AU603500B2 - Carrier for the enzymatic or chemical and enzymatic reaction of nucleic acids or nucleic acid fragments on solid phases - Google Patents

Abstract

The carrier consists of an insoluble, unswellable, porous polymer to which one or more nucleic acids or nucleic acid fragments, which are possibly provided with protective groups customary in nucleic acid chemistry, are attached, the pores in the polymer essentially having a defined size between 1400 and 10,000 ANGSTROM .

Description

603500 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION NAME ADDRESS OF APPLICANT: Boehringer Mannheim GmbH Sandhofer Strasse 112-132 D-6800 Mannheim-Waldh f Federal Republic of Germany NAME(S) OF INVENTOR(S): Heinz Hartmut SELIGER Gabriele GROEGER ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Carrier for the enzymatic or chemical and enzymatic reaction of nucleic acids or nucleic acid fragments on solid phases

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The following statement is a full description of this invention, including the best method of perforting it known to me/us:- .4 t 4 .4 o 4 44 4 4, I 4 4 *4 9*4444 4.

4.

464 44,444 The present invention is concerned with insoluble, polymeric carriers for the enzymatic or chemical and enzymatic reaction of nucleic acids or nucleic acid fragments on solid phases.

For the precise synthesis of nucleic acids, in general nucleic acid fragments, i.e. fragments of whole nucleic acids, oligomers or monomers, are linked with one another by means of a combination of chemical and enzymatic reactions. Thus, for example, for the build 10 up of deoxyribonucleic acid (DNA), oligodeoxyribonucleotide fragments of about 15 60 units chain length can be chemically synthesised and these then, after phosphorylation on the 5'-end, mixed and assembled in enzyme-catalysed reactions by means of DNA ligase to 15 give a double-stranded nucleic acid, the correct positioning of the individual fragments thereby being achieved by mutual Watson-Crick base pairing on the basis of the hybridisation and thus at least partial overlapping of the fragments. Oligoribonucleotides 20 chemically synthesised or obtained by enzyme-catalysed reactions can be linked correspondingly t.ith RNA ligase to give ribonuzleic acid (RNA). Analogously, nucleic acids which contain not only DNA but also RNA fragments can be obtained as a single strand.

In, the following, by nucleic acids are to be understood all possible types and forms of nucleic acids.

They can be not only deoxyribonucleic acids but also -3ribonucleic acids, as well as mixed polynucleotides, which consist not only of monomers of deoxyribonucleic acids but also of monomers of ribonucleic acids.

Furthermore, the nucleic acids can be present as a single or double strand.

Parts or fragments of nucleic acids can be not only monomers, such as nucleosides or nucleotides, but also oligomers of 2 100 monomer units and larger polymers with several hundred monomer units are also possible.

For the enzyme-catalysed linkage of nucleic acid fragments, it has proved to be advantageous when one of the fragments is bound to a polymeric insoluble carrier.

Thus, Cozzarelli et al. describe in Biochem. Biophys.

Res. Comm., 28, 578 586/1967 the linkage of DNA t fragments by means of a DNA ligase, one of the fragments thereby being bound to cellulose.

T.M. Jovin and A. Kornberg describe in J. Biol.

Chem., 243, 250 259/1968 the use of an oligomer of j o, 20 deoxyribothymidine bound to cellulose particles as primer and matrix for DNA polymerase.

U. Bertazzoni et al. described in Biochim.

Biophys. Acta, 240, 515 521/1971 the chain elongation of oligodeoxyribonucleotides covalently bound to cellulose by means of terminal deoxyribonucleotidyl transferase.

A. Panet and H.G. Khorana described in J. Biol.

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-a- -4- Chem., 249, 5213 5221/1974 the binding of polydeoxyribothymidine to cellulose and elongation of this carrier-bound nucleic acid by mneans of ligase by a further DNA fragment. With the .so produced cellulosebound polynucleotide and a short primer, a part of the polynucleotide was replicated with DNA polymerase.

The advantages of the use of immobilised nucleic acid fragments for the enzymatic nucleic acid build u,, as well as quite generally for their enzymatic reaction, are to be seen, in particular, in the easy separation of the immobilised reaction product from the other materials needed for the reaction and resulting during the reaction. On the basis of this simple possibility for the isolation of a desired reaction product, by use At 15 of excesses of enzyme and/or non-immobilised substrate, a displacement of the equilibrium to give higher yields of immobilised reaction product, for example of chainelongated product, can also be achieved without the subsequent purification thereby being made substantially difficult.

Whereas gels, such as swellable insoluble polysaccharides, can be used as carriers for the enzymecatalysed reaction of immobilised nucleic acids or nucleic acid fragments, it has, however, been shown that such materials are only poorly suited for building up or changing nucleic acids by chemical methods on polymeric insoluble carriers.

~arnr~~~~aart~u,4~~~ Thus, for example, in the case of the chemical synthesis of nucleic acids, which usually starts from corresponding monomers, such as nucleosides or nucleotides, cycles must usually be passed through which consist essentially of the steps: activation of the nucleic acid part to be elongated, possible isolation of the'product, addition and condensing on of a new nucleoside or nucleotide and isolation of the product.

The partial or complete automation of such chemical processes for the build up of nucleic acids was achieved after introduction of the solid phase methods, the nucleic acid fragments .to be elongated thereby being fixed on to insoluble carriers.. The o'j o ,immobilisation of the "growing" nucleic acid part thus makes possible the particular purification of the desired reaction product by simple washing of the solid phase. Soluble reaction components are brought into contact with the solid phase, and after completion of 'the reaction, are removed by washing.

R. Frank et al., Nucl. Acids Res., 11, 4365-4377/ 1983 described the use of cellulose in the form of J paper leaflets ("disks") as solid phase for the chemical synthesis of short DNA fragments. However, paper, cellulose and generally all polysaccharides have the fundamental disadvantage that all reactive groups not needed for the fixing of the nucleic acid or nucleic acid fragments, such as hydroxyl residues, must be -6blocked in order that no disturbance of the synthesis cycles takes place. However, a complete blocking of all reactive groups of Pulysaccharides used as carrier materials not needed as nucleotide anchors is impossible, especially as, in the case of multiple repetition of the reaction cycles, the macroscopic structure of such solid swellable carriers often changes, for example by the formation of cracks, and thus previously inaccessible reactive groups are then accessible to the reactants and can lead to disturbances fit t of the course of the reaction or to side reactions.

