Separation of nucleic acid fragments

(42) Start of patent period. 15. 7.1994 (45) Date of issue: 27. 2.1995

<td>(56) Documents:</td><td>(73) Patent owner:</td>

<td>CA 113 (25), 1990; 227352S, EP-A 507591</td><td>BONN GÜNTHER DR. A-6170 ZIRL, TYROL (AT). HUBER CHRISTIAN MAG. A-6064 RUM, TIROL (AT). OEFNER PETER DR. A-6020 INNSBRUCK, TIROL (AT).</td>

(54) METHOD OF SEPARATING NUCLEIC ACIDS (57) Non-porous poly (ethylvinyibenzene-divinylbenzene) particles are used to describe the alkylation of polymers for the purpose of separating and isolating single and double-stranded nucleic acids. Practical applications include, but are not limited to, the chromatographic analysis of oligonucleotides, DNA restriction fragments and PCR products.

CQ

AT 398 973

INR 0078018

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The separation of nucleic acids is an area of ​​great scientific and technical importance and timeliness. Among the chromatographic methods used here are, in particular, anion-exchange chromatography and ion-pair reverse-phase chromatography.

In the separation of double-stranded nucleic acids, a significant disadvantage of anion-exchange chromatography is the different retention behavior of GC and AT base pairs, which makes separation by molecular size impossible. This considerably restricts the use of anion exchange chromatography in the analysis of nucleic acids, quite apart from the fact that the elution in a salt gradient required in this process makes further investigations of the DNA molecules more difficult.

Although W. shows Bloch in his European patent application (EP 0 507 591 A2) that by the addition of tetramethylammonium chloride (TMAC) to the eluent a substantial separation of DNA fragments according to their size on non-porous anion exchangers is possible, but one can Figs. 5 of the patent application that the fragments with 434 or 458 Base pairs (34 base pairs difference) have a different retention, but that the fragments with 458 or 504 Base pairs (46 base pairs difference) elute at the same time, so that here too no strict length-dependent separation is given. In addition, the addition of TMAC represents a significant decrease in resolution. Since this method is in principle an anion exchange chromatography, direct further investigations by the presence of non-volatile salts are also difficult here because of the Saizgradienten used.

The ion-pair reversed-phase chromatography has also been described by Eriksson et al. for practical application to the separation of double-stranded DNA fragments still significant drawbacks. These are, apart from the low selectivity, especially in that especially shorter restriction fragments are found only very incomplete and also that the analysis time is in the hour range (see. Eriksson, S., Glad, G., Pernemalm, PA., Westman, E., (1986) J. Chromatogr. 359, 265-274).

With respect to those of Y. Ohimya et al. (Y. Ohimia, Y. Kondo, T. Kondo, anal. Biochem. 189 (1990) 126130) for the separation of DNA fragments by ion exchange chromatography on a trimetylammonium-functionalized column, the same objections apply as for the separation of DNA fragments on a diethylaminoethyl-modified column (Y: Kato, Y , Yamaski, Akane Onaka, T. Kitamura, T. Hasimoto, T. Murotsu, S. Fukushige, K. Matsubara, J. Chromatogr., 478 (1989) 264-268): A strict separation of the DNA fragments according to their size is not possible in both cases (see. Kato et al., Fig. 1 and Ohimiya et al. Illustration 2), further investigations are hampered by the high salt content of the eluted samples.

Assuming that the disadvantages of the method described by Eriksson et al. Huber et al. first suggested the use of non-porous polystyrene particles in ion-pair reversed-phase chromatography (Huber, CG, Oefner, PJ and Bonn, GK (1992) J. Chromatogr. 599.113-118). It has been found that with the use of such column material, the separation of single-stranded nucleic acids can be substantially improved. Further improvement resulted from the incorporation of polyvinyl alcohol during the synthesis of the polymer particles.

Experiments, the Huber et al. Proposed methods to apply to double-stranded nucleic acids, were unsuccessful until it was found that the initially detected poor resolution occurs only in nucleic acids with more than 150 base pairs. Thus, in its most general form, the invention consists in carrying out the separation of double-stranded DNA fragments by ion-pair reversed-phase chromatography using a column of non-porous polymeric particles having a diameter between 1 and 100 μm, preferably 2 and 3 μm.

Relevant for the chromatographic effectiveness of the particles is their respective near-surface region, which is why material data in the text and in the claims are in each case based on this range of particles whose core can consist of different materials.

In general, without the restriction to short fragments, the Huber et al. indicated column material for the analysis of double-stranded DNA by ion pair reversed-phase chromatography usable when the surface of the polymeric particles is modified for the purpose of reducing their polar character, in particular by alkylation with alkyl radicals longer than ethyl.

