CA1248895A

CA1248895A - Nucleic acid reagents, a method for their preparation and their use

Info

Publication number: CA1248895A
Application number: CA000474112A
Authority: CA
Inventors: Hans Soderlund; Marjut Ranki; Airi M. Palva
Original assignee: Orion Yhtyma Oy
Current assignee: SANGTEC MOLECULAR DIAGNOSTICS AB
Priority date: 1984-02-17
Filing date: 1985-02-12
Publication date: 1989-01-17
Also published as: DK58985D0; NO166543B; RO92633B; JPH0463679B2; FI71768C; BE901671A; DE3505287C2; FI840655A; SE8500733L; GB2156074A; GB8503900D0; US4731325A; ZA85511B; HUT37459A; HU194938B; SE8500733D0; NL193663B; FR2559783B1; AU3842285A; SE463103B

Abstract

ABSTRACT OF THE DISCLOSURE

The invention is related to improved nucleic acid reagents comprising arrays of nucleic acid fragments and com-binations of such fragments. The preparation of such fragments by recombinant DNA techniques and their use in hybridization methods is also described. By making different combination of the arrays of nucleic acid fragments - some labeled and some affixed to solid carriers, it is possible to create kits for the identification of e.g. venereal diseases. The improved nucleic acid reagents comprise two series, one labeled and one affixed to a solid carrier of at least two but preferably more arrays of alternating nucleic acid fragments, which are sufficiently homologous to sequences in the nucleic acid to be identified. Nucleic acid fragments belonging to different series must not be homologous to each other. The invention is especially related to arrays of nucleic acid fragments prepared from the recombinant plasmid pXTH1220 (DSM 2825), comprising DNA from the Chlamydia trachomatis L2 serotype and from the recombinant plasmid pKTH1271 (DSM 2825) compris-ing DNA from the cytomegalovirus (AD 169, ATCC VR-538)-(CMV).
Sandwich hybridization tests performed with arrays of nucleic acid fragments are at least four times as sensitive as sand-wich hybridization tests performed with reagents belonging to the prior art.

Description

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Improved nucleic acid reage2lts and methods for their preparation The invention relates to improved nucleic acid reagents com-05 prising an array of nucleic acid ragments and ~o combinations of such improved reagents. The invention also relates to meth-ods for the preparation of nucleic acid reagents comprised of an array of clones and combinations of such nucleic acid rea-gents, by recombinant-DNA techniques, and to their use for the identification of nucleic acids by hybridization methods Various hybridi~ation methods have commonly been used for the identification and study of nucleic acids. Some examples are the direct hybridization methods in which the sample contain-ing the nucleic acid to be identified is ~ither in a solution (Brautigam-et al., J.Clin.Microbiol. 1980, 12 226-234 and the British Patent Publication No. 2,019,408) or affi~ed to a solid carrier (US-Patent Nos 4,139,346 4,302,204, 4 358 535 20 4 395 486, the British Patent Publications Nos 2,034 323

2,095,833, the European Patent Publications Nos 62 286, 62,237 and 61,740), and is detected by using one labeled nucleic acid reagent which hybridizes with the nucleic acid to be identi-fied.
Other known nybridization methods include the two-step sandwich hybridization method presented by Dunn and Hassell in Cell 12 23-36, 1977 and the one-step 6andwich hybridization methods presented in the European Patent Publication No. 79 139. For the ident~fication of the nucleic acids by the sandwich methods two separate nucleic acid reagents are needed to detect the nucleic acids present itl the sample solution. One of these reagents is affixed to a solid carrier and the other is labeled.

