WO2006132588A1

WO2006132588A1 - Method for the purification of synthetic oligonucleotides containing one or several labels

Info

Publication number: WO2006132588A1
Application number: PCT/SE2006/000687
Authority: WO
Inventors: Anders Hanning; Jenny HELLSTRÖM
Original assignee: Quiatech Ab
Priority date: 2005-06-10
Filing date: 2006-06-12
Publication date: 2006-12-14

Abstract

Liquid chromatographic purification methods, based on differences in partition between a solid phase and a liquid phase, for a product oligonucleotide containing one or several labels and additionally containing at least one cleavable separation tag, are improved by carrying out the combined three steps in sequence; step 1 is chromatographic separation based on the presence or absence of the at least one separation tag; step 2 is cleaving off of the at least one separation tag from the oligonucleotide; step 3 is chromatographic separation based on the presence or absence of at least one of the one or several labels.

Description

METHODS FOR THE PURIFICATION OF SYNTHETIC OLIGONUCLEOTIDES CONTAINING ONE OR SEVERAL LABELS

TECHNICAL FIELD The present invention relates to an improvement in the methods and compounds used for the manufacturing and purification of synthetic oligonucleotides containing one or several labels.

BACKGROUND The chemical synthesis of oligonucleotides is a well established technology.

The commercially most important route for oligonucleotide synthesis is the β- cyanoethyl phosphoramidite route [MJ. Gait (ed.), "Oligonucleotide Synthesis: A Practical Approach, IRL Press, 1984; S.L. Beaucage, M.H. Caruthers, "The Chemical Synthesis of DNA/RNA", Chap. 2 in S.M. Hecht (ed.), "Bioorganic Chemistry, Nucleic Acids", Oxford University Press, 1996; CB. Reese, Tetrahedron, 2002, 58, 8893], but other routes are also well known. In solid support based oligonucleotide synthesis via the phosphoramidite route, the direction of synthesis is most commonly from the 3' end to the 5' end, but synthesis in the reverse direction is also well established. In the following discussion of problems in oligonucleotide synthesis, phosphoramidite-based synthesis in the 3' to 5' direction will be used as a non-limiting example. However, deficiencies in other synthetic strategies are obvious to the skilled person.

In phosphoramidite-based synthesis, a number of impurities in the shape of different oligonucleotides (other than the expected product oligonucleotide) are formed as side products during the synthesis. One such impurity is truncated fragments that are due to the limited coupling yield per phosphoramidite unit. Such truncated 3' fragments are usually capped in order to prevent them from growing further and to form internally deleted oligonucleotides. Another impurity is cleaved apurinic fragments. During the strongly acidic steps of the synthesis cycle some purine bases are cleaved off from the growing oligonucleotide chain. Such apurinic chains are subject to chain cleavage during subsequent alkaline synthesis steps, during which cleavage both 3' and 5' fragments are formed. Another problem is late start of the synthesis (not starting with the intended first phosphoramidite). This may be due to inefficient coupling during the first few synthesis cycles, followed by incomplete or reversible capping, incomplete detritylation, and/or incomplete oxidation [J. Temsamani et al., Nucleic Acids Res., 1995, 23, 1841; The Glen Report, 2004, 17(1), 1, Glen Research, Sterling, VA]. In this case, incorrect truncated 5' fragments are formed. Such fragments may start to grow at the intended oligonucleotide starting sites or at unspecific surface sites on the solid synthesis support. A problem that is specific to the synthesis of labeled oligonucleotides is the possible absence or instability of the label itself. Most commonly labels, e.g. dye labels, are incorporated into the growing oligonucleotide chain during regular synthesis in the form of phosphoramidite derivatives of the label (so called direct labeling method). If this phosphoramidite derivative is not perfectly pure with respect to the label, label- less phosphoramidite will be incorporated. Also, if the label as such or the linker between the label and the phosphoramidite moieties is not stable during the rather aggressive synthesis and deprotection conditions used, oligonucleotide without label or with broken down label may be formed. Also, bulky label phosphoramidites may show a lower coupling yield than standard nucleoside amidites. If low coupling of label phosphoramidite is followed by inefficient or reversible capping, label-less fragments may again be foπned. This problem is especially pronounced during the first few synthesis cycles [J. Temsamani et al., Nucleic Acids Res., 1995, 23, 1841; The Glen Report, 2004, 17(1), 1, Glen Research, Sterling, VA]. There are alternative ways of incorporating labels into oligonucleotides, e.g. in a post-synthetic derivatization reaction [Vinayak et al., US Patent 6,255,476; J.A.M. Vet, S.A.E. Marras, "Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays, Chap. 17 in P. Herdewijn (ed.), "Oligonucleotide Synthesis, Methods and Applications", Humana Press, 2005], but similar considerations concerning the purity are again applicable.

The presence of those impurities may impair the performance of labeled oligonucleotide in demanding applications. As an example, when dual dye- labeled oligonucleotide probes are used in real time PCR (RT-PCR), the presence of impurities may add background fluorescence to the assay as well as cause unspecific hybridization. In both cases, the sensitivity of the assay is lowered [WA. Rudert et al, Biotechniques, 1997, 22, 1140; A.T. Yeung et al, Biotechniques, 2004, 36, 266]. In order to remove impurities, labeled oligonucleotides are most often purified. .

The most common purification method is reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC removes most of the impurities efficiently, but the method is slow (due to slow separation and the need for re- equilibration between runs), expensive (since a lot of hands on time is required), and needs to be modified to suit different kinds of labels. Further, HPLC is an inherently serial method which is not amenable to parallelization, and consequently large scale manufacturing becomes tedious and requires large investments in HPLC equipment. One other problem with HPLC is the risk for cross-contamination of samples, since one column is re-used for many different samples. Moreover, labeled oligonucleotides are often required in desalted form, and HPLC purification with a buffer solution then needs to be complemented with an extra desalting step.

Purification may also be performed by a simpler RP cartridge procedure. This procedure is usually based on the presence of a cleavable hydrophobic protecting group, e.g. a dimethoxytrityl group, at the 5' end of the oligonucleotide. However, simple cartridge purification fails to remove most of the impurity fragments. As an example, the obtained purity of dual labeled probes is usually not good enough for accurate RT-PCR assays.

