IL95537A - Process for the multiplication of nucleic acids using at least two adaptors - Google Patents

Description

/29/187 95537/2 PROCESS FOR THE MULTIPLICATION OF NUCLEIC ACIDS USING AT LEAST TWO ADAPTORS 95537/2 -2- The present invention is concerned with a process for the preparation of nucleic acids, with the use thereof in a process for the detection of nucleic acids and with reagents which are suitable for use in this process.

The detection of nucleic acids in samples has recently found increasing use in molecular biological and genetic fundamental research, in clinical diagnosis and in biotechnology.. The purpose of this detection is, for example, the finding of pathogens in biological samples or of specific nucleotide sequences in genomes. The possibility of being able to detect very lo.w concentrations is thereby especially important. For such uses, it has proved to be necessary to multiply the nucleic acids to be detected in a preceding step and only then to detect according to conventional methods.

Such a procedure is suggested in published European Patent Specification No. A-0 , 201 , 18 , corresponding 78284. The nucleic acid to be multiplied is mixed with two single-stranded oligonucleotide primers which are each complementary to a different strand of the nucleic acid to be multiplied. The primers are each elongated to a complementary strand of the nucleic acid. Each of the double ''strands thereby formed can again be used as nucleic acid to be multiplied but only after it has been separated into individual strands. Each of the two complementary nucleic acid individual strands can now be reacted in the following cycles with new oligonucleotide primer and multiplied analogously.. This process has the disadvantage thet large amounts of oligonucleotide primers must be used. Furthermore, the nucleic acid double strands formed must each time be physically separated from one another in an additional reaction step between the actual * multiplication steps. For this purpose, there are necessary either elevated temperatures or additional reagents.. Both are incompatible with the simplicity of carrying out the reaction.

In World Intellectual Property Organisation's Patent Specification No. WO 88/10315 is also described such a process which, hacwever, does not solve the descjribed problems; in particular, there are here also required more than equimolar amounts of promoter oligonucleotide., referred to the resulting amount of nucleic acid. Furthermore, the processes of the prior art have the disadvantage that they operate relatively slowly because of the large number of successive reaction steps.

Therefore, there is a need for a process for the multiplication of nucleic acids which takes place quickly in few reaction steps without a succession of several .identical reaction cycles and especially of temperature cycles and can take place with as few as possible simple reagents.

Thus, according to the present invention, there is provided a process for the preparation of nucleic acids with the use of a nucleic acid A,, wherein the nucleic acid A is reacted with at least two adaptors,, each of which has a nucleotide sequence which, in each case,, can be hybridised with a part of a strand of the nucleic acid A and of which at least one-contains a nucleotide sequence specific for a replication system,, under conditions in which is farmed a nucleic acid substantially complementary to at least a part of the nucleic acid A which, furthermore, contains at least one adaptor which has a nucleotide sequence specific for a replication system and the complex so formed of nucleic acid A and the nucleic acid formed in the previous step is reacted under conditions appropriate for a replication reaction with one or more proteins of thejreplicati^on system which alone or together catalyse the formation of nucleic acid sequences which contain at least the nucleotide, sequence of an adaptor,; which contains a nucleotide sequence specific for a replication system,, as well as a nucleotide sequence^ which is either substantially homologous or essentially complementary to at least a part of the nucleotide sequence of the nucleic acid. A# The present invention also provides reagents for carrying out this process* In the following,., as a nucleic acid substantially complementary to another nucleic acid there is designated a nucleic acid, the nucleotide sequence of which can hybridise with another nucleotide sequence even though the base pairing in one or more nucleotides does not,, as a rule, correspond to the rule according to Watson and Crick (mismatch).

As a nucleic acid substantially homologous to another nucleic acid., there is designated a nucleic acid, the nucleotide, sequence of which differs from the nucleotide sequence of the other nucleic acid in one or more nucleotides but which, nevertheless, can hybridise with a nucleotide sequence complementary to the other nucleic acid.

In the process according to the present invention, as nucleic acid A there can be used all kinds of nucleic acids-. These: include not only RNA but also DNA, both in single- or double-stranded form, of natural as well as synthetic origin.. When the nucleic acids are present in double-stranded form, they must be converted into single strands.This can take place in conventional manner,, for example by heating or with the help of appropriate reagents, which also include double strand-unwinding enzymes, for example helikase. Enzymatic strand separation can also be induced by enzymes, for example recA, in the presence of ATP' or analogous enzymes. The strand separation can be increased by single strand-binding enzymes, for. example T4 gene 32 protein or Escherichia coli SSB.. The nucleic acids A can also be pre-treated with chain-shortening enzymes, for example restriction enzymes.

The origin of the nucleic acids is of no importance. Preferably, nucleic acids can be multiplied . in solutions. - or suspensions but also fixed to solid phases or n cell-containing media, cell smears, fixed cells or tissue? segments or on isolated. chromosomes. The multiplication of dissolved nucleic acids is especially preferred.. Examples thereof include viroid,, viral, bacterial and cellular nucleic acids.. ¾e medium in which the single-stranded nucleic acids are present is, in the following, referred to as sample medium.

Besides- the nucleic acid A,, this medium can also contain other components,., especially nucleic acids, which are not to be? multiplied..