Thus, especially in the case of the use of paper as eft 6 carrier material for the chemical synthesis of nucleic acids, a large number of faulty sequences can arise.

Furthermore, the deficient mechanical stability of paper in the case of comparatively long chemical ft stressing gives rise to difficulties in the mechanical synthesis, for which reason sequences of more than to 25 bases are hitherto not prepared in pralcis.

In the case of swellable carriers, depending upon the reaction medium, considerable washing and reaction times can occur due to swelling and deswelling of the material. Furthermore, diffusion problems can arise.

Because of these disadvantages, non-swellable carriers are usually employed for the chemical reactions and especially for the synthesis of nucleic acids or nucleic t.

acid ftagments. A conventional material for this -7purpose is silica gel.

Silica gel is an often used insoluble carrier for the chemical build up of nucleic acids. This material possesses a broad distribution of different pore sizes and an irregular pore structure.

For the synthesis of double-stranded DNA with the use of not only chemical but also enzymatic reaction I steps, Fractosil 1000 a silica gel with a pore size of 1000 R, and Sephacryl-500 a hydrophilic, gelforming i.e. swellable material obtainable by threedimensional cross-linking of linear dextran chains with N,N'-methylene-bis-(acrylamide), were compared with one I another for their suitability as carrier materials by Z. Hostomsky J. Smrt in Nucleic Acids Research Symp.

15 Ser., 18, 241 244/1987. It was thereby ascertained that the non-swellable silica gel, in contradistinction I to the gel-forming carrier, partly or completely inhibited the enzymatic reactions and the ligation or liberation of the final nucleic acid from the carrier 4 20 material.

j Therefore, there is still a need for carrier fr materials for the combined use of chemical and enzymatic methods for the reaction of nucleic acids or nucleic acid fragments on insoluble, polymeric carrier materials which, for chemical reactions, avoids the disadvantages of swellable carriers and can be used advantageously for enzymatic reactions.

8 a- ThvA, it is an object of the present invention I to provide suitable carriers for the enzymatic or chemical and enzymatic reaction of nucleic acids or nucleic acids or nucleic acid fragments on solid phases which fulfil the above requirements.

Thus, according to the present invention, there is provided a carrier for the enzymatic or chemical and enzymatic reaction of nucleic acids or nucleic acid fragments on a solid phase consisting of an oil r insoluble, non-swellable, porous polymer on which are 4' fixed one or more nucleic acids or nucleic acid fragments optionally provided with protective groups usual nucleic acid chemistry, wherein the pores of the ,polymer essentially possess a definite size within a definite range. on the one hand the pores must be so 4 large, that enzymes have essentially unhindered access to those fixed nucleic acids or nucleic acid fragments which shall be reacted. on the other hand the pores must be so small, that the carrier shows a large surface, which offers many reactive sites to be in the position to react as many nucleic acid molecules as possible on as little carrier material as possible. In this sense carriers have proved to be preferred which possess pores of a definite size of from 1400 to a 10,000 A.

4\ Those polymers are preferred, the pores of which essentially have definite sizes of from 2000 to 0 5000 and especially of from more than 2500 to 5000 A, "definite" thereby being understood to mean a maximum deviation of 10% from the given value.

surprisingly, we have found that those polymers which fulfil these pcre size conditions and which! with regard to their composition, are resistant towards the agents necessary for the chemical and enzymatic reaction of nucleic acids are very suitable not only for chemical reactions but also for enzymatic reactions, for example those for the synthesis of nucleic acids, which can be carried out manually or automatically.

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4 4** 4, I 4 144 1 I It I 4 4 4 44 44 I 4 4 4 4 44 I, I; 44 $4 4 4 4* 4 4 4* 14444

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'4 44 4 444 444414

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r A- tAT~~ A-A> 9- 4 C 4 4 C C

CC

*4 4 C 4 CC 4.44 4 4 4, #4 44 4 4 #4 4*4*4 4 .4 .4 4 *4* C C 4 I C Especially when the carrier polymer is a material which does not exclude from its hollow spaces aqueous solutions, especially buffer solutions, as media for enzyme reactions, enzymatic reactions, such as ligations with ligases or the splitting off of nucleic acids or nucleic acid fragments, for example with restriction enzymes, take place quickly and completely. Preferred carrier polymers are inorganic materials and especially those, which are essentially built up of silicon and 10 oxygen atoms.

0 Glass with a controlled pore size of about 3000 A has proved to be quite especially suitable for chemical reactions and particularly also for enzymatic reactions of nucleic acid or nucleic acid fragments.

15 The nature of the polymeric carrier material is decisive for the advantageous possibility of use of the carrier according to the present invention for the combined use of chemical and enzymatic methods for the reaction of nucleic acids or nucleic acid fragments.

20 Thus, for example, it is not necessary, after the conc~lusion of a chemical synthesis of nucleic acid chains, to separate these from the solid phase employed therefor and to select another carrier material for the enzymatic chain elongation. Loss-rich separation and coupling-on reactions can be avoided with the carriet according to the, present invention. The carrier accotding to the present invention is quite especially All -A.A,

I,

Ioutstandingly suitable for enzymatic reactions.

The immobilised nucleic acids or nucleic acid fragments can be fixed on to the carrier material in greatly differing ways. However, it is especially advantageous when the carrier for the solid phase synthesis of nucleic acids contains the nucleic acid fragrments, optionally provided with protective groups I usual in nucleic acid chemistry, covalently bound to the insoluble, non-swellable, porous polymer. For the 110 chemical synthesis of nucleic acids, optionally protected monomers of the corresponding nucleic acids are mostly used as starting materials in known manner but oligoiners can also be used. In the case of 1 enzymatic reactions, comparatively large nucleic acid fragments are often bound to the polymeric carrier I material. However, the enzymatic reaction of small nu'nleic acid fragments is, of course, often just as I possible.

In the case of a preferred embodiment of the carrier according to the present invention, the nucleic acid fragments are bound to the polymer via a spacer.