Several different oligonucleotide purification procedures, most of them concerning unlabeled oligonucleotides, exist and are well established [D.T. Gjerde et al., "DNA Chromatography", Wiley-VCH, 2002; P.I. Oefiier, CG. Huber, J. Chromatogr. B, 2002, 782, 27]. Further, Blocker et al. [US Patent 4,997,927] described a method where an oligonucleotide with a lipophilic protecting group is adsorbed on a solid carrier and subsequently washed, after which the protecting group is cleaved off and the product oligonucleotide is eluted. However, this method is not well suited for labeled oligonucleotides since it does not take the presence or stability of the label as such into account. Thus, as is apparent to the skilled person, the label may be missing due to e.g. impure labeling reagents, label breakdown during chemical synthesis, or hydrolysis of label linker. Oligonucleotides with the correct sequence but lacking the label are not removed in the cited method. Hirano et al. [US Patent 5,413,762] described a device comprising switching between an affinity cartridge and an HPLC column. However, in effect this is no more than a combined cartridge and HPLC purification system. Kunihiko et al. [JP2000344791] described a system for optimized and automated HPLC purification of oligonucleotides. However, this system does not overcome the basic drawbacks of HPLC purification. Gjerde et al. [US 2002/0102563 Al] described a method for polynucleotide purification based on ion-pairing chromatography. However, this method is mainly a size- based separation method and is not well suited to purify labeled oligonucleotides. Johansen [WO 03/080834 A2] described a method based on gradient anion exchange chromatography. Again, however, the method is mainly a size-based separation method and is not well suited to purify labeled oligonucleotides.

Purification methods based on the presence of a hydrophobic moiety at the 5' end fail to remove 5' fragments of apurinic fragments, since such fragments possess the correct 5' end. Methods for removal of 5' apurinic fragments have been described, e.g. based on the use of selectively cleavable linkers [Kwiatkowski et al., Nucleic Acids Res. 1996, 24, 4632; Holmberg, US Patent 5,589,586; Kwiatkowski et al., US Patent 6,291,669; Kwiatkowski et al., US Patent 6,429,309; Kwiatkowski et al., US Patent 6,646,118]. Combination of such methods with purification methods based on the presence of a hydrophobic moiety at the 5' end has the potential to yield very pure unlabeled oligonucleotides. However, also such combined methods fail to take the stability or purity of the label itself into account.

Methods for separating oligonucleotides by means of two separation tags attached to both ends of the oligonucleotide have also been described

[Kwiatkowski, WO 03/066651 Al]. Such methods yield product oligonucleotide with correct 3' and 5' ends. The described methods are limited to cleavable separation tags, and in particular to tags yielding a 3' hydroxyl moiety upon cleavage. However, such methods do not take the presence or stability of the label as such into account. Thus, as is apparent to the skilled person, if the label is missing due to e.g. impure labeling reagents, label breakdown during chemical synthesis, or hydrolysis of label linker, the product oligonucleotide may be contaminated by fragments with correct ends but lacking the label. Labeling reagents are not generally 100% pure [Product Catalog, ChemGenes Corp., Wilmington, MA]- the few percent impurities in the labeling reagent may directly be reflected in a few percent impurities in the labeled oligonucleotide. Also, it is well known [R. Vinayak, Tetrahedron Lett. 1999, 40, 7611; M.H. Lyttle et al., J. Org. Chem., 2000, 65, 9033; Product Catalog 2004, Glen Research, Sterlin, Va] that many labels, e.g. dye labels, are not completely stable to the harsh synthesis and deprotection condition encountered during oligonucleotide manufacturing. Thus, many labels, especially dye labels, may be partly broken down during the manufacturing process.

A process for separating and deprotecting oligonucleotides with one or two cleavable separation functions has also been described [Kwiatkowski, WO 2004/020449 Al]. This process, based on deprotection in an organic solvent, provides mild and efficient deprotection methods. Again, however, it does not provide a separation method based on the presence or stability of the label as such. In the cited patent application [p. 17, lines 10-23], an example is presented where a dual dye oligonucleotide probe is purified by a simple trityl-on purification with an extra hydrophobic, modified trityl moiety, in combination with synthesis using a selectively cleavable disiloxyl linker to remove apurinic fragments. This method may work well in special cases, however, for reasons stated above, the method should not be applicable to the purification of dual dye probes in general. To show this, a number of different dual dye probes were purified with the method described in the cited example (See Example 1 below for experimental details). In all cases, a significant amount of oligonucleotide lacking the quencher moiety was obtained; for Dabcyl quencher 4-12% and for TAMRA quencher 11-12%.

Thus, there exists a need for improved purification methods for labeled oligonucleotides, based on the actual presence, stability, and purity of the label or labels per se.

SUMMARY

The present invention is based on the discovery that liquid chromatographic purification methods, based on differences in partition between a solid phase and a liquid phase, for product oligonucleotides containing one or several labels and additionally containing at least one cleavable separation tag can be improved by carrying out the combined three steps in sequence; step 1 is chromatographic separation based on the presence or absence of the at least one separation tag; step 2 is cleaving off of the at least one separation tag from the oligonucleotide; step 3 is chromatographic separation based on the presence or absence of at least one of the one or several labels. Thus, in one aspect, the present invention provides a method where, in step 1, the oligonucleotide containing the at least one separation tag has a higher affinity for the solid phase than any oligonucleotide lacking the at least one separation tag. Thus, the oligonucleotide containing the at least one separation tag can be retained on the solid phase while any oligonucleotide lacking the at least one separation tag can be selectively eluted. In another aspect, the present invention provides a method where, in step 3, the oligonucleotide containing the at least one label has a higher affinity for the solid phase than any oligonucleotide lacking the at least one label. Thus, the oligonucleotide containing the at least one label can be retained on the solid phase while any oligonucleotide lacking the at least one label can be selectively eluted.

The essence of the idea is to conduct all three steps in sequence. In step 3, the separation is based on the presence and stability of the at least one label moiety itself- this selectivity is a prerequisite for any purification method for labeled oligonucleotides. In this way, fragments lacking the at least one label is efficiently removed. In order to perform step 3, the at least one separation tag must first be cleaved off from the oligonucleotide; this is performed in step 2. However, before being cleaved off, the at least one separation tag is utilized for the first separation in step 1. By conducting these three steps in sequence, it was discovered that purified product oligonucleotide containing one or several labels could be obtained.

In Figure 1, a simplified schematic drawing of the method of the invention is presented.

The advantages of the invention will be better understood from the following discussion of the beneficial influence of different aspects and embodiments of the invention. Clarifying examples will mainly refer to oligonucleotides labeled with dye moieties, but, as will be apparent to the skilled person, the invention is not limited to such labels.

DESCRIPTION OF THE DRAWINGS

Reference is being made to the accompanying drawings, wherein: Figure 1 is a simplified schematic drawing of the method of the invention; Figure 2 is a simplified schematic drawing of an exemplary manufacturing of a dual dye labeled oligonucleotide; Figure 3 shows a chromatogram of an oligonucleotide labeled with Dabcyl + FAM, manufactured according to Example 1; Figure 4 shows chromatograms of four different oligonucleotides, each containing two dye labels (BHQl + FAM, BHQl + TET, BHQl + HEX, Dabcyl + FAM), manufactured according to Example 4.

Figure 5 shows a chromatogram of an oligonucleotide labeled with TAMRA + TET, manufactured according to Example 5.