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Nucleic acid reagerlts, both those affixed to a solid carrier and those which are labeled, are characterized in that their ~ase sequence i6 compleme~ltary, or nearly complementary, to the nucleic acid to be identified, i.e. homologous. The nucleic 05 acid reagents used are either natural nucleic acids as such or as fragments of them. The fragments are produced, for example, by using restriction enzymes. Nucleic acid reagents have also baen prepared synthetically or by recombinant-DNA techrliques.
Natural plasmids (US-Patent No. 4,358,535), nucleic acids from 10 bacteriophages (US-Patent No. 4,543,535), ribosomal RNA and messenger R~A (US-Patent ~o. 4,302,204), or nucleic acid from different viruses (Stalhandske et al., Curr. Top.Microbiol.
Virol. 1 , 1983) have been used as the nucleic acid reagents.
The whole virus genome has been used for identifying, for e~ample, parts belonging to the diferent viruses in the mes-senger RNA of a hybrid virus (Dunn and Rassell, Cell, 12, 23-3S, 1977). Nucleic acid reagents have also been prepared by using recombinant-DNA techniques (US-Patents Nos 4,395,486 and 4,359,535, the European Patent Application No. 79,139 and the 20 British Patent Publication ~o. 2,034,323 and the European Pa-tent Application ~o. 62,286). Nucleic acid reageIIts produced by recombinant-DNA techniques have been used either in such a way that the replicated defined DNA fragment has been purified out from the DNA of the vector, or as recombinant-DNA molecules linked to different vectors. The previously used nucleic acid reagents produced by recombinant-DNA techniques are made up of one continuous identifying nucleic acid fragment or of several separate clones.

We have developed new, more sensitive nucleic acid reagents, comprising at least two series of alternating array~ of nucleic acid fragments prepared from either one or several segments homologous to the nucleic acid to be identified.

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81~3~5 Nucleic acid reagents which comprise such arrays of nucleic acid fragments are in sandwich hybridization tests at least twice as sensitive as the previously used nucleic acid rea-gents. By using the nucleic acid reagents according to the invention, or their combinations, it is possible to identify smaller amounts of nucleic acids than previously, and they are especially well applicable for sandwich hybridization methods.
The charactPristics of the invention are shown in the distinguishing features of the claims, and the invention will now be described in greater detail in the following description and the acompanying drawings, in which Figure 1 shows an array of sandwich hybrids, Figure 2 depicts a sandwich hybrid of the prior art, Figure 3 shows the sites of two alternating series of nucleic acid fragments in a nucleic acid which has been selected for the preparation of an array of nucleic acid reagents according to the invention, Figure 4 shows the corresponding sites of three alternating series of arrays of nucleic acid fragments, Figure 5 shows an array of nucleic acid fragments according to Figure 3 separate (a?, joined together (b) and both separate and joined together (c?, Figure 6 shows an array of sandwich hybrids, Figure 6a shows an array of sandwich hybrids which is formed when separate fragments are used, Figure 6b shows an array of sandwich hybrid which is formed when joined b-fragments are used, Figure 6c shows an array of sandwich hybrids which is formed when both separate and joined b-fragments are used, Figure 7 shows an array of nucleic acid reagents which iden-tify different nucleic acids, Figure 8 shows an array of sandwich hybrids which are formed when the array of nucleic acid reagents according to:Figure 7, ~: ~ ` '', , :
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~z~ s identifying different nucleic acids, are used, Figure 9 shows an array of hybrids formed by a direct hybri-dization method, Figure 10 shows the recombinant plasmid pDTH1220, Figure 11 shows an array of sandwich hybrids which is formed when an array of nucleic acid fragments prepared ~rom the re-combinant plasmid pKTH1220 are used, Figure 12 shows the recombinant plasmid pKTH1271, Figure 13 shows an array of sandwich hybrids which is formed when arrays of nucleic acid fragments prepared from the re-combinant plasmid pKTH1271 are used.
As afore said, the nucleic acid reagents according to the invention have a very high sensibility in sandwich hybridization methods. This higher sensibility is in part based on the fact that the use of several probes increases the quantity of labeled hybrids on the solid carrier. There may be labeled vector-derived nucleic acid along with every hybridizing probe (Figures 1 and 2). In Figures 1 and 2, v represents vector-derived DNA, x the nucleic acid to be ~0 identified, b the labeled probe, a the identifying nucleic acid reagent affixed to the solid carrier, and F the filter.
When several probes are used, the quantity of labeled, vector-derived nucleic acid parts increases, and more label is bound to the hybrids being formed. The hybrids are thus more ~5 easily detectable.
When the array of nucleic acid fragments according to the invention are used in sandwich hybridization methods, at least two, or as shown in Figure 1, three, identifying nucleic acid fragments are affixed to the solid carrier. In this case the different areas of the nucleic acid strand x to be detected may hybridize to the nucleic acid fragments affixed to the solid carrier, for example al, a2j and a3, at one or several points, depending on the degree of reaction. ~When the reaction reaches ltS final stage, a situation according .