For the preparation of nucleic acids, the sample medium is mixed, with at least two adaptors. The addition of two adaptors per nucleic acid strand A is preferred. Both adaptors must be hybridisable with a strand of the nucleic acid A. either internally or on end sequences. This is fulfilled when each adaptor contains a . nucleotide sequence which is substantially complementar to a nucleotide sequence of one strand of the nucleic acid A.. These regions can lie anywhere on the nucleic acid A,. Preferably, at least one of the regions lies on one end of the nucleic acid.- Especially preferably, both regions lie on the ends of the nucleic acid A. It has proved to be preferable when one end or the ends of the nucleic acid A have nucleotides which are complementar to the single strand sequence of the adaptor or of the adaptors and can be hybridised with these. The adaptors can contain not only ribo- but also deoxyribonucleotides. They preferably contain deoxyribonucleotides-. At least two of the adaptors hybridise with the same strand of the nucleic acid to be multiplied in regions separate from one another. These hybridisation regions preferably lie at 1 to about 20,000 and especially preferably 100 to 8000 nucleotides apart from one another and, therefore, do not overlap with one another, It has proved to be advantageous when the adaptors are present single-stranded in the region in which they are to hybridise with the nucleic acid.

The nucleotide sequence of the single-stranded hybridising region of the adaptors is so chosen that free ends of these single-stranded regions, afte the hybridisation of these single-stranded regions with the nucleic acid, point at one another.. Therefore, there participatesa t least one adaptor with a single-stranded 5' -end and one with a single-stranded 3 '-end. Preferably, the 3' -end of one adaptor facing the other adaptor is a hydroxy1 end and the 5' -end of the second adaptor facing the first adaptor has a phosphate group.. Other 5' -ends of the adaptors can carry phosphate groups.. Other 3' -ends of the adaptors which do not participate in the gap-filling reaction can be modified, for example can carry a 2' ,.3 ' -dideoxy-nucleotide on the 3 '-end. The single-strandedregion of the adaptors hybridising with the nualeic acid A preferably has a nucleotide length of 15 to 60 and especially preferably of 20 to 40..

Furthermore, at least one and preferably two of the adaptors has a region which essentially cannot, hybridise with the nucleic acid to be multiplied. This region is preferably double-stranded. In this region, this adaptor or these two adaptors contain a nucleotide sequence specific for a replication system. In the case of a double-stranded region, the region can be formed,, for example, by ttedouble-stranded sequence as such or its secondary structure.. By replication systems are to be understood the reagents necessary for the replication of a nucleic acid. To this belongs especially a replication enzyme, preferably a DNA polymerase. Such replication systems are known, for example, from the phages PRDL, 015, M29, Nf , GA-1, Cpl and 029, as well as eukaryotes, such as adenoviruses.. Nucleotide sequences which are specific for the replication systems are preferably the "origin of replication" (ori) sequences., (see: B. Lewin, Genes III, pub.

John Wiley,, 1987) . In the case of the 029 phage , these preferably" have a length of 12 to 59 nucleotides (nt).. Each of the 029-ori- regions preferably contains a double-stranded region with a length of 6 nucleotides-.

Preferably, at least one of the adaptors has a nesidue bound to one or both nucleotide strands in a region which is preferably not the single-stranded region provided for the hybridisation with the nucleic acid. This residue is preferably a protein or a component from cell extracts* The protein can have a plurality of functions. A replication-initiating function has proved to be especially advantageous.

In the case of the use of the replication system of 029 » the protein p3 of the 029 phage has proved to be especially preferred* The binding of the residue; to the adaptor is preferably covalent. In the case of the protein p3 , the binding by means of a reaction with the 029 protein p2 and dATP has proved to be suitable. In the case of p3 > it is no.t absolutely necessary but, nevertheless, preferred that an adaptor is present covalently bound > already at the beginning of the hybridisation reaction of the adaptor and of the nucleic acid, with p3„ The binding can also first take place after the hybridisation. Especially preferred is the case th3t two adaptors are used which, on the 5 ' -ends of the ori-specific region, each have bound a protein p3.. In this case, the process according to the present invention is especially effective. However, it is, for example, also possible to use an adaptor with a double-stranded specific region and an adaptor with a single-stranded region. The adaptors can have additional sequences, especially between the hybridising and replication enzyme-specific region.

The nucleic acid A. is reacted with the adaptors' under conditions in which the adaptors hybridise with the corresponding regions of a strand of the nucleic acid A.. The process according to the present invention then provides for the formation of a nucleic acid B which, at least in part, is substantially complementar to the nucleic acid L· and includes a^t least one adaptor which contains a sequence specific for the replication system.. The length of the nucleic acid B is determined by the length of the adaptors and the length of the nucleotide sequence of the nucleic acid A to be multiplied lying between the single-stranded ends of the hybridised adaptor.. The formation of the nucleic acid B preferably takes' place in a so-called "gap—filling" reaction.. The conditions for this are. known, for example, from Maniatis et al... (Molecular Cloning, 1982, pub. Cold Spring Harbor Laboratory) or Davis et al.. (Basic Methods in M0lecular Biology , 1986, pub. Elsevier),, Enzymes which are used in the "gap-filling" reaction are those polymerases which are: able to synthesise a complementary strand between 3'- and 5' -ends of double-stranded regions. Depending upon -Lithe nature of the nucleic acid A.,, these are RNA-dep^endent DNA polymerases, for example virus-coded enzymes, such as reverse transcriptase, or DNA-dependent DNA polymerases-, such as' T7, T3 or T4- DNA polymerase, lenow enzyme or Taq-DNA polymerase..