DETAILED DESCRIPTION

In this context, the expression "liquid chromatography" is used to denote any separation method based on differences in partition between a solid phase and a liquid phase. Both liquid-solid chromatography and liquid-liquid chromatography, where an insoluble liquid is adsorbed onto a solid phase, are included. The term "solid phase" also includes gel media, like e.g. swellable organic polymers or silica gel. The "chromatography column" is the physical device that contains the solid phase. The solid phase may e.g. have the form of small particles, beads, a membrane, a frit, a sintered cake, or a monolith. The expressions "high performance liquid chromatography" or "HPLC" and "low pressure cartridge chromatography" are used — the border between those is generally not well defined. In this context "HPLC" refers to closed column systems where the liquid is pumped at several bars, several tens of bars, or even hundreds of bars pressure; such columns may offer hundreds or thousands of theoretical plates. "Low pressure cartridge chromatography" refers to column systems where the liquid is driven by low pressure, i.e. on the order of a few bars or less; such columns generally offer a small number of theoretical plates. The term "label" is used to denote any non-nucleic acid moiety coupled to an oligonucleotide. A common class of labels is dyes, i.e. chemical moieties that absorb light in the visible or near infrared wavelength range. Dyes include fluorophors and quenchers. However, labels are not limited to dyes, but may include e.g. affinity ligands, protecting groups, antigens, enzyme substrates, lipophilic labels, crosslinking functionalities, or hybridization-stabilizing moieties. An oligonucleotide may contain one or several labels - of these are at least one utilized for the separation in step 3. If more than one label is utilized for the separation, these labels may be of identical or different kinds. Further, the oligonucleotide may contain one or several additional labels that are not as such utilized in the separation. Labels are functional, integral parts of the product oligonucleotide and are not to be cleaved off during the manufacturing of the product oligonucleotide. The expression "dual labeled probe" is used to denote an oligonucleotide labeled with at least one fluorophor and at least one quencher. Such probes are commonly used in nucleic acid assays, e.g. real time PCR. Different PCR probe variants exist, including but not limited to 5' exonuclease probes, molecular beacons, TaqMan^® probes, Amplifluor^® primers, Quantitect^® probes, and Scorpion^® primers. There are also PCR probes based on oligonucleotides labeled with a single dye, e.g. LUX^® primers and Light Cycler Hybridization Probes. Some PCR probes may have a combined primer and probe function. The expressions "outer end" and "inner end" of an oligonucleotide refer to the direction in which the oligonucleotide is being synthesized. Oligonucleotides are most commonly synthesized from the 3' end to the 5' end - in this case, the 5' end constitutes the outer end and the 3' end constitutes the inner end. However, synthesis in the reverse direction is also well established. The term "separation tag" denotes any chemical moiety that is coupled to the oligonucleotide that allows separation based on the presence or absence of the tag, and that is selectively cleavable from the oligonucleotide. Thus, the separation tag as such is not a functional part of the product oligonucleotide. In order to connect the separation tag to the oligonucleotide chain, the tag may be attached to some kind of reactive linker or spacer moiety. The reactive group may be e.g. a phosphoramidite. Upon cleavage of the separation tag the linker or spacer may remain attached to the product oligonucleotide. The linker or spacer may e.g. be a short aliphatic hydrocarbon or polyol chain. It may also be e.g. a nucleotide, which may then constitute an integral part of the product oligonucleotide. A separation tag may comprise a trityl moiety modified with 1-3 alkoxy groups (e.g. a monomethoxytrityl, a dimethoxytrityl, or a trimethoxytrityl moiety), or a pixyl (9-phenylxanthenyl) moiety modified with 0-1 alkoxy groups. For example, the separation tag may comprise a 4-hexyloxy-4'-methoxytrityl, A- decyloxy-4'-methoxytrityl, 4-hexadecyloxy-4'-methoxytrityl, (4- octadecyloxyphenyl)-9-xanthyl, 4,4'-bis-hexyloxytrityl, 4,4'-bis-decyloxytrityl, 4,4'-bis-hexadecyloxytrityl, 4-octadecyloxytrityl, 4-hexadecyloxytrityl, A- decyloxytrityl, or a 4-hexyloxytrityl moiety. A separation tag may comprise an aliphatic hydrocarbon chain or one or several diol moieties. The cleavable function of a separation tag may further comprise e.g. an acetal group, a thioacetal group, a siloxyl group, a disiloxyl group, a hydrocarbyldithiomethyl group, or a photocleavable group. The term "oligonucleotide" includes oligomers of ribonucleotides and deoxyribonucleotides that have a 3 '-5' phosphodiester backbone, as well as oligomers with other backbone structures, e.g. methyl phosphonate and phosphorothioate linkages. The oligomers may also contain non-standard monomers like e.g. inosine, nubularine, modified sugar moieties, and modified base moieties like 7-deazapurine, isocytidine, pseudo-isocytidine, isoguanosine, and 8-oxopurine. Also peptide-nucleic acids (PNA) and locked nucleic acids (LNA) are included, as well as combinations of LNA or PNA with oligomers of ribonucleotides and deoxyribonucleotides.

One aspect of the present invention is characterized in that all three steps are performed with the oligonucleotide retained on a liquid chromatography column. This is a simple and straightforward aspect. However, sometimes step 2, the cleavage of the separation tag, may disrupt the chromatographic partition equilibrium. Step 2 may e.g. involve drying steps or involve the use of liquids in which the product oligonucleotide is highly insoluble, and may therefore cause bulk precipitation of the oligonucleotide. Step 2 may also, e.g. alter the counterion state of the oligonucleotide. The reestablishment of a chromatographically advantageous counterion state may be kinetically hindered. In such cases, it was found unexpectedly beneficial to use another aspect of the invention in which the oligonucleotide is eluted from a liquid chromatography column after step 2. The sample may then be reapplied (reloaded) to a chromatography column under more advantageous chromatographic conditions. In one embodiment of this aspect, the establishment of such conditions may involve altering the composition of the sample solution, e.g. changing the proportions of aqueous buffer versus organic solvent, changing the pH, changing the composition or concentration of ions, or diluting or concentrating the sample solution. In most cases, cleavage off of the at least one. separation tag in step 2 can be performed with the sample still adsorbed on the solid phase. As an example, particularly efficient methods for cleavage of separation tags are described by Kwiatkowski [WO 2004/020449]. However, in some instances it may be desirable to first elute the sample and then perform the cleavage. Therefore, one aspect of the invention provides a method wherein step 2 is performed with the oligonucleotide in solution.

In one simple and straightforward aspect of the invention, steps 1 and 3 are performed on the same chromatography column. This aspect is used when the sample is not eluted until the end of the purification process, but it can also be used in cases where the sample is eluted after step 1 or step 2 - the sample is then reapplied to the column before step 3. However, in some instances, e.g. for reasons of chromatographic efficiency or for reasons of simple and streamlined processing, it may be desirable to use another chromatographic column in step 3. Therefore, in another aspect of the invention, steps 1 and 3 are performed on different liquid chromatography columns.