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to Figure 1 may be produced, in which the sample strand forms a loop or loops to which the probe or probes, for example, bl and b2 in Figure 1, hybridize. At this time the distance of the vector-derived nucleic acid parts from the hybridization joining point (1) decreases (Figure 1), and the hybrid is more stable than the hybrid formed by one reagent pair ~prior art) shown in Figure 2, this hybrid being of the same size as the total area of the array of nucleic acid fragments. The vector-derived parts of a hybrid formed from one reagent pair are easily broken by, for example, mechanical strain, such as shaking. In such a case the label already bound to the hybrid escapes.
Since the improved nucleic acid reagents according to the invention are more sensitive than previously used nucleic acid reagents, they are suitable for demonstrating chromosomal rearrangements and hereditary diseases.
Thus, the present invention relates to nucleic acid reagents comprising an array of nucleic acid fragments, their combinations, their preparation, and their use for the detection of nucleic acids in hybridization methods.
The invention also relates to nucleic acid reagents composed of an array of nucleic acid fragments.
These arrays of nucleic acid reagents comprise at least two, but preferably several, alternating nucleic acid fragments, up to 20 fragments, which are derived from one or several nucleic acids sufficiently homologous to the nucleic acid which is to be identified. Thereby there are obtained at least two series of alternating arrays of nucleic acid fragments, which must not be homologous to one another.
In summary therefore, the present )invention provides a nucleic acid reagent comprising at least two series ~or sets) of at least two arrays of alternating nucleic acid fragments homologous to a nucleic acid which is to be identified but not homologous to one another.
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The invention also provides nucleic acid reagents comprising arrays of alternating nucleic acid fragments comprising the recombinant plasmid pKTH1220 or its derivatives and which recom~inant plasmid contains the DNA
of Chlamydia trachomatis L2 bacterium and is cloned into the host Escherichia coli K12, HB101, and the deposit number of this host comprising the recombinant plasmid pKTH1220 is DSM
2825 .
Further, the invention provides nucleic acid reagents comprising two or more series of at least two alternating arrays of nucleic acid fragments sufficiently homologous to the nucleic acid which is to be identified but not homologous to one another, comprising the recombinant plasmid pKTH1220 or its derivatives and which recombinant plasmid contains the DNA of Chlamydia trachomatis L2 bacterium and is cloned into the host Escherichia coli K12, ~B101, and the deposit number of this host comprising the recombinant plasmid pKTH1220 is DSM 2825.
Additionally, the invention provides a nucleic acid reagent for use in a sandwich hybridization array to detect the identity of a nucleic acid, said nucleic acid reagent comprising at least two sets of at least two alternating nucleic acid ~ragments which are homologous to the nucleic acid which is to be identified, said alternating ~5 nucleic acid fragments being not homologous to one another, wherein at least one of the sets of nucleic acid fragments are labeled and at least one set of nucleic acid frayments are affixed to a solid carrier.
Another aspect of the invention provides a method for condu`cting a sar.dwich hybridization array using a nucleic acid reagent to detect the identity of a nucleic acid, said method comprising exposing said nucleic acid to said nucleic acid reagent, said nucleic acid reagent comprising at least two sets of at least two alternating ~Z~3895 - 5b -nucleic acid fragments which are homologous to the nucleic acid which is to be identified, said nucleic acid fragments being not homologous to one another, wherein the nucleic acid fragments from each of said sets alternate in said nucleic aeid reagent and at least one of said sets of nueleie aeid fragments are labeled and at least one set of nueleie fragments are affixed to a solid earrier.
The arrays of nueleie aeid reagents ean be prepared synthetieally. In this ease the fragments from the two alternating ssries of arrays of nueleie aeid fragments, must not be homologous to eaeh other. But they must be suffieiently homologous to alternating sites in the nueleie aeids to be identified. These fragments can easily be preparad by fully automatie machines after characterization of the nucleic acid sequenee of the nueleie aeid to be identified.

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' 6 ~ 35 The nucleic acid reagents according to the inver~tion are com-posed of separate, or joined, or both 6eparate and joined array of nucleic acid fragments.