As spacers, there can be used a plurality of substances which can be selected by the expert according to the -reactions in question. Points to be taken into account for such a choice are especially the stability towards the particularly chosen reaction conditiond in the cas of not only chemical reactions but also of enzymatic

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reactions. Amongst the methods of chemical nucleic acid synthesis in which the phosphite method today prevails, it has proved to be quite especially advantageous when the nucleic acid to be brought to reaction, optionally provided with protective groups usual in nucleic acid chemistry, or the part of a nucleic acid is bound to the carrier material with an

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aminopropyl-silyl spacer (-Si-(CH 2 3 Especially carriers of the general formula:- OR 0 0 1 1I I polymer-0-Si-CH 2

-CH

2 -CH 2

-NH-C-CH

2

-CH

2 -C-0-nucl

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UK,

(I)

it 11 in which "polymer" signifies a glass with a controlled pore size of from more than 2500 to 5000 9 and prefer- 0 ably of about 3000 A, R and which can be the same or different, are CI-C 5 -alkyl or polymer and "nucl" is a fragment of a nucleic acid optionally provided with protective groups usual in nucleic acid chemistry, have proved to be outstandingly suitable for the combined -chemical and enzymatic synthesis of nucleic acids.

Enzymatic reactions can be carried out thereon especially advantageously.

The fragment of a nucleic acid in the meaning of "nucl" can be not only a single nucleotide but also an oligomer of 2 100 monomer units or a comparatively large polymer with several hundred monomer untts.

.i_ -12- As fragment of a nucleic acid in the definition of "nucl", oligonucleotides of 6 30 nucleotides have proved to be especially suitable for enzymatic reactions and those oligonucleotides of 15 25 nucleotides are quite especially preferred.

Analogously to the process described by T. Atkinson and M. Smith in M.J. Gait in "Oligonucleotide Synthesis a practical approach", IRL Press, Oxford, Washington D.C. (1984), pp. 45 49, such a quite especially preferred carrier can be produced by aminopropylation of controlled pore glass 3000 A (CPG 3000, Serva, Heidelberg, Federal Republic of Germany) and reaction with a nucleic acid fragment correspondingly substituted on the 3'-end by 2-nitrophenylsuccinyl and optionally provided with protective groups usual in nucleic acid chemistry. There can thus be produced loading densities of about I pmole to pmole of nucleic acid or nucleic acid fragment per gram of polymeric carrier material. Especially preferred are carriers with a loading density of 5 to 11 pmole/g.

The present invention also provides a process 'for the synthesis of nucleic acid according to a solid phase-method, in which, as solid phase, there is used an insoluble, non-swellable, porous polymer, the pores of which essentially possess a definite site of from 0 1400 to 10,000 A. The pores preferably have a -13controlled size of from 2000 to 5000 and especially of from more than 2500 to 5000 IBy solid phase methods are here to be understood all those processes for the synthesis of nucleic acids which proceed heterogeneously, i.e. where a nucleic acid part, be it a monomer, such as a nucleoside or nucleotide optionally provided with protective groups usual in nucleic acid chemistry, or a nucleic acid fragment consisting of several monomer units optionally also protected, is fixed on to an insoluble carrier material, elongated by mneans of chemical and enzymatic reactions or by means of enzymatic reactions alone by one or more monomer units and, after conclusion of the synthesis, the final nucleic acid is optionally removed from the solid phase.

For the chemical synthesis of nucleic acids, the phosphodiester, phosphotriester and the phosphite process are usual, the two latter methods and of these especially the phosphite method having proved to be advantogeous for the solid phase synthesis of nucleic acids.

Whereas with chemical processes, oligonucleotides of about up to 60 monomer units can best be synthesised, these can be elongated by enzymatic methods to give nucleic acids of several hundred monomer units, as has already been stated in the desawiption of the prior art. Usual enzym~es include, for example, kinases,

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ii -14polymerases and ligases. Enzymes, for example restriction endonucleases, can also be used for the splitting off from the carrier matrix of the nucleic acids finally synthesised on a solid phase.

Hitherto, an efficient, possibly automatable synthesis of nucleic acids, which contained chemical and enzymatic process steps, was only possible when, in the case of the use of swellable solid phases, the disadvantages of deficient mechanical stability, longer washing and reaction times, as well as possible side reactions or, in the case of the use of non-swellable solid phases, possible inhibitions of enzymes used and thus incomplete reactions were taken into account. For the avoidance of some of these disadvantages, the chemical synthesis steps could be carried out on a non-swellable material and the enzymatic synthesis steps on a swellable material. However, this means that, during the synthesis, the carrier material must be changed and loss-rich separation and coupling-on reactions must be carried out.

The process according to the invention consists of the steps: Fixing of nucleic acid fragments optionally provided with protective groups on to an insoluble, nonswellable, porous polymer, elongation of the immobilised nucleic acid fragment by further nucleic acid fragments and optional splitting off of final nucleic acids from .1 the insoluble polymer does not display these disadvantages. It can be used very advantageously for the combined chemical and enzymatic build up of nucleic acids. A change of the carrier material in the case of i 5 combination of chemical and enzymatic synthesis steps Sis not necessary. The process is quite especially Ssuitable for enzymatic nucleic acid synthesis.

SAccording to the present invention, as carrier material it is preferred to use a polymer consisting essentially of silicon and oxygen atoms which is particularly characterised by its great mechanical stability and by its high permanent porosity and low saelling porosity. This applies quite especially to glass of a definite pore size between 1400 and 10,000 a.

Especially glass of a controlled pore size of about 2000 5000 R, preferably of from more than 2500 to 5000 a and quite especially one of a controlled pore size of about 3000 R shows outstanding properties which recommend it as a non-swellable, porous material for possibly automatable solid phase processes. Hereby i to be especially stressed is the suitability for the immobilisation of nucleic acids or nucleic acid fragments and the suitability of such solid phase-bound substances as enzyme substrates.

Nucleic acid fragments can be fixed to the carrier material in the most varied manner. However, it is especially advantageous when the nucleic acid fragments,

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-16which can optionally be provided with protective groups, are bound covalently to the insoluble, non-swellable polymers, preferably via a spacer. As spacers, a plurality of substances can be considered which can be selected by the expert depending upon the reaction conditions in question. It has proved tobe quite especially advantageous when the nucleic acid fragments, which can optionally be protected by a protective group usual in nucleic acid chemistry, are bound to the carrier material with an aminopropylsilyl spacer (-Si-(CH 2 3 for example by means of II (C 2

H

5 0) 3 Si-O-(CH2 3

-NH

2 In this regard, the abovedescribed carriers of general formula are especially suitable for the process according to the present invention.

S' An inhibition of enzymes is hereby not observed.

This applies especially for polynucleotide kinase and t DNA polymerase, as well as RNA and DNA ligase and restriction endonucleases. Therefore, the process according to the present invention is quite especially suitable for the enzyme-catalysed reactions of immobilised nucleic acids or nucleic acid fragments.