In one aspect of the invention, the separation is based on reverse phase chromatography. This chromatographic separation mode is the most common in oligonucleotide separation by means of separation tags. One such example is the well-established trityl-on purification method. In one embodiment of this aspect, the at least one cleavable separation tag is chosen to comprise a trityl moiety modified with 1-3 alkoxy groups, a pixyl moiety modified with 0-1 alkoxy groups, a 4-hexyloxy-4'-methoxytrityl, 4-decyloxy-4'-methoxytrityl, A- hexadecyloxy-4 ' -methoxytrityl, (4-octadecyloxyphenyl)-9-xanthyl, 4,4 ' -bis- hexyloxytrityl, 4,4'-bis-decyloxytrityl, 4,4'-bis-hexadecyloxytrityl, 4- octadecyloxytrityl, 4-hexadecyloxytrityl, 4-decyloxytriryl, or a 4-hexyloxytriryl moiety.

In one aspect of the invention, steps 1 and 3 are based on HPLC. HPLC has the potential to yield very good separation efficiency. However, for reasons of cost, process time, and simplicity, another aspect of the invention, based on low- pressure cartridge chromatography, may be preferred. This aspect has inter alia the advantage that the liquid may be driven by some low pressure mechanism, like gravity, vacuum, compressed gas, a syringe pump, or a peristaltic pump. Vacuum or gas flow may be used for any intermediate column drying step. Another advantage of this aspect is that the purification of several oligonucleotides may be performed more or less simultaneously in parallel. The purifications may e.g. take place in a 6, 12, 24, 48, 96, 384, or 1536 well plate or in loose cartridges placed in a 6, 12, 24, 48, 96, 384, or 1536 hole plate. The procedure may be performed in fully parallel mode, such that liquid is dispensed to and pumped through all columns simultaneously, or in semi-parallel mode, such that liquid is dispensed to and pumped through single columns or groups of columns in rapid sequence. Other ways of altering the degree of parallelization are obvious to the skilled person. The degree of parallelization may depend on, inter alia, the hardware system used to carry out the procedure. Another advantage of using non-expensive, disposable cartridges is that any risk of cross- contamination between samples on the column is eliminated, as is the need for washing and re-equilibration of the column between runs.

In one aspect of the invention are steps 1 and 3 based on gradient elution. Gradient elution often yields clean and rapid separations, and is well suited to separate compounds with large affinity differences, e.g. compounds with and without separation tags, respectively. One particularly simple kind of gradient is the step gradient, where the composition of the eluent is changed in one or several steps (as opposed to the ramp gradient, where the composition changes gradually over time). It is well known that the hydrophobicity (and consequently the affinity in RP chromatography) of oligonucleotides depends on the base sequence. In order to compensate for these hydrophobicity variations, a hydrophobic ion-pairing counter-cation is often added to the eluent, e.g. triethylamine. The consequence is then that the affinity becomes length- dependent (depending on the number of counterions) rather than sequence- dependent [D.T. Gjerde et al., "DNA Chromatography", Wiley- VCH, 2002]. Because of these affinity variations, it has not been considered possible to purify oligonucleotides of different length and sequence with a general, pre-defined step gradient. However, it was unexpectedly discovered that for oligonucleotides labeled with at least one hydrophobic label, the affinity in RP chromatography depends mainly on the hydrophobicity of the at least one label, and to a much smaller degree on the length and sequence of the oligonucleotide as such. Therefore, based only on the nature of the at least one label, a suitable step gradient can be pre-defined, more or less independent of the length and sequence of the oligonucleotide. This makes the step gradient method much more generally useful than what has been previously believed. One advantage of step gradient elution is that the different eluents can be ready-made or pre-mixed offline. Another advantage of a well-designed step gradient elution is that fraction collection can quite easily be performed by simply collecting the eluates of the different steps. The exact retention volume of a certain compound can vary slightly depending on the number of theoretical plates of the column. In this respect, cartridge columns with their low number of theoretical plates offer an extra advantage in terms of rapid elution. Step gradients may be designed in different ways. For example, in step 3 of the present invention, the composition of the first eluent is chosen so that oligonucleotide containing the at least one label is retained on the solid phase while any oligonucleotide lacking the at least one label is selectively eluted. Then a second eluent is applied which elutes the oligonucleotide containing the at least one label. The composition of this second eluent may be chosen such that any impurity, which has a higher affinity for the solid phase than the oligonucleotide containing the at least one label, is not eluted. An alternative to pre-mixing of eluents is to have the gradient mixed by the liquid chromatography system. This alternative is often used with HPLC₅ and it can be used for step as well as ramp gradients. The most common way of forming a gradient, especially in reverse phase chromatography, is to mix an organic solvent with an aqueous buffer at varying proportions. For gradient elution of oligonucleotides, aqueous buffers containing tertiary amines or quaternary ^"ammonium ions, like e.g. trimethylamine, triethylamine, tripropylamine, tributylamine, tetramethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium may be used (including different isomers of the propyl and butyl species). For the purification of labeled oligonucleotides the said cations, functioning as counterions to the anionic phosphate groups of the oligonucleotide, offer a further advantage, i.e. the possibility to balance the hydrophobicity of the oligonucleotide backbone itself in order to optimize the separation efficiency and robustness.

In one aspect of the invention the at least one label, on which the separation in step 3 is based, is one or several dye moieties. If more than one label is utilized, the labels may be of identical kind or of different kinds. Dye labels are often used on oligonucleotides. The label may be a fluorescent label, either alone or in combination with a quenching label; in the latter case the oligonucleotide is a dual labeled probe. The label may also be a quencher label alone. Sometimes oligonucleotides are labeled with several dye moieties, e.g. more than one fluorophor label or more than one quencher label. In addition to their functional use for optical detection, dye moieties are often large, hydrophobic molecules, well suited for separation by reverse phase chromatography. The separation in step 3 may be based on the quencher, the fluorophor, or both. In most cases, it is advantageous to let the at least one label, on which the separation in step 3 is based, comprise a quencher moiety, since quencher-less fragments are most detrimental to the performance of dual labeled probes in RT-PCR. Suitable quenchers may belong e.g. to the classes of azobenzenes, modified azobenzenes, nitrothiazoles, or rhodamines. Suitable quenchers include, but are not limited to, Dabcyl, Dabsyl, TAMRA^®, ROX^®, Black Hole Quencher^® 0, Black Hole Quencher^® 1 , Black Hole Quencher^® 2, Black Hole Quencher^® 3, Eclipse^® Dark Quencher, Elle^® Quencher, NFQ, QSY-7^®, and Methyl Red. Suitable fluorophors may belong e.g. to the classes of fluorescein dyes, rhodamine dyes, sulfonated rhodamine dyes, cyanine dyes, or 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes. Suitable fluorophors include, but are not limited to, fluorescein, tetramethylrhodamine, FAM^®, TET^®, HEX^®, JOE^®, NED^®, VIC^®, TAMRA^®, ROX^®, Cy^® 3, Cy^® 5, Cy^® 3.5, Cy^® 5.5, Cy^® 7, Oregon Green^® fluorophors, Texas Red^®, Light Cycler^® Red 640, Rhodamine Red, Rhodamine Green, R6G, Yakima Yellow^®, Redmond Red^®, Cascade Blue^®, Pulsar 650^®, Bodipy^® fluorophors, Alexa^® fluorophors, CAL^® Fluor fluorophors, and Quasar^® fluorophors. In Figure 2, a simplified schematic drawing of an exemplary manufacturing of a dual dye labeled oligonucleotide is presented.

