide. Just start the process from the beginning. With
each use, yield will drop and backpressure will increase.
HOW ABOUT C18 SILICA CARTRIDGES?
C18 silica RP cartridges are used successfully for the purification of
synthetic oligonucleotides. We even use them here (from Alltech)
COMMON QUESTIONS
since they are available with up to 0.9g of packing material which
gives a good capacity. They are not, however, pH stable and at the
very least the ammonium hydroxide must be evaporated first.
CAN THIS PROCESS DAMAGE OLIGOS?
Not at all! The polymeric packing, frits and cartridge body are totally
inert. However, the solutions must be prepared using the best quality
reagents. Procedures for the formulation of the reagents are described
on Page 23. The two reagents which must be pure are triethylamine
and trifluoroacetic acid. If you forget to dilute the trifluoroacetic
acid, it will destroy the DNA!
HOW CAN LARGE NUMBERS OF SAMPLES BE
PROCESSED?
Poly-Pak cartridges can be used manually or on a vacuum manifold
whereas Poly-Pak barrels are designed for use on vacuum manifolds
only. Using a vacuum manifold, up to 12 (or even 24) samples can be
processed in parallel. We dispense the liquids from wash bottles and,
in this way, 12 samples can be processed in about 20 minutes. Most
users load the samples onto and elute the product from the cartridges
by hand and use the manifold for washes only. Others load and elute
on the manifold. Experimentation will determine which mode is best
for you.
WHEN DO I USE CARTRIDGES AND WHEN BARRELS?
26
Cartridges are useful at any time since they can be used individually
by hand or attached to a vacuum manifold for parallel sample
processing. Barrels are really only intended for parallel processing on
a vacuum manifold. Barrels are a little cheaper since they are less
complex to produce and they do contain a little more packing
material so isolated yields may be about 25% higher using them.
Traces of packing material may occasionally leak from barrels since
the frit system is a simple press fit. If this happens, the traces of
packing can be centrifuged and separated. To our knowledge,
packing has never leaked from the new design of Poly-Pak cartridge.
COMMON QUESTIONS
HOW DO POLY-PAK CARTRIDGES COMPARE WITH
COMPETING PRODUCTS?
We think Poly-Pak cartridges are the best of this type and we are
totally unbiased. Seriously though, all cartridges containing similar
polymeric matrices will purify oligonucleotides to the same extent.
The main differences are in convenience of use. Poly-Pak cartridges
contain very fine polydivinylbenzene beads which we believe have the
highest affinity for DMT-ON oligonucleotides. Poly-Pak cartridges
contain the packing in a tight bed allowing smaller volume washes
than some competitive products. Finally, Poly-Pak cartridges have
comfortable backpressure. We believe this all contributes to making
Poly-Pak cartridges the very best polymeric reverse phase cartridge.
TROUBLESHOOTING
Call or fax with DMT yields and RP HPLC data on the crude
DMT-ON product, if possible, and crude and purified A260 units.
The more data you can provide, the more likely we are to be able to
troubleshoot the problem.
27
CATALOG NUMBERS
Item Catalog No. Pack
Packing and Cartridges
Poly-Pak鈩? Packing Material 60-1000-05 5g
60-1000-25 25g
Poly-Pak鈩? Cartridges 60-1100-01 each
60-1100-10 Pack/10
Poly-Pak鈩? Barrels 60-2100-30 Pack/30
Poly-Pak鈩? II Cartridges 60-3100-01 each
60-3100-10 Pack/10
Poly-Pak鈩? II Barrels 60-4100-30 Pack/30
Reagents and Kits
Wash Solvent Kit 60-4900-01 each
(Sufficient for use with up to 10 cartridges)
2.0M Triethylammonium Acetate 60-4110-52 200mL
(TEAA) HPLC grade 60-4110-57 450mL
60-4110-60 960mL
2% Aqueous Trifluoroacetic Acid 60-4040-57 450mL
28
Vacuum Manifold
12-Port Vacuum Manifold 60-9000-01 each
(Includes glass chamber with rack to hold collection
tubes, aluminum lid with 12 shut-off valves)
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