Examples of such enzymatic reactions include the enzymatic phosphorylation of oligonucleotide fragments, 3 for example oligodeoxythymidylate with T 4 -polynucleotide kinase, the ligating on of DNA fragments to oligonucleotides, for example oligodeoxythymidylate with the -17- .help of DNA ligase in the presence of a counter-strand fragment, chain elongation by single-stranded linking of DNA fragments with 3'-ribo terminus on to an immobilised oligonucleotide, for example oligodeoxythymidylate, with the help of RNA ligase, replication of an immobilised RNA-DNA hybrid strand by Klenow DNA polymerase in the presence of a universally usable starter oligonucleotide, for example oligodeoxyadenylate, or cutting off of an immobilised oligonucleotide double strand by the use of restriction enzymes.

The present invention is also concerned with the i use of an insoluble, non-swellable, porous polymer, the pores of which have essentially a definite size of from 1400 to 10,000 R, as carrier material for the immobilisation of nucleic acids or nucleic acid fragments, especially when such carrier-fixed compounds are used for the enzymatic or chemical and enzymatic reaction of nucleic acids or nucleic acid fragments on a solid phase. Such polymers can also be used for use for synthesis automats for the build up of nucleic S' acids since they are characterised by high mechanical stability and prolonged loadability with changing reagents and solvents. However, the above-mentioned polymers are quite especially suitable as carrier materials for enzymatic reactions since these proceed thereon unhindered.

Inorganic polymers are preferred, especially s -18those which are built up essentially from silicon and I oxygen atoms. Such polymers advantageously have pores of a definite size of from 2000 to 5000 R and especially of from more than 2500 to 5000 S. Glass with a controlled pore size of about 3000 has proved to be quite especially advantageous.

I Such wide-pored, non-swellable polymers can be 1 used for chemical syntheses because they can easily be washed out so that excess reagents and solvents can be easily removed. Furthermore, the carrier material according to the present invention provides very high, almost quantitative yields in the case of the chemical condensation steps.

The use according to the present invention of wide-pored, non-swellable polymers has proved to be especially advantageous for eazymatic reactions for the build up or for the decoupling of nucleic acids from the carrier material. Enzymes are not inhibited by the material and accept as substrate the nucleic acids or nucleic acid fragments immobilised therewith.

j The following Examples are given for the purpose of illustrating the present invention: 4- J Abbreviations used in the Examples: Tris tris-(hydroxymethyl)-aminomethane DTT dithiothreitol ATP EDTA ethylenediamine-tetraacetic acid

A

-19- C pi

HEPES

I

A

i

I

I

DMSO

PEG

TE

BSA

DMTrdT counts per minute 4- (2-hydroxyethyl piperazine-etlhanesulphonic acid dimethyl suiphoxide polyethylene glycol tris/EDTA buffer bovine serum albumin deoxythymidine substituted in the by a dimethoxytrityl radical MSNT l-(mesitylenesulphornyl)-3-nit-ro-l,2,4-triagole dNTP desoxynucleoside triphosphate TEA triethylamine Example 1.

Synthesis of immobilised (dT) 20 oligonucleotides.

A) Synthesis of immobilised monomeric deoxythymidine.

a) Carrier material: controlled pore glass (CPG) (Serva, Heidelberg, Federal Republic of Germany) Aminopropylation.

The aminopropylation of the carrier material took.

place analogously to the literature reference tMatteucci and Caruthers, 103 3185 3191/1981.

Batch: 5 g. GPG 1400 or GPG 3000 2.6 ml. 3-aininopropyltriethoxysilane 3 nil. trimethylchlorosilane CPG 1400 or CPG 3000 is functionalised by reaction with 3-aminopropyltriethoxysilane in 50 ml.' toluene.

The reaction mixture is shaken for 12 hours at ambient -atemperature and subsequently boiled under reflux for 18 hours. The carrier is filtered off with suction and washed three times with, in each case, 20 ml.

toluene, three times with, in each case, 20 ml. methanol and twice with, in each case, 20 ml. 50% aqueous methanol. The CPG is then shaken overnight in aqueous methanol solution. The liquid phase is separated off and the CPG washed twice with, in each case, 20 ml.

methanol. Subsequently, it is dried first in the air and subsequently in a vacuum.

44 Non-reacted hydroxyl groups of the glass are blocked by reaction with trimethylchlorosilane in 10 ml.

anhydrous pyridine. The suspension is shaken over- S, night, subsequently filtered off with suction and the glass washed five times with, in each case, 20 ml.

methanol and three times with, in each case, 20 ml.

diethyl ether. After air drying, the aminopropylated CPG is completely dried in a vacuum.

5'-0-DMTr-3'-0-succinylnucleoside.

Analogously to the process of Matteucci and Caruthers, 103, 3185 3191/1981 there are reacted mmole DMTrdT mmole (200 mg.) succinic anhydride 300 mg. 4-N,N-dimethylaminopyridinG

(DMAP)

ml. anhydrous pyridine.

deoxythymidine is dissolved in -21pyridine, dehydrated twice and mixed with DMAP, as well as with succinic anhydride. The batch is stirred for 12 hours at ambient temperature and then tested by thin layer chromatography. In the case of a sufficient degree of reaction, the pyridine is removed and the residue dissolved in 30 ml. dichloromethane. The dichloromethane solution is shaken out against fecooled 10% aqueous citric acid solution and the organic phase is subsequently washed twice with, in each case, 15 ml. of water. After drying the dichloromethane layer over anhydrous sodium sulphate, the solution is concentrated on a rotary evaporator and precipitated out in 250 ml. petroleum ether. The precipitate is filtered off with suction and also after-washed twice with, in each case, 20 ml. petroleum ether. The yield is 90% of theory.

Preparation of the p-nitrophenyl ester.

Analogously to the process of Matteucci and Caruthers, 103, 3185 3191/1981 there are reacted 1 mmole 3'-0-succinylated nucleoside (prepared as described above) 4 1 mmole (207 mg.) dicyclohexylcarbodiimide (DCC) 1 mmole (140 mg.) 4-nitrophenol 3 ml. anhydrous dioxan 0.2 ml. anhydrous pyridine.

5'-0-Dimethoxytrityl-3'-O-succinyldeoxythymidine is r i. -22dissolved in the pyridine-containing dioxan solution and the DCC added thereto. From the initially clear solution, dicyclohexylurea precipitates out in a short time. After a reaction period of two hours, the reaction mixture is tested by thin layer chromatography.