In table 1 suitable labels are listed. These are selected from the classes of fluorophors such as fluorescein dyes, rhodamine dyes, sulfonated rhodamine dyes, cyanine dyes, 4,4-difiuoro-4~bora-3a,4a-diaza-s-indacene dyes, quenchers such as dyes azobenzenes dyes, modified azobenzenes dyes, nitrothiazole dyes. In one aspect of the invention, the at least one separation tag is attached to the outer end of the product oligonucleotide. In this way, oligonucleotides with correct outer ends are obtained in step 1. Any truncated fragments are removed in the purification. Also, the inner parts of any apurinic, cleaved fragments are removed. In most cases, oligonucleotides are synthesized in the 3' to 5' direction; in this case the outer end corresponds to the 5' end. In one aspect of the invention, the at least one label, on which the separation in step 3 is based, is attached to the inner end of the product oligonucleotide. In this way, oligonucleotides with correct inner ends are obtained in step 3. The outer parts of any apurinic, cleaved fragments are removed. Moreover, oligonucleotides starting to grow late in the synthesis - hence not incorporating the first, labeled phosphoramidite - are removed. Further, fragments where the label is missing due to e.g. impure labeling reagents, label breakdown during chemical synthesis, or hydrolysis of label linker, are removed. With 3' to 5' synthesis, the inner end corresponds to the 3' end. Purification based on separation tag at the outer, 5' end and label at the inner, 3' end is especially well suited for manufacturing of 3' exonuclease probes with a quencher at the 3' end and a fluorophor at the 5' end.

In one aspect of the invention, the cleaving off of the at least one separation tag is performed in an organic solvent. As an example, particularly efficient methods for cleavage of separation tags in organic solvents are described by Kwiatkowski [WO 2004/020449]. In the very common case of trityl-on purification, the trityl (or related separation tag) may be removed by e.g. dichloroacetic acid or trichloroacetic acid in dichloromethane, dichloroethane, or toluene. In one embodiment of this aspect, the separation tag is eluted in step 2. In reverse phase chromatography, the separation tag is hydrophobic. Such a cleaved off separation tag can easily be eluted in an organic solvent, while the elution in a water-based cleavage solution may be more problematic. As an example, cleaved of trityl tags are easily eluted in dichloromethane. The main advantages of eluting the separation tag in step 2 are that the tag will not interfere with the subsequent separation in step 3, and there is no risk of the tag contaminating the final product oligonucleotide solution.

In addition, the present invention is based on the idea that a phosphoramidite reagent comprising a cleavable separation tag for use in any of the above described inventive methods, aspects, or embodiments, may be chosen to comprise a trityl moiety modified with 1-3 alkoxy groups, a pixyl moiety modified with 0-1 alkoxy groups, a 4-hexyloxy-4'-methoxytrityl, 4-decyloxy-4'- methoxytrityl, 4-hexadecyloxy-4 ' -methoxytrityl, (4-octadecyloxyphenyl)-9- xanthyl, 4,4'-bis-hexyloxytrityl, 4,4'-bis-decyloxytrityl, 4,4'-bis- hexadecyloxytrityl, 4-octadecyloxytrityl, 4-hexadecyloxytrityl, 4-decyloxytrityl, or a 4-hexyloxytrityl moiety. Further, the present invention is based on the idea that the solid separation phase for use in any of the above described inventive methods, aspects, or embodiments, may be based on polystyrene, partly crosslinked polystyrene, modified polystyrene, a polystyrene copolymer, polyethylene, fluorinated polyethylene, charcoal, graphite, derivatised silica, or hydrocarbyl Sepharose^®. The silica may be derivatised by e.g. C4, C8, or Cl 8 groups. The hydrocarbyl group on the Sepharose^® may be e.g. butyl, octyl, or phenyl.

It is essential that the chromatographic properties of the separation tag and of the solid phase are matched such that the affinity of the solid phase for oligonucleotide derivatized with at least one separation tag is higher than the affinity of the solid phase for any oligonucleotide (without label or containing the said one or several labels) lacking the at least one separation tag. Thus, for a specific kind of labels, it is essential to select the combination of the separation tag and the solid phase such that the labels themselves do not interfere with the separation tag-based separation step. As an example, in the purification of dual labeled probes by reverse phase chromatography, it is important that the hydrophobic contribution of the at least one separation tag is significantly higher than the combined hydrophobic contribution of the labels. To this end, the common dimethoxytrityl separation tag may not do, but a significantly more hydrophobic separation tag may have to be chosen. Here it must be noted that the selection of separation tag depends on what solid phase is being used; the reasoning based on hydrophobicity may actually represent an over-simplification in this respect. It is the actual affinity of the separation tag at hand to the solid phase at hand - as compared to the affinity of the label or labels at hand to the solid phase at hand - that has to be considered.

The expression "comprising" as used herein should be understood to include, but not be limited to, the stated items.

All references cited herein are hereby incorporated by reference in their entirety. The idea of the invention will now be illustrated by the following, non- limiting examples.

EXAMPLES

Reagents and Analytical and Preparative Methods Used in the Examples

Unless otherwise stated, the following reagents and methods were used in the Examples that follow this methods section.

2-[9((4-octadecyloxy)phenyl)xanthen-9-yloxy]-ethanol (Cl 8Px-O-EG) and its phosphoramidite derivative l-[9-((4-octadecyloxy)phenyl)xanthen-9-yloxy]-4- diisopropylamino-7-cyano-3,5-dioxa-4-phospha-heptan were prepared for use as separation tag. Solid synthesis support for oligonucleotide synthesis was either a commercially available controlled pore glass CPG (Thymidine succinyl functionalized 0.2 μmol, 1000 A, Applied Biosystems, Foster City, CA) or a Thymidine disiloxyl functionalized CPG (25-35 μmol/g, 1000 A, Quiatech, Uppsala, Sweden). TAMRA-dT phosphoramidite and 6-Fluorescein (FAM) phosphoramidite were obtained from Glen Research, Sterling, VA. Dabcyl, EDEX and TET phosphoramidites were obtained from ChemGenes, Wilmington, MA. BHQl and BHQ2 phosphoramidites were obtained from Biosearch Technologies, Novato, CA. All commercial chemicals were of synthesis quality and were used without further purification.