Especially preferred are DNA polymerases which do nat have a strand-separating activity, for example T4 DNA polymerase or leiiow polymerase* Furthermore , a DNA ligase, for example Escherichia coli or T DNA ligase, is preferably used.. If the reaction is to be. carried out at elevated temperatures,, then there is preferably used a thermostable DNA ligase such as is known from published European Patent Specification No„ A-O,.373*962'..

The result of the r "gap-filling" reaction is the formation of a nucleic acid strand substantially complementary to the region of the nucleic acid A lying between the adaptors, ; which is covalently bound to the single-stranded end of at least one adaptor which contains a nucleotide sequence specific for a replication system.. This newly formed nuOleic acid is,; in the following, called, nucleic acid B..

The case is preferred in which the nucleotide sequence of two adaptors are incorporated into the newly formed nucleic acid B. This can be achieved, for example, by the additional use of DNA ligase.

An important step of the process according to the present invention is the reaction of the complex formed from nucleic acid A and nucleic acid B with one or more proteins of a replication system which, under suitable conditions, via an in vitro replication reaction which comprises an enzymatic strand separation, can form a nucleic acid C or D complementary to nucleic acid B or nucleic acids at least partly complementary to it, with the use of suitable co-factors snd/or cell extracts. Preferred proteins are. those with DM polymerase activity. Especially preferred are those proteins which are capable of this without renewed addition of adaptors.. In the case of the use of 029-ori sequences-, such an enzyme; is, for example, the protein p2 of the phage 029, together with p3 and dATP, The components of the replication system can be-added, for example, individually, as a mixture or in the form of an extract. The addition of purified components is. preferred.

For. this purpose, the enzyme binds to the recognition region of the adaptor and forms, with the cooperation of nucleoside triphosphates,, the complementary nucleic acid. C or D, A protein of the replication system has the property of carrying out a strand displacement. In the case of 029, p2 has not only DNA polymerase but also strand displacement activities. Because of the enzymatic strand separation during the multiplication reaction, a separate denaturing, step between individual multiplication steps can be omitted. Thus, the process according to the present invention has the advantage that it can take place continuously and quickly. For the case that two adaptors have been used, each of which has a recognition site for the enzyme, the process is especially advantageous, since: in principle the formation of the nucleic acids C or D can begin at both adaptors Therefore, the multiplication reaction is greatly accelerated.

The reaction for the formation of nucleic acids C or D from the complex can now take place several times in succession since the freshly formed nucleic acids C or D can also form complexes, the reaction of which with the enzyme leads to further nucleic acids D and C without separate intermediate reection steps* In the ideal case,, an exponential increase of the amount of nucleic acids C and D is achieved. No further adaptors are needed for the formation of nucleic acids G and D. The nucleic , acids C or D formed contain especially, in each case, at least one nucleotide: sequence of the adaptors and at least one nucleotide sequence which is substantially complementary or homologous to the nucleic acid A or a part thereof. The length of this nucleotide sequence corresponds to nucleic acid A. in the region which lay be-tween the single-stranded ends of the adaptor.. Therefore,, the nucleic acids B, C and D formed preferably contain only a part of nucleic acid A, When the desired number of nucleic acids C and D is formed r the reaction can be terminated. This preferably takes place by the addition of a stop reagent y for example ethylenediamine-tetraacetic acid (EDTA)„ The reaction time necessary for the preparation of the nucleic acids is, in comparison with the processes of the prior airfe, greatly reduced by the process according to the present " invention. As in the case of the known processes, it depends, inter alia , upon the length of the nucleic acids formed.

The nucleic acids C and D and the complexes formed from them can be the subject of further reactions.. For example* double strands formed from them can be cleaved with restriction enzymes. In this case,, adaptors- are preferably used which have a cleavage position for the restriction enzyme in their double-stranded region.

The adaptors can also contain a sequence for hybridisation with M13 universal sequencing primers and/or ΓΊ13 universal reverse sequencing primers..

The nucleic acids C and D can also be the subject of reactions for the introduction of radioactive or non-radioactive labellings so that the nucleic acids C and D can easily be detected.

Whether the desired number of nucleic acids C and D has been formed can be determined by known methods, for example by gel- chromatography.

In principle, with the process according to the present invention, besides the nucleic acid Δ, further nucleic acids or parts thereof with differing nucleotide sequence can be used in the same sample medium for the preparation of nucleic acids. It is a prerequisite that, for each nucleic acid single strand, the corresponding adeptors are pres-ent.. When the differences in the nucleotide sequence of the further nucleic acids are such, for example in the case of a difference; only in a base in the inner region of the region comp!Iementary to the adaptor, that the* adaptor under the hybridisation conditions with nucleic acid A also hybridises, with the further nucleic acids', then no adaptors specific for the further nucleic acids are necessary.. If the nucleic acid in the sample was originally a dauble-strended nucleic acid, then both individual strands can be multiplied when they are present as individual strand. If the nucleoside sequences do not have regions with the same nucleotide sequence, then at least four adaptors are needed which preferably bind to different,, non-complementar sequences..

The process according to the present invention has various advantages: for example, in the case of the use of the replication system of the phage, 029j the reaction sequence can be carried out at one low temperature ; high temperatures for denaturing are not necessaryv The in vitro replication functions with long DNA fragments of up to 19 kb (with.029 D.NA) and at least up to 7 kb (M13 genome) with heterologous DNA.. In comparison therewith, the size of the sequences which can be amplified according to published European latent Specification No» A-0 201,184-, ponding to IL 78281 and IL 78284, is at most 3 - 4 kb.