The dicyclohexylurea is filtered off and the filtrate immediately reacted with the prepared carrier material.

Reaction of the active ester with the aminopropylated carrier.

Analogously to Matteucci and Caruthers, J.A.C.S., 103, 3185 3191/1981, there are reacted 1 mmole 5'-0-DMTr-3'-0-(4-nitrophenyl)-succinyldeoxythymidine g. carrier material 8 ml. anhydrous dimethylformamide 1 ml. anhydrous triethylamine 1 ml. acetic anhydride ml. anhydrous pyridine mg. 4-N,N-dimethylaminopyridine (DMAP).

Aminopropylated CPG is suspended in dimethylformamide.

IThe solution of the nitrophenyl active ester of the monomer is added thereto, triethylamine is added thereto and the reaction mixture is carefully shaken. After a reaction period of 12 hours, the carrier is filtered off with suction and washed with dimethylformamide, methanol and diethyl ether. After drying in the air, the substance is stored in a vacuum.

-4* -23- Unreacted amino groups are masked by reaction with acetic anhydride in anhydrous pyridine. As catalyst, DMAP is added thereto. After 30 minutes, the carrier is filtered off with suction, washed with methanol, dried in the air and then in a vacuum.

b) Carrier materials cellulose or Sepharose.

Loading of f-he organic carrier materials with nucleoside.

Analogously to R. Frank et Nuci. Acids Res., 11, 4365 4377/1983, there are reacted 44 2 g. carrier material (Whatmann filter platelets No.3, diameter 0.9 mm. or Sepharose 4B CL in dioxan) 1 rnmole 3'-0-succinylated deoxythymidine 2.08 g. (7 mmole) MSNT 0.2 ml. 1-methylimidazole 4 150 ml. anhydrous pyridine 4 4. 50 ml. pyrid'ine 0 50 ml. chloroform 4 50 ml. diethyl ether 20 1.acetic anhydride 1 g. 4-N,N-dimethy~amn.n:pyridine

(DMAP).

The carrier material is mixed three times with, in each case, about 20 ml. anhydrous pyridine and made anhydrous. The 3'-0-succinylated nucleoside, MSNT and 1-methylimidazole are then added thereto and the batch shaken for 3 nours at ambient temperature. The reaction solution, which becomes black during this time, is arM wS W< 1 *j i^ iiwww if-VM-iIX -24suction filtered substantially free from the carrier in a Schlenk's frit and the carrier material again washed white with pyridine, chloroform and diethyl ether and dried. For the blocking of the still unreacted hydroxyl groups, the carrier is shaken for two hours with 40 ml. of a mixture of 80 ml. pyridine, ml. acetic anhydride and 1 g. DMAP. Thereafter, the material is again washed with pyridine, chloroform and diethyl ether and dried.

c) Determination of the carrier loading.

The monomer loading of the carrier is determined by the spectrophotometric quantification of the colour of the dimethoxytrityl (DMTr) cation: A 1 mg. sample is taken, the DMTr cation is liberated by acid treatment and the absorption determined at 498 nm. The desoxyribonucleoside loading is calculated according to the formula: E (m x V (ml) x 14.3 x pmole nucleotide/g.

weight of sample (mg) carrier F498 7 x 104 cm 2 mmole" 1 The following loadings are ascertained: i-^ t carrier monomer loading (pmole7g.) CPG 1400 DMTrdT 15.8 CPG 3000 DMTrdT 10.4.

cellulose DMTrdT 96.2 Sepharose DMTrdT 91.3 B) Synthesis of CPG-(dT 20 cellulose-(dT 20 and Sepharose.-(dT 20 a) Synthesis of CPG-(dT 2 0) In each ca~se, 20 50 mg. of CPG material loaded with the deoxythyrnidine (-'\-terminus) prepared under A) are filled into a steel column or cartridge which is fixed by a clamping system and connected with a SAM I synthesiser (firm ]iosearch).

Reagents: detritylation: 3% trichioroacetic acid in dichloromethane condensation: DMTrdTp III R VMe) with R =isopropyl 350 mg. tetrazole in 10 ml. anhydrous acetonitrile anhydrous acatonitrile oxidation: 500 mg. iodine in 125 ml. tetrahydrofuran, 12.5 ml. water in 1 ml. pyridine capping: 12.5 ml. acetic anhydride in 12.5 ml. TEA, ml. acetonitrile and 4 ml. 1-methylimid a zole washing step: anhydrous acetonitrile.

The individual steps of a cycle take place as follows: 1. Detritylation: For splitting off the dimethoxytrityl radical, 3% trichloroacatic acid is pumped through the carrier material for 2 to 3 minutes. The red-coloured detritylating solutions are collected in a sample 91, -26collector and can be used for the yield determination.

2. Washing: Acid traces are removed by washing with acetonitrile.

3. Condensation: Of the activatable nucleoside (DMTrdTpIII(NR 2 Me) with R isopropyl), per condensation cycle, 50 mg. are placed in a concentration of 50 mg./ml. in a storage vessel from which, during the condensation cycles, mixed with the tetrazole solution, the monomer is pumped through the column within one minute. For the improvement of the yield, the monomer capable of reaction is subsequently pumped round for 8 minutes through the column via a loop.

4. Washing: Excess reactants used are rinsed out with acetonitrile by wabhing lasting several minutes.

Oxidation: By pumping the iodine solution through the column for 2 minutes, the trivalent phosphorus of the phosphorous acid triester is oxidised to pentavalent phosphorus.

6. Washing: The oxidation solution is again washed out with acetonitrile. i 7. Capping (masking of unreacted hydroxyl groups): The capping solution is pumped through the carrier material for 2 minutes.

-27- 8. Washing: The capping solution is removed from the reaction mixture by washing with acetonitrile.

The runoff of a synthesis cycle in the SAM synthesiser requires an expenditure of time of about minutes.

b) Synthesis of cellulose-(dT 20 and Sepharose-(dT 20 In the case of the cellulose and Sepharose materials, the washing steps mentioned Cnder a) are prolonged to about double the time (from usually between 2 and 3 minutes to about 5 minutes). After the detritylation, the introduction of an additional washing step with dichloromethane before the washing with acetonitrile is necessary in order to be able completely to remove acid traces still present in the carrier material. (2 minutes washing with dichloromethane).

c) Loading density.