Oligonucleotide syntheses were performed on an Applied Biosystems 394 DNA/RNA synthesizer instrument. The first phosphoramidite coupled to the solid synthesis support was one of the quenchers listed above followed by the oligonucleotide synthesis. One of the fluorophores listed above and the C 18Px-O- EG amidite were coupled last. The Cl 8Px acts as a cleavable separation tag during the purification procedure while the ethylene glycol moiety remains attached to the oligonucleotide. All couplings were performed under conditions recommended by the manufacturer for 0.2 μmol scale synthesis. The amidites used for the oligonucleotides were protected by benzoyl (dA, dC) and isobutyryl or dimethylformamidme (dG) at the exocyclic amine functions. Purification column resin was a poly(styrene-divinylbenzene) polymer used in a cartridge column. The automatic liquid handling system used for the purification procedure was a Gilson Aspec instrument.

Analytical liquid chromatography of the purified labeled oligonucleotides was performed on an Agilent HPLC system, equipped with a Chromolith

Performance RP 18 column (Merck) or a Gemini RP C18 column (Phenomenex), diode array detector, using a linear gradient of solvent A: acetonitrile 5% v/v in triethylammonium acetate 0.1 M, pH 7 and solvent B: acetonitrile 80% v/v in triethylammonium acetate 0.1 M, pH 7.

EXAMPLE l

Synthesis and Purification of Dual Dye Labeled Oligonucleotide by Simple

Trityl-on Purification

Synthesis of the dual dye labeled oligonucleotide was done as described previously [Kwiatkowski, WO 2004/020449 Al₅ p. 17, lines 10-23], except that an amino functionalized CPG was used as solid synthesis support (Millipore, Lincoln Park, NJ). Two standard thymidine amidites were first coupled to the support followed by a disiloxyl amidite and then the oligonucleotide synthesis continued as described. The support was incubated in ImL tert-butylamine 50% v/v in ethanol for 2h in room temperature for removal of cyanoethyl protecting groups and cleavage of apurinic sites. The supernatant was discarded and the support washed with acetonitrile, acetonitrile in water 50% v/v and finally acetonitrile again to eliminate all cleaved apurinic 5 ' fragments. The acetonitrile was discarded and the oligonucleotide was cleaved from the support by incubation in 300μL l-methyl-2-pyrrolidone 50% v/v in triethylamine 33% v/v and triethylamine-3HF 17% v/v for 2h in room temperature. The supernatant was removed and transferred to a Sarstedt tube, the support was washed with 300μL water which was added to the supernatant. 1.4mL tert-butylamine 33% v/v in water 33% v/v and methanol 33% v/v was added to the supernatant. Deprotection continued in an oven for 15h in 55°C. The purification column was conditioned with acetonitrile and acetonitrile in triethylammonium acetate buffer. The sample was applied to the purification column and the column was washed with acetonitrile in triethylammonium acetate buffer extensively to remove truncated and apurinic 3 ' fragments. The column was dried with compressed nitrogen and washed with ImL dry acetonitrile to make the system water free. Removal of the C 18Px was done by applying 2mL trichloroacetic acid 2% w/w in dichloromethane followed by ImL acetonitrile. The acid treatment and acetonitrile wash was repeated one time and then the product was eluted with acetonitrile in triethylammonium acetate buffer. The product was analyzed by RP HPLC. Several attempts to purify Dabcyl + FAM probes using this method was conducted and never resulted in a pure product. Contaminating species lacking the Dabcyl quencher was always present in the range from 4-12%. For TAMRA + FAM and TAMRA + TET the contaminants lacking the TAMRA quencher was 11-12%. In Figure 3, a chromatogram of an oligonucleotide labeled with Dabcyl + FAM is presented. The presence of impurities is obvious, and the presence of less hydrophobic quencher-less fragments before the main product peak is especially noticeable.

EXAMPLE 2 Solid Support Based Synthesis of Labeled Oligonucleotides

All syntheses were done using the ABi 394 DNA/RNA synthesizer operated at standard 0.2 μmol synthesis cycle with reagents and under conditions recommended by the manufacturer. The synthesis support was either thymidine succinyl or thymidine disiloxyl derivatized CPG. The same oligonucleotide sequence was used in all labeled oligonucleotides (length 29 bases) except for Dabcyl + FAM where the oligonucleotide was 26 bases long and for TAMRA + FAM where the oligonucleotide was 21 bases long. Labeled oligonucleotides with BHQl + FAM or HEX or TET, BHQ2 + TAMRA, Dabcyl + FAM, and TAMRA + FAM or TET were synthesized. The couplings of the labels were done according to the recommendations of the manufacturer. EXAMPLE 3

Cleavage and Deprotection of the Synthetic Oligonucleotides

Different cleavage and deprotection schemes for oligonucleotides synthesized on different synthesis supports were used. In the case of thymidine succinyl support the oligonucleotide was cleaved from the support by incubating the support in a Sarstedt screw-lock tube in either 1 mL ammonium hydroxide (32%) 1 h in room temperature plus 15 h in 55⁰C or in 1.4 mL tert-burylamine 33% v/v in water 33% v/v and methanol 33% v/v during 15 h in 55°C. In the case of thymidine disiloxyl support the oligonucleotide was cleaved from the support by incubation in 300μL l-methyl-2-pyrrolidone 50% v/v in triethylamine 33% v/v and triethylamine-3HF 17% v/v for 2 h in room temperature and deprotected in either ammonium hydroxide (32%) 15 h in 55°C or in 1.4 mL tert-butylamine 33% v/v in water 33% v/v and methanol 33% v/v during 15 h in 55⁰C.

EXAMPLE 4

Purification of the Synthetic Oligonucleotides by Step Wise Elution Variant 1

¹A to ¹A of each of the full syntheses were used in the purification procedure. The oligonucleotide samples were dissolved in a mixture of organic solvent and triethylammonium acetate buffer. The solvent was either acetonitrile or methanol. The purification column was conditioned first with acetonitrile and then with the organic solvent and buffer mixture. The sample was applied to the column and oligonucleotide fragments lacking the C 18Px-O-EG tag were washed out with one additional volume of the same buffer mixture. The column was dried with compressed nitrogen and washed with 1 mL dry acetonitrile to make the system water free. Removal of the C 18Px was done by applying 2 mL trichloroacetic acid 2% w/w in dichloromethane followed by 1 mL acetonitrile. The acid treatment and acetonitrile wash was repeated one time and then the column was dried again with compressed nitrogen. Step wise elution was done with 2 mL of a lower concentration of solvent in buffer before eluting the pure product. The product was analyzed by RP-HPLC. Figure 4 shows the RP-HPLC chromatograms of BHQl + FAM₅ BHQl + TET, BHQl + HEX and Dabcyl + FAM probes. In all cases, the amount of quencher-less oligonucleotide fragments was at or below the quantification limit of 0.5%.