This effect of alkylation was quite surprising to the person skilled in the art. Although the use of (albeit porous) alkylated polystyrene particles was by Morgan and Celebuski (Morgan, R. L. and Celebuski, J. E. (1991) J. Chromatogr. 536, 84-93) have already been described. In this work it was shown that oligonucleotides labeled with fluorescein or biotin become stuck on unmodified porous poly (styrene-divinyl-benzo!) Particles (PRP-1 column, Hamilton, Reno, NV, USA) and only in subsequent cases

AT 398 973 Β ing running are eluted as so-called ghost gangs. This undesirable effect could be eliminated after modification of the octadecyl porous poly (styrene-divinylbenzene) particles (Polyspher RP-18, EM Science, Cherry Hill, NJ, USA). However, an improved resolution did not occur.

The mentioned work of Huber et al. by no means indicated the improvement that can be achieved by the alkylation of the polystyrene. The improvement in the resolution of oligonucleotides achieved by the incorporation of the polyvinyl alcohol into the polymer particles was due to the presence of hydroxyl groups, inter alia attributed to their polar character. It was therefore quite surprising that alkylation with completely apolar groups such as octadecyl residues results in an even greater improvement in the resolution of oligonucleotides and even allows the separation of double-stranded DNA fragments.

By use of the invention, fragment length separation by ion pair chromatography is possible even with a difference of only one base pair. The advantage of the method is therefore the use of a volatile ion pair reagent in conjunction with the high resolution for the separation of DNA fragments and PCR products. Isolated fractions are directly accessible to further experiments after removal of the volatile eluent by simple freeze-drying.

The improvement achieved by the invention of the ion pair reversed-phase chromatography extends the. Scope of this analysis method on the clinical diagnosis of diseases by the detection of oncogenes or viral genes.

The detection of oncogenes or viral genes today takes place in such a way that polymerase chain reaction products of these genes are separated by gel electrophoresis. However, gel electrophoresis is a collection of labor-intensive and non-automatable techniques.

Katz et al. (Katz, ED, Haff, LA, Eksteen, R. (1990) J. Chromatogr. 512, 433-444), the chromatographic separation of polymerase chain reaction products has already been described. However, the method used, namely the anion exchange HPLC, not only leads to the disadvantages outlined in principle, but also to a gradual increase in the background signal, which together with the relatively long analysis times makes clinical use difficult. In Katz's work, the possibility of detecting oncogenes or viral genes, although theoretically composed, are not mentioned.

Thus, the invention also provides a diagnostic method for detecting malignant tumors and / or viral diseases in the human or animal organism, wherein oncogenes or viral genomes in particular by a polymerase chain reaction propagated and separated in a chromatographic process of other products, which characterized in that the ion-pair reversed-phase chromatography is used as the chromatographic method.

The use of modified polymer particles according to the invention is not limited to the separation of double-stranded DNA fragments. There is also no restriction on ion-pair reversed-phase chromatography, but it is possible to use it in various chromatographic methods and in particular also for solid-phase extraction.

Subsequently, the preparation of the polymer particles according to the invention will be described with reference to an embodiment and their effectiveness explained in connection with three chromatograms. It shows

Fig. 1 shows the separation of DNA restriction fragments by unmodified and modified non-porous polystyrene particles, Fig. 2 shows a demonstration example of the high resolution separation of DNA restriction fragments, and Fig. 3 shows an example of the high speed analysis of PCR products by means of a Chromatography column prepared according to the invention particles.

The preparation of non-porous, spherical poly (ethylvinylbenzene-divinylbenzene) particles was carried out in a two-stage process which guarantees a very narrow size distribution. In the first step, polystyrene nuclei having an average diameter of 0.8-1 μm are synthesized here by means of emulsion polymerization of styrene. In the second step, these seeds are then mixed with a mixture of mono- and Divinylmonomeren (z. B. Ethylvinylbenzene and divinylbenzene) to a size of 1-10 .mu.m and finally polymerized by a stepwise increase in temperature intended to prevent the particles from clumping together.

The emulsion polymerization was based on a publication by Goodwin et al. (Goodwin, JW, Hearn, J., Ho, CC and Ottewill, RH (1974) Coiloid &amp; Polymer Sei. 252, 464-471) from 1974 according to the following protocol: 0.236 g of sodium chloride was dissolved in 354 ml of deionized water in a 1 liter reaction vessel, further equipped with reflux condenser, stirrer and gas inlet tube, under argon atmosphere, and heated to 87 · C (stirrer speed: 350 rpm). Then, 33.7 g of freshly distilled styrene and, after one minute, 0.2184 g of potassium peroxodisuifate dissolved in 50 ml of deionized water were added. After addition of the reagents, the

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End of the gas inlet tube withdrawn above the liquid level. The reaction mixture was for

Stirred for 6.5 hours at 87 'C and then cooled to room temperature and, taking into account the adhering to the stirrer amount of polystyrene (generally between 5 and 10 g) so diluted that the concentration of polymerized styrene about 54.6 g per 1000 ml of suspension scam. The diameter of the spherical particles was determined by light microscopy and was about 1 μm.