The rate of amplification is theoretically higher or the multiplication of the nucleic acids is quicker than in the case of other target amplification methods.. Since the method can preferably be carried out in the temperature range of from 25 to 50C. and especially preferably of from 50 to 37°C., it can be used for amplification in in situ hybridisation experiments', for example in cell smears,, fixed cells or tissue; section? or an isolated chromosomes. The relatively short adaptors diffuse into tissues' just as well as oligonucleotides..

An advantage of the process according to the present invention is that the replication step, because of the high processivity and strand displacement activity of the polymerase, is not dependent upon the secondary structure of the total complex. It is, therefore, possible also to prepare long nucleic acids.

By means of the introduction of restriction enzyme cleavage positions in the adaptors between single- stranded region (specific for nucleic acid A) and the region specific for the replication system, the resultant products can, for example be cloned or sequenced directly or, as sample, be cleaved free from the adaptor sequences. This is not possible with the products from transcription amplification processes.

The process according to the present invention for the preparation of nucleic acids is extremely time-saving and extremely sensitive.. Inclusive of adaptor hybridisation, gap filling and replication, for a 100 nucleotide-containing partial piece of the nucleic acid A, there is given a time requirement of up to about 1 hour with a theoretical amplification rate of 2692 ; processes according to the prior art would, in the most favourable case,, require 2 to 4 hours for this. The process according to the present invention can be carried out as a one-vial reaction, preferably via the successive addition first of the components of the gap-filling reaction and then the components for the in vitro replication.

The nucleic acids B,. C and D resulting in the multiplication reaction preferably have the same; length. This is advantageous since a homogeneous; nucleic acid population makes possible more uniform conditions for the detection thereof ,, for example in the case of hybridisations.

The nucleic acids B, C and D are DNA's. This has the advantage that the nucleic acids C and D formed from them can again be used a template for the formation of D and C in the same reaction without intermediate separate reaction steps.In that the newly formed nucleic acids B„ C and D already have at least one seqiuence specific for the replication system, the renewed, addition of adaptors is superfluous.

The process according to the present invention can be used for the preparation of a plurality of copies- of nucleic acids A or of parts thereof or of nucleic acids complementary thereto.. This is especially important in molecular-biological and genetic fundamental research, in clinical diagnosis and in biotechnology and in the study of the structure and functions of rare genes..

The subject of the present invention is also a process for the detection of nucleic acids A which includes- the above-mentioned, process for the preparation of nucleic acids.

For. this purpose,, a sample,of which it is presumed that it contains the nucleic acid to be detected, is subjected to the above-described process for the: preparation of nucleic acids, whereby the nucleic acid to be detected is treated in just the same way as described above for the nucleic acids .

The nucleic acids B,. C and D can then be prepared with thehelp of modified or non-modified nucleoside triphosphates., Modified nucleoside triphos hates are preferably used. Such modified nucleoside triphosphates are known.. The modification can consist, for example,, in the replacement of one or more residues of the nucleoside triphosphate by a radioactive „ fluorescent, coloured, immune-reactive ,. biospecifically bindable or chemically reactive residue.. Appropriate immune-reactive residues include, for example, haptens, such as digoxigenin or sulphonic acid residues, bio-specif ically-bindable residues are, for example, vitamins,- such as biotin, and a chemically-reactive residue is, for example, an additional amino group or sulphhydryl group which is possibly attached to the nucleotide triphosphate via a spacer. jn ¾ηβ case of the use: of modified nucleoside: triphosphates, for the detection of the presence or amount of the nucleic acid A there is simply determined the amount or the presence of the nucleic acids B,. C or D via the; incorporated modification after separating off unreacted nucleoside triphosphates. The procedure via the incorporation of aliready modified nucleotides is especially advantageous since usual further hybridisation steps with labelled nucleic acid probes or: elongation reactions can then be omitted, Processes for the detection of nucleic acids into which modified nucleotide phosphates are incorporated are known, in the case of hap en labelling for example from published European Patent Specification No.

A-0,324-,4-68 and in the case of biotin labelling from published Federal Republic of Germany Patent Specification No. A-29 15082, For this purpose , the hybridised nucleic acids-are: separated from unreacted components on molecular sieves or affinity materials which recognise and bind the dctuble-strand-ed nucleic acids or a residue of the. nucleic acid, for example p3« Subsequently , the amount of labelling is determined.

However, it is also possible, especially in the case of non-modified nucleoside triphosphates, to detect the nucleic acids C or D formed by hybridisation with a modified nucleic acid substantially complementary to at least a part thereof. These nucleic acids can be detectable or can be made detectable. Such processes are also known. Such a process is described, for example, in U.S.. Patent, Specification No.. 4, 358 , 535 and in published European Patent Specification No, A-0 , 192 ,1.68 , corresponding to IL 77926.

Because of the possible incorporation of modified nucleoside . triphosphates,: the process according to the? present invention for the preparation of nucleic acids is also outstandingly suitable as a process for the preparation of modified nucleic acids and especially of modified. D , Such modified nucleic acids find use' as so-called probes in DNA diagnosis. If the modification is a detectable group or a group which can be converted into a detectable group, then nucleic acids are obtained which can be used as detection probes in the process described in U.S. Patent Specification No, 4 , .358, 35. If the modification is a bindable group, for example an immune-reactive group or biotin, then nucleic acids are obtained such as are described 95537/2 -21-in published European Patent Specification No.