EXAMPLE 5

Purification of the Synthetic Oligonucleotides by Step Wise Elution Variant 2

¹A to 1/1 of each of the full syntheses were used in the purification procedure. The oligonucleotide samples were dissolved in a mixture of organic solvent and triethylammonium acetate buffer. The solvent was either acetonitrile or methanol. The purification column was conditioned first with acetonitrile and then the organic solvent and buffer mixture. The sample was applied to the column and oligonucleotide fragments lacking the C 18Px-O-EG tag were washed out with one additional volume of the same buffer mixture. The column was dried with compressed nitrogen and washed with dry acetonitrile to make the system water free. Removal of the C 18Px was done by applying trichloroacetic acid 2% w/w in dichloromethane followed by acetonitrile. The acid treatment and acetonitrile wash was repeated one time and then the column was dried again with compressed nitrogen. The full sample was eluted by applying the solvent- containing triethylammonium acetate buffer. The sample was diluted with triethylammonium acetate buffer to decrease the solvent concentration. The column was reconditioned with acetonitrile and a lower concentration solvent- containing triethylammonium acetate buffer. The diluted sample was reapplied to the column. Step wise elution was done with a lower concentration solvent- containing buffer before eluting the pure product. The product was analyzed by RP-HPLC. Figure 5 shows the RP-HPLC chromatograms of TAMRA + TET, BHQ2 + TAMRA, and TAMRA + FAM probes. For the first two probes, the amount of quencher-less oligonucleotide fragments was below the quantification limit of 0.5%. For the TAMRA + FAM probe, the amount of quencher-less probe was 0.8%. EXAMPLE 6

Study of the retention behavior of labeled and unlabeled oligonucleotides Two sets of oligonucleotides were synthesized. Both sets contained four different sequences: one 15-mer with 53% AT content, one 29-mer with 62% AT content, one 45-mer with 33% AT content, and one 45-mer with 69% AT content. One set was unlabeled; one set was labeled with a TET fluorophor at the 3' end. Further, the 29-mer was synthesized with two labels: TET at the 3' end and TAMRA at the 5' end. The oligonucleotides did not contain any separation tag and were not purified.

In order to study the retention behavior of the oligonucleotides, a Merck- Hitachi HPLC system with a low pressure gradient mixer and a diode array detector was used. A short HPLC column packed with a poly(styrene- divinylbenzene) solid phase was used. The oligonucleotide samples were applied to the column and eluted with a ramp gradient. Solvent A was 0.1 M triethylamine acetate pH 7 and solvent B was methanol. The ramp gradient started with isocratic 20% B for 5 minutes, and then increasing to 70% B in 30 minutes. The retention time for the different samples was registered and the % methanol at which the different oligonucleotides were eluted was estimated. The result is presented in the table.

For unlabeled oligonucleotides, the retention time increases monotonously and significantly with the length. This is in line with established knowledge, and is due to more TEA cations ion pairing with longer oligos, making them more hydrophobic. The higher hydrophobicity of A and T as compared to G and C is also obvious. For TET-labeled oligonucleotides, all samples elute within a 1% methanol range. This is due to the hydrophobic TET moiety dominating the hydrophobicity of the whole oligonucleotide, making the influence of the base sequence and the triethylamine counterions less pronounced. The elution difference between TET-labeled oligonucleotide and

TAMRA/TET-labeled oligonucleotide is on the order of 7-8% methanol.

From the above data, it is obvious that a robust step-gradient method can be designed for the separation of TET-labeled oligonucleotide and TAMRA/TET- labeled oligonucleotide. The invention is, of course, not restricted to the aspects, embodiments, and variants specifically described above, or to the specific examples, but many changes and modifications may be made without departing from the general inventive concept as defined in the following claims.

Table 1

• DYES o Fiuorophors

■ Fluorescein dyes

• FAM (= fluorescein with carboxyl linker)

• TET

• HEX • JOE

• Yakima Yellow

• Oregon Green

■ Rhodamine (incl. sulfonated) dyes

• TAMRA • ROX

• Texas Red

• Rhodamine Red

• Rhodamine Green

• R6G • Alexa Fluor 488

• Alexa Fluor 514

• Alexa Fluor 546

• Alexa Fluor 568

• Alexa Fluor 594 • Alexa Fluor 610

■ Cyanine dyes

. Cy 3

. Cy 5

• Cy 3.5 ^■ • Cy 5.5

• Cy 7

• Quasar 570

• Quasar 670

^■ 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes / BODIPY dyes • BODIPY family dyes, many different

^■ Other fiuorophors

• Alexa Fluor family dyes, many different (all structures not published)

• Redmond Red

• Cascade Blue • Pulsar 650

• CAL Fluor family (structure not published)

• NED (structure not published)

• VIC (structure not published)

• LightCycler Red 640 (structure not published) o Quenchers

■ Azobenzene {incl. modified) dyes

• Dabcyl

• Black Hole Quencher 1

• Black Hole Quencher 2 • Black Hole Quencher 0

• Black Hole Quencher 3

• Dabsyl

• Methyl Red

• Eclipse Quencher ■ Rhodamine dyes • TAMRA

• ROX

• QSY-7 Nitrothiazole dyes Other quenchers

• EIIe Quencher (structure not published)

• NFQ (structure not published)

Exemplary dye structures. Several dyes have different isomers but all of these may not be pictured. Also, most dyes have different variants with different linkers, reactive groups, or protective groups. The purpose of the figure is only to show structures of the chromophores as such.

FAM, carboxyfluorescein

TET, 2',4,7,7'- tetrachlorofluorescein

HEX, 2^I,4,4',5',7,7'- hexachlorofluorescein

JOE, 4',5'-dichloro-2',7'- dimethoxyfluorescein

Yakima Yellow amidite

Oregon Green, 2',7'-difluorofluorescein TAMRA, carboxytetramethylrhodamine

ROX, carboxy-X-rhodamine

Texas Red

Rhodamine Red

Rhodamine Green

Oi

R6G, rhodamine 6G

Fluor 488

Alexa Fluor 546

Alexa Fluor 568

where n = 1 ,2 or 3 for Cy3, 5 or 7

BODIPY FL

BODIPY TMR

BODIPY TR

BODIPY 530/550

BODIPY R6G

BODIPY 581/591

BODIPY 576/589

BODlPY 650/665

BODIPY 564/570

BODIPY 493/503

BODIPY 558/568

BODIPY 630/650

Alexa Fluor 430

Alexa Fluor 532

Red amidite

Alexa Fluor 405, Cascade Blue

Pulsar

650 CPG

Dabcyl, 4-((4-(dimethylamino)phenyl)azo)benzoic acid

Black Hole Quencher 1

Black Hole Quencher 0 CPG

Black Hole Quencher 3

Dabsyl amidite

Methyl red amidite

amidite

Nitrothiazole blue

Nitrothiazole orange

Claims

1. A liquid chromatographic purification method, based on differences in partition between a solid phase and a liquid phase, for a product oligonucleotide containing one or several labels and additionally containing at least one cleavable separation tag, characterized in that the method comprises carrying out the combined three steps in sequence; step 1 is chromatographic separation based on the presence or absence of the at least one separation tag; step 2 is cleaving off of the at least one separation tag from the oligonucleotide; step 3 is chromatographic separation based on the presence or absence of at least one of the one or several labels.