Since the particles produced by means of emulsion polymerization are on the one hand too small for chromatographic purposes and, on the other hand, are not pressure-stable owing to the lack of crosslinking, in a second step the polymer nuclei were modeled on that of Ugelstad et al. (Ugelstad, J., Mork, PC, Herder Kaggerud, K., Ellingsen, T. and mountains, A. (1980) Adv. Colloid Interface Be. 13, 101-140) in the activated swelling method described in 1980 to a particle diameter of 1-10 μm. The aqueous suspension of Poiystyrolkeime (200 ml) was then added first with 60 ml of acetone and then with 60 ml of a 1-chlorododecane-containing emulsion (0.206 g of sodium dodecyl sulfate,

49.5 ml of deionized water and 10.5 ml of 1-chlorododecane were 4 Stirred at 0 'C in an ultrasonic bath until a fine emulsion &lt;0.3 .mu.m was obtained) during approx. 12 Swelled at room temperature for hours. As a result, the acetone was evaporated at 80 ° C. for 30 minutes. Then, 310 g of a mixture of ethylvinylbenzene and divinylbenzene in a ratio of 1: 1.71, which further contained 2.5 g of dibenzoyl peroxide as initiator, were added and stirred under repeated light microscopic control until the desired particle diameter was reached (on average 4 up to 8 hours). The ratio of monomer to initiator was at least 100: 1 in order to achieve a degree of polymerization of&gt; 200, which gave the particles the necessary pressure stability for high pressure liquid chromatography. Subsequently, the entire batch was transferred to a separatory funnel, with the unused monomer that was discarded separated from the swollen particles. Finally, a stepwise increase in temperature (63 'C for ca. 7 Hours, 73 'C for approx. 2 Hours and 83 'C for approx. 12 Hours) (degree of polymerization&gt; 500). Particles thus prepared, as confirmed by mercury porosimetry, have no pores&gt; 30 Å, which allows size exclusion effects in chromatography to be avoided.

Although the non-porous uniformly spherical particles prepared in this or other ways can be directly used for reversed-phase chromatographic separation of nucleic acids, as mentioned above, the dissolution was probably due to the undesirable adsorption of the aromatic bases of the nucleic acids to the particle surface wish you left. In order to minimize this effect, the aromatics of the stationary phase were alkylated by electrophilic aromatic substitution in a further step. In a typical approach, 10 g of the dried particles were suspended in 100 ml of 1-chlorooctadecane and, after addition of 1 g of aluminum chloride, stirred at 100 ° C. for 12 hours (370 rpm). Subsequently, the reaction mixture was cooled to 80 'C, added 150 ml of 4 M hydrochloric acid, for approx. 2 Stirred minutes, transferred to a separatory funnel, mixed with 300 ml of n-heptane, the phases thoroughly mixed and discarded after subsequent separation of the phases, the aqueous phase. The organic phase was washed twice more with 200 ml of 1 M hydrochloric acid and then centrifuged off at 5000 rpm. The precipitated particles were washed four times with 100 ml of n-heptane, and then each twice with 100 ml of diethyl ether, 100 ml of dioxane and 100 ml of methanol and finally dried.

With the aid of Fourier transform infrared spectroscopy (FTIR), the actual alkylation of the aromatics could finally be detected. The particles produced differed little in size from each other. The mean value for the diameter was found to be 2.10 μm, with a standard deviation of 0.12 μm.

One criterion for achieving high resolution chromatographic separations of nucleic acids is the protocol used to package the column. For a 50 x 4.6 mm ID Column 1.4 g of the alkylated particles were suspended in 15 ml of tetrahydrofuran in the ultrasonic bath and filled with 50 ml of methanol at a pressure of 70 MPa in the column. Subsequently, rinsing with 50 ml of deionized water was also carried out at 70 MPa to dewell the particles and thus achieve an even higher packing density.

The actual separation of the single and double-stranded nucleic acids was carried out according to the principle of ion-pair reverse-phase liquid chromatography. As lonenpaarreagenz triethylammonium acetate was mostly used, but there are also other tertiary and quaternary ammonium salts in question. Elution was carried out by means of a linear organic solvent gradient such as acetonitrile or methanol.