A-0,097,373, corresponding to IL 69051. They can be used as capture probe in, for example, the process according to published European Patent Specification No. A-0 139T4-89.

The process according to the present invention for the detection of nucleic acids combines the advantages of the process for the multiplication of nucleic acids or parts thereof with those of labelling during the multiplication..

With this process r there can be detected not only DNA but also RITA. In the case of RNA, the detection of rENA is preferred, since:- here, especially high sensitivities or short reaction times are possible. In this way*,, species diagnosis, for example, becomes possible.

The single-stranded, hybridising regions of the adaptors can be selected according to the instructions of U.S.. Patent Specification No. 4,851,330.. However, this species diagnosis is also possible via bacteria-specific genes, for example toxin genes or patho— geneity factors.

The process according to the present invention can also be used for mutagenesis via mutagenesis adaptors or via adaptors which, in the hybridising single-stranded region,, have at least one mismatch or- fo the detection of mutations via adaptors which* in the hybridising single-stranded region, have at least one mismatch.

An especially preferred embodiment of the process according to the present invention for the preparation of nucleic acids becomes possible by the in vitro use of the replication system of the phage 029 for the formation of nucleic acid C or D from the complex of the nucleic acids A and B„ This replication system is described, for example, in B ochim.. Biophys. Acta, 951:%,· ^17- 2^ 1 88.

For the start,, two adaptors are used for the sequence-specific amplification. These adaptors contain as dcQible strand the left and right origin of replication (ori) of the phege 029 and a single-strandedr target-specific region.. The replication system of 029 was chosen since, for the in vitro replication, besides the nucleoside triphosphates, only the DNA polymerase p2 and protein p3 correctly bound, to the 5'-ends of the adaptors and free p3 , as well as dATP, are needed. After hybridisation of the adaptors, the single-stranded gap between both adaptors is closed with dNTPs and DNA polymeras or Klenow polymerase and Iligated to a complete strand by means of Escherichia coli DNA. ligase or T4- DNA ligese. Thereafter,, the replication reaction is initiated with p2 and p3 and the replication carried out with dNTPs.. The 029 DNA polymerase is inhibited with EDTA after an appropriate reaction time, dependent upon the template length.

The detection of the amplification products is possible not only in gel with hybridisation with labelled nucleic acid probes or by labelled nucleotides incorporated during the amplification reaction.

The following conditions have proved to be advantageous: Gap-filling reaction.

Concentration of adaptors 1.00 to 500 jj.fi; temperature 25 to 5°C. and preferably 30 to 37°C„;-sample volume^ 20 to 50 p . ; buffer, substance preferably T is HCl,.. pH 7..5 - 8.0 , 20 to 100 mM? salts,, preferably magnesium chloride 2 to 15 mM, ammonium sulphate 5 to 50 mM; adjuvants: DT 1 to 10 mM,, bovine;: serum albumin (BSA) 50 to 250 /A-g./ml, ; nucleoside triphosphates 0 to 250 ^-M; period of hybridisation 5 to 15 minutes; polymerases 2 to 5 U; ligase 2 to 5U , incubation 5 to 30 minutes* Replication, Temperature, preferably 25 to 45°C. and especially-preferably 30 to 37^0. and more especially preferably as in the case, of gap-filling (however,, the temperatures of gap-filling and replication can also differ: from one another);- volume: 20 to 50 μ.1. ; p3 : 20 to 00 ng..; p2: 2 to 300 ng. ; buffer:Tris, pH 7..5 , 20 to 100 mM; salts magnesium chloride 2 to 15 mM, ammonium sulphate: 20 to 50 mM; adjuvants spermidine: 1 to 5 mM; incubation time 15 minutes (for very short regions) to 90 minutes (for long regions); nucleotides as above or alternatively -dNTPs, biotin-dNTPs or digoxin-dNTPs, Detection. a) Agarose gel in the case of directly lablled nucleic acid b) Hybridisation with radioactively- or non-radio- actively-labelled sample DNA., do:t» Southern blot, for example on membnanes:: 1.) fixing with UV„ 250 nm 2) fixing via hybridisation with wall-bound complementary nucleic acids, or in microtitre plates, tubes- (of nylon NG synthetic material,, streptavidin-coated synthetic material,, columns or beads, etc).

The given values serve as- recommendations but obviously changes can be made.

In theory, the detection process according to the present invention can be carried out up to the detection completely in a solution without separation of any components. Only then is a separation of excess labelling agent necessary.

Also the subject of the present invention are the adaptors used in the above-described process which adaptors consist of at least oiie; single-stranded nucleic acid region which is substantially complementary to at least a part of the nucleotide sequence of a nucleic acid to be multiplied and a double-stranded region which contains a sequence for the recognition of a DNA polymerase;. Adaptors are preferred which additionally contain a bound protein..

In Figs. 1 and 2 of the accompanying drawings, the process according to the present invention for the preparation of nucleic acids is shown schematically using the example of the pro-tein-primed in vitro replication with the help of the replication system of the phage 029.

Fi ,. 1 shows the reaction of nucleic acid A. (1) with two adaptors (2,: 3) which, in their single-stranded regions (6, 5), hybridise with different regions of the single-stranded nucleic acid Α».

Furthermore, each of the adaptors 3 end 2 contain, in each case., a nucleotide: sequence; for the specific binding of the replication system (4·,, 7)» I addition, the case is here shown in which the adaptors contain protein p3 covalently bound on the 5' -end. By gap-fillina the complex (8) is produced.