2. The method according to claim 1, characterized in that in step 1, the oligonucleotide containing the at least one separation tag has a higher affinity for the solid phase than any oligonucleotide lacking the at least one separation tag.

3. The method according to any of claims 1-2, characterized in that in step 3, the oligonucleotide containing the at least one label has a higher affinity for the solid phase than any oligonucleotide lacking the at least one label.

4. The method according to any of claims 1-3, characterized in that all three steps are performed with the oligonucleotide retained on a liquid chromatography column.

5. The method according to any of claims 1-4, characterized in that the oligonucleotide is eluted from a liquid chromatography column after step 2.

6. The method according to claim 5, characterized in that the composition of the sample solution is altered through changing the proportions of aqueous buffer versus organic solvent, changing the pH, changing the composition or concentration of ions, or diluting or concentrating the sample solution, before being reapplied to a liquid chromatography column.

7. The method according to any of claims 1-3, characterized in that step 2 is performed with the oligonucleotide in solution.

8. The method according to any of claims 1-7, characterized in that step 1 and 3 are performed on the same liquid chromatography column.

9. The method according to any of claims 1-7, characterized in that step 1 and 3 are performed on different liquid chromatography columns.

10. The method according to any of claims 1-9, characterized in that the method is based on reverse phase chromatography.

11. The method according to claim 10, characterized in that the at least one cleavable separation tag is chosen to comprise a trityl moiety modified with 1-3 alkoxy groups, a pixyl moiety modified with 0-1 alkoxy groups, a 4-hexyloxy-4'-methoxytrityl, 4-decyloxy-4'-methoxytrityl, 4- hexadecyloxy-4'-methoxytrityl, (4-octadecyloxyphenyl)-9-xanthyl, 4,4'- bis-hexyloxytrityl, 4,4'-bis-decyloxytrityl, 4,4'-bis-hexadecyloxytrityl, 4- octadecyloxytrityl, 4-hexadecyloxytrityl, 4-decyloxytrityl, or a 4- hexyloxytrityl moiety.

12. The method according to any of claims 1-11₅ characterized in that steps 1 and 3 are based on high performance liquid chromatography.

13. The method according to any of claims 1-11, characterized in that steps 1 and 3 are based on low-pressure cartridge chromatography.

14. The method according to claim 13, characterized in that purification of several product oligonucleotides are performed in parallel.

15. The method according to any of claims 1-14, characterized in that steps 1 and 3 are based on gradient elution.

16. The method according to claim 15, characterized in that steps 1 and 3 are based on step gradient elution.

17. The method according to any of claims 15-16, characterized in that the gradient is formed by mixing an organic solvent with an aqueous buffer at varying proportions.

18. The method according to claim 17, characterized in that the aqueous buffer contains a tertiary amine cation or a quaternary ammonium cation.

19. The method according to claim 18, characterized in that the cation is selected from trimethylamine, triethylamine, tripropylamine, tributylamine, tetramethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium.

20. The method according to claim 1-19, characterized in that the at least one label, on which the separation in step 3 is based, is a dye label, more than one identical dye labels, or more than one different dye labels.

21. The method according to claim 20, characterized in that the at least one label, on which the separation in step 3 is based, is a quencher label.

22. The method according to claim 20, characterized in that the at least one label, on which the separation in step 3 is based, is a fluorophor label.

23. The method according to claim 20, characterized in that the at least one label, on which the separation in step 3 is based, is a quencher label and a fluorophor label.

24. The method according to any of claims 20-23, characterized in that the product oligonucleotide is a dual dye labeled oligonucleotide probe.

25. The method according to any of claims 20-24, characterized in that the at least one label, on which the separation in step 3 is based, is selected from the classes of azobenzenes dyes, modified azobenzenes dyes, nitrothiazole dyes, rhodamine dyes, fluorescein dyes, sulfonated rhodamine dyes, cyanine dyes, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes, or from Dabcyl, Dabsyl, TAMRA^®, ROX^®, Black Hole Quencher^® 0, Black Hole

Quencher^® I₅ Black Hole Quencher^® 2, Black Hole Quencher^® 3, Eclipse^® Dark Quencher, Elle^® Quencher, NFQ, QSY-7^®, Methyl Red, fluorescein, tetramethylrhodamine, FAM^®, TET^®, HEX^®, JOE^®, NED^®, VIC^®, Cy^® 3, Cy^® 5, Cy^® 3.5, Cy^® 5.5, Cy^® 7, Oregon Green^® fluorophors, Texas Red^®, Light Cycler^® Red 640, Rhodamine Red, Rhodamine Green, R6G, Yakima

Yellow^®, Redmond Red^®, Cascade Blue^®, Pulsar 650^®, Bodipy^® fluorophors, Alexa^® fluorophors, CAL^® Fluor fluorophors, and Quasar^® fluorophors.

26. The method according to any of claims 1-25, characterized in that the at least one separation tag is attached to the outer end of the product oligonucleotide.

27. The method according to any of claims 1-25, characterized in that the at least one separation tag is attached to the 5' end of the product oligonucleotide.

28. The method according to any of claims 1-22 or 24-27, characterized in that the at least one label, on which the separation in step 3 is based, is attached to the inner end of the product oligonucleotide.

29. The method according to any of claims 1-22 or 24-27, characterized in that the at least one label, on which the separation in step 3 is based, is attached to the 3' end of the product oligonucleotide.

30. The method according to any of claims 1-29, characterized in that the cleaving off of the at least one separation tag is performed in an organic solvent.

31. The method according to claim 30, characterized in that the cleaving off of the at least one separation tag is performed with dichloroacetic acid or trichloroacetic acid in dichloromethane, dichloroethane, or toluene.

32. The method according to any of claims 30-31, characterized in that step 2 also comprises elution of the cleaved off at least one separation tag.

33. Use of a phosphoramidite reagent comprising a cleavable separation tag in the method of any of claims 1-32, wherein the cleavable separation tag is chosen to comprise a trityl moiety modified with 1-3 alkoxy groups, a pixyl moiety modified with 0- 1 alkoxy groups, a 4-hexyloxy-4'- methoxytrityl, 4-decyloxy-4'-methoxytrityl, 4-hexadecyloxy-4'- methoxytrityl, (4-octadecyloxyphenyl)-9-xanthyl, 4₅4'-bis-hexyloxytrityl, 4,4'-bis-decyloxytrityl, 4,4'-bis-hexadecyloxytrityl, 4-octadecyloxytrityl, 4-hexadecyloxytrityl, 4-decyloxytrityl, or a 4-hexyloxytrityl moiety.

34. Use of a solid phase in the method in any of claims 1-32, wherein the solid phase is based on polystyrene, partly crosslinked polystyrene, modified polystyrene, a polystyrene copolymer, polyethylene, fluorinated polyethylene, charcoal, graphite, derivatised silica, or hydrocarbyl Sepharose.