The preparation of (dT 20 )-oligonucleotides on the various carrier materials in the SAM I synthesis apparatus gives the following loadings: i~) carrier end loading CPG 1400 11.7 pmole/g.

CPG 3000 7.8 pmole/g.

cellulosa- P. 25.4 pmole/g.

Sepharose CL 30.7 pmole/g.

-28- After splitting off of the protective groups (DMTr and methoxy), the oligonucleotide remains on the solid phase and, after removal of the reagents and organic solvents, is dried and stored in a refrigerator until used for the enzymatic reactions.

i d) Splitting off of protective groups.

Splitting off of the DMTr protective group: During a synthesis, the DMTr group is split off with 2% trichloroacetic acid in methylene chloride for 2 minutes or with 3% dichloroacetic acid in methylene chloride within 3 minutes. If the product is to be purified without affinity chromatography, then the immediate detritylation of the oligonucleotide also takes place in the synthesis apparatus. After an 15 affinity chromatographic purification, the splitting I t, ii off of the DMTr protective group from the oligonucleotide takes place by treatment for 30 minutes with 80% acetic acid. After lyophilisation has taken place, water is added twice and again lyophilised. The DMTr group is then extracted with diethyl ether and the product evaporateii.

Splitting off of the methoxy radical on the phosphate.

i The splitting off of the methoxy radical takes place by treatment of the carrier with a freshly prepared solution of thiophenol:TEA:dioxan (1:2:2 v/v/v) over a period of time of 45 minutes. The liquid phase is subsequently separated off and the carrier washed r '1 -um~*BY~imeera~~ll"- IU--~*De.

-29three times with 1 ml. methanol and three times with 1 ml. diethyl ether.

Instead of chemically synthesizing carrier bound oligothymidylate it is also possible to synthesize carrier bound oligonucleotides with mixed base capositon.

Example 2.

Enzymatic 5'-phosphorylation of an immobilised (dT 20 oligonucleotide.

To 100 pg. CPG 3000-(dT 20 from Example 1 (about 500 pmole 1 nmole 5'-OH ends) is added 1 /ul. kinase buffer (400 mM Tris-hydrochloric acid buffer (pH 7.6), 100 mM magnesium chloride, 10 mM DTT, 50% glycerol), 1 pl.4 32 P-ATP, as well as 1 pi. of a 100 pM cold ATP solution and 1 pl. T4-polynucleotide kinase and made up with double distilled water to a total volume of 10 pl.

The reaction mixture is incubated for 15 minutes at 370C. For the quantitative phosphorylation of the free 5'-hydroxyl groups, 1 pi. 1 mM ATP is subsequently added thereto and allowed to react for a further minutes at 370C. The reaction is terminated by the addition of 1 pl. 250 mM EDTA solution/10 pl. reaction solution.

Reaction batch: parent solution reagent end concentration CPG 3000 32 AT0.

1

M

P-ATP

400 mM Tris-HC1 pH 7.6 40 mM 100 mM magnesium chloride 10 mM mM DTT 1 mM glycerol 4 u/pl. T4-PN kinase 400 U/ml.

pl. total volume j ;I ii IC-. 1 *ik- ~Fs For the removal of unbound reactants and buffer, the carrier is subsequently washed five times with, in each case, 500 pl. 0.1 M dipotassium hydrogen phosphate solution. The efficiency of the washings is tested by Cerenkov measurement. In the case of this process, the amount of residual activity remaining on the carrier is from 8'x 10 5 to 2.4 x 106 cpm.

32P decays with a half life of 14 days. By means of the Cerenkov measurement, the breakdowns per minute (counts per minute, cpm) are determined. This takes place in a normal scintillation counter. The amount of radioactivity between 8 x 105 and 2.4 106 cpm permits a conclusion of the amount of incorporated radioactive phosphate in the oligonucleotide immobilised on the carrier material. Since subsequently, by the addition of "cold" ATP, it is completely phosphorylated, the amount of radioactivity indicates whether the kinase reaction has taken place at all or whether it was inhibited by any contaminations in the carrier or by 20 salts or impurities of the oligonucleotide. Higher amounts of radioactivity are favourable since the oligonucleotide can be made visible over a longer period of time. The kinasation of the carrieroligonucleotide is a prerequisite for all other enzymecatalysed reactions.

In the case of tha use of comparatively large amounts of carrier (500 pg. to I a total volume 4~,

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I .1 4.

so V V

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II

31 of 40 pl. is used, the initial ATP concentration is 100 jAM and, afer 15 minutes, 1 pl. of a 10 mm ATP solution is added thereto.

Reaction temperature and period remain the same.

Instead of the enzymatic phosphorylation of CPG bound oligothymidylate the enzymatic phosphorylation of CPG 3000 bound oligonucleotides with mixed base composition is also possible.

Example 3.

Enzymatic linkage of inmobilized DNA fragments with DNA ligase.

The 5'-terminally phosphorylated oligothymidylate from Example 2 covalently bound to CPG 3000 is first hybridized in 15 pl. of water and 2 pl. DNA ligase buffer (760 nM Trishydrochloric acid buffer (pH 10 rmM magnesium chloride, 1 rM ATP) with oligoadenylate and a further non-immobilized oligonucleotide, which has an oligothymidylate as 3'-end, by heating for 3 minutes to 100 0 C. and subsequent slow cooling to ambient temperature.

1 pl. 1M ATP and 2 pl. DNA ligase are added thereto and the reaction mixture incubated at 20 0 C. for 2 to 24 hours.

Reaction batch: parent solution reagent end concentration CPG 3000-(dT 20 )p oligo A oligo T mM ATP 150 pM mM magnesium chloride 1 mM 760 mM Tris.HC1, pH 7.6 76 mM 6 U/pl. DNA ligase 600 U/ml.

pl. total volume i$ %i Ir -32- For the. determination of the yields, a sample (2 pl. suspension) is taken, split off from the carrier by treatent with ammonia for 30 minutes and lyophilised. Subsequent gel electrophoresis on a 10 20% denaturising polyacrylamide gel (0.4 nun.), as well as cutting out of the educt and product bands after the making of the autoradiogram and Sel elution with 0. 5 M ammonium acetate, 1 mM EDTA, the degree of reaction is determined by Cerenkov comparative measuremnent.- Table: DNA ligase reactions: 4 a 4 CG 3000-(dT) 20 oligo A oligo T yield of pho sphate product 1 Mg. Wd) 20 HdT) 10 47%, 1 nmole 1.1 nmole 1 Ug Wd) 12 18 (dT) 10 47% 0.8 nmole 1.1 nmole 500 pg. p(dA) 25 30 (dT) 10 69% 2.3 nmole 5.5 omole 1.00 pg. Wd) 20 (dCTAGGT 10 1 ntaole 1 nmole Instead of CPG bound 5 '-termially phosphorylated oligothymidylate CPG 3000 bound 5 '-terninafly phosphyoryla ted oligonucleotides with mixed base ccoposition can also be linked to suitable olit-onucleotides by mans of DNA ligase.