In Fig.. 2, there: is shown the replication of the complex (8). By means of the replication system . of the phage: 029, which especially contains the proteins p2 and p3„ as well as dAT and the other nucleoside triphosphates;, the nucleic acids C and D,. respectively (1.0 and 11) are formed which again form complexes (9) which, like complex (8), can form a basis for the replication. Thus, in the case of each new replication, new nucleic acids C and D are formed.. As soon as the desired amount of nucleic acids has been achieved T the process can be stopped.

Fig.. 3 shows the arrangement of the adaptors relative to one another and to nucleic acid A.

Fig. 4 shows a further arrangement of the adaptors relative to one another and to nucleic acid A. This arrangement can be achieved by digesti the template nucleic acid before amplification, for example with a restriction endonuclease.

The following Examples are given for the purpose of illustrating the present invention: Example la F&ur oligonucleotides are used as adaptors. The left adaptor consists of a nucleic acid strand of 66 nt in which -6 nt correspond to the 5 * -end of the left origin of replication sequence of 029 from PNAS,, 28 , 2596-260.0/1981 ;: Virology, 1§5„ W-483/1986 ; Gene 5 „ 1 - 11/1986 ; NAR, 16/13., 5895-5913/1988 , and 20 nt to HBV-specific sequences (see European Patent Specification No.. B-0 ,,013 ,,828, nt.No. 1278-1297), and carrplementary strand to the ori region of 46 nt. The right adaptor consists of a nucleic acid strand of 79 nt in which 59 n correspond to the; 3 ' -end of the right ori sequence of 029 and 20 nt to HBV-specific sequences (see European Patent Specification No.„ B-0 ,.013 ,828 nt.No. 1 22) , and a complementary strand to the ori region of 59 nt. The HBV sequences are complementary to the single-stranded region in the HBV genome and cover a region of 106 nt..

HBV templates (HBV genome, 0..5 ^ ^ to 0„5 fg. ) is mixed in a volume of 20 ^l. with the adaptors in Tris-HCl, pH 7.5 „ 50 mM;magnesium chloride 10 mM; ammonium sulphate 20 mM; dithiothreitol. (DTT) 2 mM; bovine serum albumin (BSA) 200 ^g./ml. ; dATP 150 yu ; dCTP 150 ^ ; dGTP 15Q ^M; dTTP 150 ^M NAD 26 ^M, so that the adaptors, ere present in a concentration of 250 nM.. ¾is solution is incubated for 15 minutes at 300C.. for the binding of the adaptors. For filling the gap between the two bound adap tors , 2 U T4 DNA polymera se and 2 U Escherichia c oli DNA liga se are added thereto in 5 ^ .. of the above-described buffer and incuba ted for 10 minute s a t 30°C .

Thereaf ter , for the initia tion of the replica tion (PNAS, 21r 5522-5526/1982; PNAS, 80, 4282-4252/1983 ; PNAS,.81,..5374-5378/1984; PNAS,.81,.80-84/1984; PNAS,. 82,.6404-6408/1985; J. Bi0i. Chem. ,: 264/15, 8935-8940/1989) , 150 ng. of the terminal protein of 029 (p3) and 80 ng. of the 029 polymerase (p2) in 25 /χ ΐ· of Tris HCl,. pH 7.5 50 mM ; magnesium chloride 10 mM ; spermidine 2 mM ? ammonium . sulphate 20 m ? asre added thereto and incubated, for 30 minutes- a t 30°C . The reaction is stopped with 10 mM EDTA and 0„1¾ SDS„ E!or the detec tion , the replication products are separated in a 0„8¾ agarose gel and made visible with ethidium bromide staining . .

Example lb The identical adaptor oligonucleotides as in example la are used. HBV-specific tenplate sequences are used as in example la and are incubated in a volume of 30 μΐ for the gap-filling- reaction between the two adaptor sequences in Tris-HCl, pH 7.5 , 25 mM; KCl, 6.3 mM; MgCl2 , 15 mM; dithioerythritol (DIE) , 2 mM; ATP, 50 μΜ; dATP, 25 μΜ, dCTP, 25 juM; dGTTP, 25 juM; dTTP, 25 μΜ with adaptor oligonucleotides, each 250 nM, 2 U KLenow polymerase and 2 U T4 -ligase for 30 ' at 30 "C. For starting the replication reaction 150 ng of the terminal protein of 29 (p3) and 80 ng of the β 29-ENA-polymerase (p2) ar added in 20 μΐ Tris-HCl, pH 7.5 , 87.5 mM; MgCl2 , 2.5 mM; spermidine, 5 mM; ( H) 2 S04 , 50 itM; and incubatet for 30 · at 30°C. The reaction is stopped with EDIA, 10 mM and SCS, 1 % and the reaction products are detected as described in example la.

Example lc As in examples la and lb HBV-specific sequences are incubated in 30 μΐ in Tris HC1, pH 8, 50 irM; MgCLj, 5 iriM H4-acetate, 60 mM; Cm, 5 IriM; dATP, 250 μΜ; dCTP, 250 μΜ; dGTP, 250 μΜ; dTTP, 250 μΜ; NAD, 200 μΜ; and adaptor specific oligonucleotides, each 250 nM with 2 U E.coli-ligase and 2 U T4-DNA-polymerase for 30'at 30°C. In the following replication reaction 150 ng of p3 and 80 ng of p2 are added in 20 μΐ Tris-HCl, pH 7.5, 50 iriM; MgCl2, 17.5 mM; spermidine, 5 mM and incubated for 30· at 30°C. The reaction is stopped as in examples la and lb and the reaction products are detected with the same methods.