Figure 1 shows the separation of DNA restriction fragments by the described non-porous polystyrene particles under the following experimental conditions: Column: 50 x 4.6 mm ID mobile phase: 0.1 M TEAA, pH 7.0. Gradient: 7.5-13.75% acetonitrile in 4 minutes, followed by 13.75-16.25% acetonitrile in 6

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Minutes. Flow rate: 1 ml / min. Column temperature: 50 'C. Detection: UV, 254 nm. Sample: 0.5 μg pBR322 DNAHaeill restriction digest.

Curve a refers to unmodified particles, curve b to particles modified by the incorporation of polyvinyl alcohol as described by Huber et al, curve c to alkylated particles. A clean separation of the fragments characterized by the number of base pairs results over a wide range of fragment lengths only in case c, but at smaller fragment lengths unmodified particles are not completely useless, and even a slight shielding of the aromatic compounds by polyvinyl alcohol leads according to curve b improved resolution.

The nonporous polystyrene particles of the present invention are eminently suitable for a variety of chromatographies and related applications, with examples given below:

1. example

For certain molecular biological experiments such as in situ hybridization, radioactive labeling of oligonucleotides with isotopes such as ³²P or ³³P necessary. In order to avoid subsequent competitive hybridization with unlabelled oligonucleotide, purification of the radiolabeled oligonucleotides is required. This can be done quickly and easily by ion-pair reversed-phase chromatography on alkylated non-porous poly (ethylvinylbenzene-divinylbenzene). The recovery rate for oligonucleotides was at least 96%.

Second example

FIG. 2 Figure 3 shows the high resolution separation of DNA restriction fragments on octadecyl-modified non-porous poly (ethylvinylbenzene-divinylbenzene) tags. The experiment was carried out under the following conditions: Column: 50 x 4.6 mm ID Mobile phase: 0.1 Μ TEAA, pH 7.0. Gradient: 8.75 - 11.25% acetonitrile in 2 minutes, followed by 11.25-14.5% acetonitrile in 10 minutes, 14.5-15.25% acetonitrile in 4 minutes, and 15.25-16, 25% acetonitrile in 4 minutes. Flow rate: 1 ml / min. Column temperature: 50 'C. Detection: UV, 254 nm. Sample: a mixture of 0.75 mg pBR3 | 2 DNA-Haelll restriction digest and 0.65 μg ΦΧ 174 DNA Hincll restriction digest. Decisive for the resolution shown are, in addition to the stationary phase, the concentration of triethylammonium acetate, the slope of the solvent gradient, the column temperature, and the flow rate. With respect to the concentration of triethylammonium acetate in the mobile phase, it can be seen that in a range of 25 a continuous increase in resolution occurs to at least 125 mM. With regard to the steepness of the solvent gradient, a flatter gradient for the resolution of the DNA molecules is necessary with increasing fragment length. The separation of DNA molecules shows maximum resolution at 50 ° C, depending on the column temperature. At even higher temperatures, denaturation of the double-stranded DNA molecules is observed.

Third example

By minimizing the gradient volume associated with the alkylated non-porous poly (ethylvinylbenzene-divinylbenzene) phase, polymerase chain reaction products could be included. the time required to regenerate the column can be analyzed within 2 minutes. Since in the polymerase chain reaction usually only one or two products of known length occur, the resolution is somewhat less stringent than with separation of DNA restriction fragments. This therefore allows the use of steep solvent gradients and thus shorter analysis times. The recovery of a 404 base pair DNA fragment was 97.5%. A key advantage of ion-pair reversed-phase chromatography over capillary electrophoresis is the need to desalt the PCR samples prior to their analysis. Therefore, in combination with an autosampler, a fully automatic analysis of PCR samples is possible. Thus, oncogenes and viral genomes (hepatitis C virus, HIV) could be detected.

The suitability of the modified polystyrene particles according to the invention for clinical practice results from FIG. Third This refers to a test carried out under the following conditions: Column: 50 x 4.6 mm ID Mobile phase: 0.1 M TEAA, pH 7.0. Gradient: 11.25-13.75% acetonitrile in 1 minute followed by 22.5% acetonitrile for 6 seconds and 11.25% acetonitrile for 54 seconds. Flow rate: 3 ml / min. Column temperature: 50 × C. Detection: UV, 256 nm. Sample: 20 μl of a PCR sample, 1 = non-specific product, 2 = 120 base pair PCR product, 3 = 132 base pair PCR product, and 4 = 167 base pair PCR product.

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The repeated repeated with the PCR products of the same oncogenes showed, as you can see, excellent reproducible results. In addition, it was particularly important to note that the result of the individual experiments is not influenced by previous loadings of the column, which is another indispensable prerequisite for the clinical use of the method.