Example 4.

enzymatic linkage of immobilised MNA fragmnents and desoxyoligonucleotides with ribo-tar~iinus using~ R~NA ligase.

All buffers anid solutions which are used are i CC-~C~-II_ i a

F

I

r d i

I

-33first autoclaved and sterile filtered.

To 100 pg. immobilised 5'-terminal phosphorylated eicosathymidylate from Example 2 is added desoxyoligonucleotide with 3'-ribo terminus and lyophilised.

5 Subsequently, 15 pi. 100 pM ATP solution, 4 pi. 10 mM spermine, 4 pl. 100 mM magnesium chloride solution and 4 pl. 100 mM DTT solution are added thereto and lyophilised. 2 pi. 500 mM HEPES and 3 pl. DMSO are added thereto, briefly shaken and then made up with 10 10 pi. 40% polyethylene glycol solution, as well as with 5 pi. RNA ligase. The reaction mixture is incubated for 48 hours at ambient temperature.

Reaction batch: parent reagent end solution concentration oligonucleotiderA 100 pM ATP 75 M mM spermine 2 mM 100 mM magnesium chloride 20 mM 100 mM DTT 20 mM 500 mM HEPES buffer, pH 7.5 50 mM 100% DMSO polyethylene glycol (MW 6000) 11 mg./ml. RNA ligase 28 pg./pl.

pl. total volume 4444 *r 4~ 4 *ri 44I S i i

I

L-

I

-34- Reactions carried out: batch dissolved amount yield oligonucleotide 1 d(T 7

A

2 )rA 1 nmole 2 d(T 7

A

2 )rA 2 nmole 3 d(T 7

A

2 )rA 4 nmole 63% 4 d(T 7

A

2 )rA 4 nmole 66% d(T 7

A

2 )rA 5 nmole 82% 6 d(GATCCA)rA 5 nmole 7 d(GATCCA)A 2 nmole 66% 8 d(N) 4 8 rA 500 pmole 9 d(N) 4 8 rA 500 pmole d(N) 4 8 rA 700 pmole 46% After the reaction has taken place, the carrier is washed three times with, in each case, 500 pl. TE (10 mM Tris-hydrochloric acid buffer (pH 2.5 mM EDTA), a sample is taken, split off from the carrier by means of ammonia treatment and separated by gel electrophoresis on a 10 20% polyacrylamide gel.

Analogously to the process in the case of the DNA 20 ligase reactions from Example 3, the yields are determined after the gel elution by Cerenkov comparative measurements.

If, after the first RNA ligase reaction, further RNA ligase reactions are to be carried out, a quantitative phosphorylation of the DNA termini with hydroxyl Ii

I

It wi 1.

Sends must first take place. For this purpose, the carrier must be phenolised for the removal of reagents from the RNS ligase reaction: addition of 500 pi. phenol, mix for 30 seconds, centrifuge for 1 minute, remove phenol phase; addition of 500 pl. phenol:chloroform (1:1 v/v), mix for 30 seconds, centrifuge for 1 minute, remove organic phase; addition of 500 pl. isoamyl alcohol:chloroform (1:25 mix for 30 seconds, centrifuge for 1 minute, remove organic phase, briefly evaporate.

By means of a subsequent renewed carrying out of the phosphorylation reaction with T4-polynucleotide kinase, the radioactivity of the carrier again lies at 1 2 x 106 cpm and the 5'-termini are phosphorylated. A further single strand linking carried out subsequently gives, with the example of the carrier sample 10, which contains, as immobilised oligomers, the oligonucleotides (dT) 20 p and dT 2 0 (dN) 4 8 Ap, the following reactions: dissolved carrier Scarrier oligonucleotide CPG-Tg 2 rAd(N) 48 P d(N) 32 rA product by-product yield yield CPG-dT 20 rAd(N) 48 rAd(N) 32 CPG-dT 20 iAd(N) 32 19% 11% -36- Abbreviations: d(N) 48 rA 5' CGC CAT CAT CAA GAA CGC CTA CAA GAA GGG CGA GTG ATA ACT GCA GCA rA d(N) 32 rA 5' GAG CCA GAC GCC CCT GGT GAC GCT GTT CAA AA rA Example Replication of an immobilised oligonucleotide with the help of DNA polymerase (Klenow fragment).

The universally usable starter oligonucleotide (dA) 10 18 is hybridised with the immobilised eicosathymidylate from Example 1 in 16 pl. double distilled water and 3 pl. nick translation buffer (500 mM Trishydrochloric acid buffer (pH 100 mM magnesium sulphate, 1 -M DTT 500 pg./ml. BSA). For this purpose, heating is carried out for 3 minutes at 100 0 C. and then allowed to cool carefully to ambient temperature. After the addition of 5 pl. 10 mM desoxynucleoside triphosphate (contains, in each case, 2.5 mM dATP, dGTP, dCTP and dTTP) and 3 pl.a 2 P-dATP, as well as 3 pl. of the Klenow fragment of the DNA polymerase, the replication is initiated of the oligonucleotide immobilised on the carrier.

Reaction batch: i iL-- a ~r~FE 3ZL -37- (I St S I I 41 *4 8 9i *7 8*80 01 *8rr parent reagent end solution concencration oligo.

oligo A 2 pM 32 P-ATP 0.2 pM mM dNTP 1.6 pM 500 mM Tris-HCI pH 7.2 50 mM 100 mM magnesium sulphate 10 mM 1 mM DTT 0.1 mM 500 pg./ml. BSA 50 pg./ml.