Example Id The reaction is carried out for the initiating gap-filling-reaction as in examples la - lc, the concentration of each adaptor is 1 μΜ. The consecutive replication reaction is carried out as described in examples la - lc.

Example le The experiments are carried out as described in examples la - Id. Before adding the adaptor oligonucleotides in the gap-filling- reaction the adaptor oligonucleotides are treated for 10· at 95"C and are afterwards chilled on ice. The following replication reaction is carried out as described in examples la - Id.

Example If The starting gap filling-reaction and the replication reaction are carried out as in examples la - le, the reaction temperature is 37°C for gap filling and replication reactions.

Example: 2, For this purpose, the adaptors are coupled on to the 5' -ends of the 029-specific ori sequences with the 029 protein p3. (from PNAS, 2Z» 6*25-6428/1.980; NAR, 13/21, 7715-7728/1985). The coupling of p3 to the specific 5' -end is carried out by in vitro replication of the oligonucleotide in question together with its complementary strand under the replication conditions described in Example 1» As double strand, the oligonucleotides contain the ori-specific sexjuences only on one end. Therefore,. p3 can only bind here on the 5'-end and the second strand does not contain any p3 molecule. By means of affinity chromatography, for ecample with antibodies against p3„ these strands are bound from the mixture on a surface and eluted after washing out of the p3-free strands. The surface can thereby be saturated with streptavidin and the (p3 antibody coupled with biotin. The adaptor-p3 molecules are then used as in Example 1 in the amplification reaction with an appropriate template UNA. However, the p3-containing strands can also be separated off on Sephadex AcA 500, Example 5 The reaction can be started with adaptors which have not only p3 covalently bound or also do not contain p3 as in Example 1 with adaptor hybridisation. However, the dNTP concentrations for filling the gap between the left and right adaptor amaunts to 33 -uM for each deoxynucleotide.. After elongation for 10 minutes at 30°C.., for the initiation of the replication besides the substances mentioned in Example 1, there are. added thereto 25 yu-Ci Za5¾7 <^ΤΡ> (= 3000 Ci/mM) and dCTP, dGTP and dTTP, each in an amount of 150 μ. M, and again incubated as in Example 1..

The reaction is stopped as in Example 1» The reaction products are detected either after gel electro phoresis as specific blackening on an X-ray film or after dropping on to nylon membranes, fixing by UV irradiation and exposure of an X-ray film with the dried membrane.. Before dropping on to nylon membranes, the amplification products are separated from non-incorporated dNTPs by gel filtration with a Sephadex G50 column and concentrated by ethanol precipitation.

Example 3b The reaction is started with the same adaptor sequences under the same buffer condition as in examples la - If, the dNTP- concentrations are 33 μΜ for each deoxynucleotide. After finishing the gap filling-reaction the reagents as described in examples la - If are added to start the replication reaction including digoxigenin-11-2 '-desoxy-uridin-5 '-triphosphate (DIG-ll-dUTP) at a final concentration of 50 μΜ, dTTP at a final concentration of 100 μΜ and dATP, dCTP and dG P at a final concentration of 150 μΜ. After stopping the reaction the products are either electrophoresed in agarose gels and transferred to a nylon- or nitrocellulose membrane or are spotted on a membrane and crosslinked to the membrane by UV-light exposition or by heat treatment. Afterwards the DIG-labelled DNA is detected as described in the Boehringer Mannheim Biochemical Manual, p. S 96-115 (Boehringer Mannheim Biochemicals for Molecular Biology 1990) or in C. Kessler et al. , Non-radioactive Labeling and Detection of Nucleic Acids: 1. A Novel DNA-Labeling and Detection System.Based on Digoxigenin: Anti-Digoxigenin ELISA Principle (Digoxigenin System) , 1991, Biological Chemistry Hoppe-Seyler, in press.

Example ?a The experiments are carried out as in Examples 1 to 3 but for the hybridisation reactions of the adaptors there are only added thereto the two oligonucleotides which correspond to the left adaptor (+/- p3 covalently bound) and the oligonucleotide which contains the HBV-specific sequence and the 3'-end of the right 029 ori region.. After the gap-filling reaction as in Examples 1 to 3, for the start of the replication reaction with the reaction mixture^ the camplementary strand to the oriregion is added to the right adaptor (+/- p3 covalently bound) so that the concentration of this oligonucleotide amounts to 250 nM.

Incubation, reaction stop and detection are carried out as in Examples 1 to 3.

Example 4b The experiments are carried out as described in examples la -If, 2 and 3. In the gap filling-reaction the left adaptor is added without the coirplementary strand corresponding to the ori-region and the right adaptor is added together with the corresponding compleinentary strand. This complementary strand contains a dideoxynucleotide at the 3 '-end.

FvTrm lp 4c The experiments are carried out as described in examples la -If, 2 and 3. For the gap-filling-reaction the complementary strand to the ori-region of the right adaptor, the right adaptor and the corresponding camplementary strand to the ori-region of the left adaptor each containing a dideoxynucleotide at the 3 '-end are added. van^lf* 4d Again the experiments are carried out as described in examples la - If , 2 and 3. For the gap filling-reaction the left adaptor is added without the complementary strand to the ori-region and the right adaptor and the corresponding complementary strand to the ori-region are added containing each a dideoxynucleotide at the 3 '-end.

Example 5> ^^-labelled amplification products are separated from non-incorporated deoxynucleotides as in Example 3 by gel filtration with Sephadex G-50. Thereafter, the amplification products are denatured by incubation for 10 minutes at 95°C. and hybridised for H- hours with 20 ng, of an oligonucleotide of 40 nt, sequence- homologous to the amplified region,, which is labelled with biotin (published European Patent Specification No.. A-0 , 097 , 373). n 200 μ, Ι. with formamide, 10%, SSC 5x ; Denhardt's solution, lx; sodium phosphate, pH 6..8,., 50 mM, in streptavidin-coated tubes at 4-5°C. The concentration of the . oligonucleotide is thereby 8 riM.. After hybridisation/ solid phase binding washing i carried out twice for 30 minutes at 37°C» with 200 ^.1. SSC„ 2x; and SDS, 0..2% , and the bound radioactive amplification products measured in a scintillation counter..

Example.6..

Unlabelled amplification products prepared as in Example 1.,. 2 or are, after gel filtration as in Example 5 * hybridised not only with a biotin-labelled oligonucleotide of 4-0 nt homologous with a part of the amplified region and a second oligonucleotide of 0 nt homologous with another part of this region at 4·5°0«. These oligonucleotides thereby show no cross-hybridisation., 200 l.. of the hybridisation batch contain the two oligonucleotides each 8 nM, formamide 10%; SSC, 5x; Denha-rdt' s solution, lx;sodium phosphate, pH 6..8, 50 mM.. Hybridisation is carried out for 4· hours in streptavidin-coated microtitre: plates and thereafter washed once for 10 minutes at 37°C. with conjugate buffer (T?is-HCl, pH 7..5 , 100 mM; sodium chloride: 0„ 9% ; bovine serum albumin 1% ; Pluronic T 68 0, 5 ) . Subsequently, incubation is carried out for 30 minutes at 37°C.. with 0 U digoxigenin alkaline phosphatase - - conjugate and. thereafter washed five times with, in each case,.200 j^l. Tris-HCl, 100 mM? and sodium chloride, 150 mil. Thereafter, incubation is carried out for 30 minutes at 37°0. with 4-methylumbelliferyl phosphate (o.l mM) and measurement is made in a Dynatech Microfluor-Reader.

Claims (12)

95537/3 CLAIMS:

1. Process for the preparation of nucleic acids with the use of a nucleic acid A, wherein the nucleic acid A is reacted with at least two adaptors, each of which has a nucleotide sequence which, in each case, can be hybridized with a part of a strand of the nucleic acid A and of which at least one contains a nucleotide sequence specific for a replication system, under conditions in which is formed a nucleic acid substantially complementary to at least a part of the nucleic acid A which, furthermore, contains at least one adaptor which has a nucleotide sequence specific for a replication system and the complex so formed of nucleic acid A and the nucleic acid formed in the previous step is reacted under conditions appropriate for a replication reaction with one or more proteins of the replication system which alone or together catalyse the formation of the nucleic acid sequences which contain at least the nucleotide sequence of an adaptor, which contains a nucleotide sequence specific for a replication system, as well as a nucleotide sequence which is either substantially homologous or essentially complementary to at least a part of the nucleotide sequence of the nucleic acid A.

2. Process according to claim 1, wherein the replication is initiated by at least one protein which is specific for the replication system used.

3. Process according to claim 1 or 2, wherein the conditions which lead to the formation of the complementary nucleic acid comprise the use of nucleoside triphosphates, of a polymerase and of a ligase.

4. Process according to any of the preceding claims, wherein the adaptors are not complementary to the same nucleotide sequences.

5. Process according to any of the preceding claims, wherein the adaptors are complementary to nucleotide sequences of the nucleic acid A to be multiplied which are spaced from one another by at least one nucleotide.

6. Process according to any of the preceding claims, wherein the adaptors each have a single-stranded nucleic acid region which is substantially complementary to a part of the nucleic acid A to be multiplied and can hybridize with this part of the nucleic acid and wherein at least one of the adaptors has a double-stranded nucleic acid region which essentially cannot hybridise with the nucleic acid to be multiplied but has a sequence for the recognition of the above-mentioned enzyme.

7. Process according to claim 6, wherein the region not complementary to the nucleic acid is an ori sequence for the replication.

8. Process according to any of the preceding claims, wherein at least one of the adaptors has, in the double-stranded region, at least one restriction enzyme cleavage site.

9. Process according to any of the preceding claims, wherein at least one of the adaptors has, in the double-stranded region, at least one sequence for the hybridisation with Ml 3 universal sequencing primers and/or Ml 3 universal reverse sequencing primers.

10. Process for the detection of nucleic acids by multiplication of the nucleic acids to be detected or of parts thereof and determination of the nucleic acid formed, wherein, as multiplication process, there is used a process according to any of claims 1 to 9.

11. Process according to claim 1 wherein two adaptors are used, each of which has a nucleotide sequence which can be hybridized with a part of a strand of the nucleic acid A and each of which has a nucleic acid region which essentially cannot hybridize with the nucleic acid A and which contains a sequence specific for a replication system.

12. Adaptor nucleic acid comprising a single stranded region which is substantially complementary to a sequence of the nucleic acid to be detected and further comprising a double stranded region which contains a nucleotide sequence which is specific for a replication system, wherein at least one protein is bound to the adaptor nucleic acid which has a replication initiating function for said replication system.