30 pl. total volume Reactions carried out: a) 200 pmole (dA) 16 are elongated in the presence of 2U Klenow polymerase after hybridisacion on CPG 3000dT 20 -rA(dA 2

T

7 from Example 4. Determination of the success of the reaction by Cerenkov comparison measurement after washing five times with, in each case, 500 pl. TE (10 mM Tris-HC1 buffer, 0.25 ~4 EDTA; pH cpm carrier before replication: 41,400 cpm carrier after replication: 243,850 cpm carrier after denaturing: 52,570 cpm supernatant after denaturing: 184,330 b) About 1 nmole of the oligoadenosine product from the supernatant after denaturing from Example (0.25 as oligonucleotide primer and 15U Klenow li 4

L"

RN

-I

-38polymerase are used analogously to Example Determination of the success of the replication by gel electrophoresis. Two samples A and B are applied.

Band bands after ammonia treatment of a carrier sample Result: homologous series from about 14 mer to 30 mer.

mer and 30 mer show stronger bands.

Band bands after carrier denaturing (only super- 10 natant).

V V t j Result: homologous series from about 14 mer to 30 mer.

Example 6.

p lI t Use of restriction enzymes for cutting off an 1* oligonucleotide double strand from the carrier.

CPG-3000-dT 2 o-rA(dA 2 T7) after the Klenow reaction of Example 5 is used.

S.-The batch is carried out in a total volume of 20 pl. An about 25 pg. carrier sample (a quarter of i the batch from the DNA polymerase reaction fromi Example 5) i a mixed with 16 pI. of water, 2 il. low salt restriction endonuclease buffer (10 mM Tris- J hydrochloric acid buffer (pH 10 mM magnesium chloride, 1 mM DTT) and 2 pI. Eco RI added thereto.

The batch is incubated for 1 hour at 37°C.

Reaction batch: 1 -39-

I

I

parent reagent end solution concentration CPG 3000-(dT) 20 oligo 10 mM Tris-HCl pH 7.5 1 mM 10 mM MgCI 2 1 mM 1 mM DTT 0.1 mM pI. total volume After splitting off the samples from the carrier, the result of the cleavage is tested by gel electrophoresis, the sample first being divided up. Part 1 is heat-denatured, part 2 is immediately split off by ammonia treatment (contains the immobilised sequences, as well as those bound by hybridisation). The split off oligonucleotides are desalinated over a Sephadex G 50 column (Pasteur pipette) with double distilled water (pH 8) as elution agent. The samples eluted in the dead volume are lyophilised and phosphorylated according to the method given for oligonucleotides in solution. A polyacrylamide gel is used for the testing of the result of the cleavage.

Products are obtained which are all shorter than the 30 mer used. No inhibition of the restriction enzyme is observed.

-L1 Example 7.

Ligating of a DNA fragment with the help of DNA ligase on an oligonucleotide bound to a swellable carrier.

Reaction batches: I, f carrier/amount oligo A dT 1 0 yield ligation product total CPG 3000 p(dA)253 500 g 2.3 nmole 5.5 nmole 69% 51% cellulose Fp p(dA) 2 5 3 0 1 mg. 2.3 5.5 nmole 68% 43% Sepharose p(dA) 25 3 0 CL4B 2.3 nmoe 5.5 nmole 65% 41% I2.3 nmole 1 mg.

The given yields are referred to the reaction of the radioactively-labelled oligonucleotide on the carrier.

All three carrier materials used are comparable in the case of the kinasation with T4-PN kinase and of the DNA ligase reaction. However, the two swellable carriers are much more difficult to free from the reactants used (clearly visible in the case of washing out of the radioactively-labelled ATP used). Even after thorough washing steps, in the case of the gel electrophoresis, parts of non-covalently bound radioactive phosphate are still present.

I

-41- In the case of the DNA ligase reaction, the yields in the case of all three carriers are comparable. However,-on.the basis of the better chemical synthesis on CPG 3000, the yield of desired ligation product (dT) 3 0 is distinctly better in the case of CPG 3000.

In the case of the chemical synthesis, cellulose filter platelets display the problem that, due to the facition of new hydroxyl groups by mechanical stressing of the carrier material, sequences of shorter chain length are formed. This can be observed by the increase of the trityl colour.

Most problematical is a chemical synthesis in the case of Sepharose CL. Since the carrier is extremely swellable, washing steps must be carried out between the reaction steps for a significantly longer period of time. The automation of the synthesis can only be carried out with great difficulty. Furthermore, as in the case of cellulose, an increase of the trityl colour can be observed so that, here too, a homologous product series results even though with clearly more strongly appearing end product.

1 1; d

Claims (8)

1. Carrier for the enzymatic or chemical and enzymatic reaction of nucleic acids or nucleic acid fragments on a solid phase consisting of an insoluble, non-swellable, porous polymer on which are ccvalently fixed one or more nucleic acids or nucleic acid fragments optionally provided with protective groups usual in nucleic acid chemistry, wherein the pores of the polymer each have a definite size in the range 2500 to 5000 A.

2. Carrier according to claim 1, wherein the polymer is essentially built up of silicon and oxygen atoms,

3. Carrier according to claim 2, wherein the polymer is a glass.

4. Carrier according to any preceding claim wherein the pore size is approximately 3000 A. Carrier according to any of the preceding claims, wherein the nucleic acid fragments are provided with protective groups.

6. Carrier according to any preceding claim, wherein the nucleic acid fragments optionally provided with protective T: groups are fixed on to the polymer via aminopropylsilyl spacers (-Si-(CH2) 3 I t "A 7. Carriers according to claim 1, substantially as hereinbefore described and exemplified. i

8. Process for the enzymatic or chemical and enzymatic solid phase synthesis of nucleic acids, in which nucleic acid fragments optionally provided with protective groups are fixed on to an insoluble, non-swellable, porous polymer, the immobilised nucleic acid fragments are made 4' 1 .i- Af 43 longer by further nucleic acid fragments and the final nucleic acids are subsequently optionally dissolved off from the insoluble polymer, wherein a polymer is used, the pores of which essentially possess a definite size of from 1400 to 10,000 A.

9. Process according to claim 8, wherein the polymer is glass with a definite pore size of from more than 2500 to A. Process according to claim 9 in which the pore size is approximately 3000 A.

11. A process according to claim 8, substantially as hereinbefore described with reference to the examples. 4, 12. Nucleic acids, whenever produced by the method of any one of claims 8 to 11. DATED this 15th day of August 1990. BOEHRINGER MANNHEIM GmbH By Its Patent Attorneys DAVIES COLLISON I I lit ft I I I JJ 9008 15,lnmdaLO3Sn:\28572boJsp,43