05 The arrays of nucleic acid fragments may ~e joined to a vector, contain parts of vectors, or be totally devoid of ~ector parts.

The nucleic acid fragments used have a minimum length of 15 nucleotides. There is no actual upper limit for length, but it is advantageous to use fragments having a length of 20-5000 nucleotidPs. The nucleic acid fragments according to the in-vention are derived either from the genome to be identified or from one part of the genome, for example from a relatively lar-ge clone representing a certain part of the genome. The arrays of nu~leic acid fragments according to the invention can thus be prepared from several independent genome areas which are not directly adjacent. The arrays of nucleic acid fragments thus prepared are combined and used for the same reagent. The arrays of nucleic acid fragments can also be isolated from a DNA which is not iden ical to the nucleic acid to be identified but suficiently homologous, 80 that a stable hybrid is formed between the reagent and the nucleic acid to be identified. The preparation of suîtable arrays of nucleic acid fragments: is ~y no means limited to the isolation of suitable nucleic acid fragments from the genome. There are available many equally u~eful methods to prepare such array~ of fragments. The man skilled in the art can prepare arrays of nucleic acid fragments by synthetic or semisynthetic methods.

The reagents are isolated in such a way that at least two se-ries of alternating nucleic acid fragments al, a2, a3, etc., and ~1~ b2~ b3, etc., are obtained. The nucleic acid fragments belonging to the series al, a2, a3, etc. are composed of frag-ments situated close to but not adjacent to one another. The nucleic acid fragments belonging to the series b1, b2, b3, etc.
are also composed of nucleic acid fragments situated close to '~-, ~ ;
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but not adjacent to one ariother. The nucleic acid fragments belonging to the series al, a2, a3, etc. and those belonging to the series bl, b2, b3, etc. must not be homologous to each other. It is preferable that the nucleic acids belongirlg to the 05 series al, a2, a3, etc. and those belonging to the series bl, b2~ b3, etc. are isolated in ~uch a way that every second fragment belongs t~ the a series and every second to the b-series, as shown in Figure 3. In Figure 3, al, a2, a3, and bl, b2, b3 are arrays of nucleic acid fragmen~s ~ufficiently homologous to the nucleic acid to be identified. It is, of course, possible that even a third nucleic acid fragment se-ries, cl, c2, C3, etcO, is isolated from the same nucleic acid, as ~hown in Figure 4. It i~ preferable that the alternating two nucleic acid reagents follow one another directly, but this is no absolute prerequisite for the invention.

The nucleic acid fragment series described above can be used either as ~eparate fragments al, a2, a3, etc., and bl, b2, b3, etc. (Figu`re 5a) or joined together into longer strands al-a2-a3, etc., and bl-b2-b3, etc. (Figure Sb). It is, of course, possible to prepare all kinds of intermediate forms such as, for example, an a-series in which al is a separate fragment and a2-a3 are joined together, and in the b-series, for example, bl-b2 are joined together and b3 is separate, etc., as shown in Figure 5c.

Figure 6 depicts various arrays of sandwich hybrids. Figure 6a shows an array of sandwich h~brids in which the arrays of nuc-leic acid fragments are separate. Figure 6b shows an array of hybrids in which the labeled array of nucleic acid fragments are joined together. Figure 6c depicts a case in which an array of sandwich hybrids is formed from both joined and separate labeled arrays of nucleic acid fragments. In Figure 6, x represents the nucleic acid to be identified; bl, b2, and b3 represent the labeled probe, and al, a2, and a3 represent arrays of nucleic acid fragments affi~ed to a solid carrier.

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~ucleic acid fragments which belong to the b-series can, for example, be labeled in such a way that a labeled nucleic acid reagent is obtained, i.e. the probe B. The nucleic acid rea-gents which belong to the a-~eries can be affixed to a solid 05 carrier in such a way that a nucleic acid reagei,t A bound to a solid carrier is obtained. It i~, of course, alternatively pos-sible to prepare a labeled nucleic acid reagent A, and a cor-responding nucleic acid reagent B b~und to a solid carrier.

Such nucleic acid pairs A and B, or B and A, labeled and res-pectively affixed to a solid carrier can be prepared for seve-ral different nucIeic acids to be identi~ied. They can be com-bined into suitable nucleic acid reagent combinations, which are composed of different nucleic acid reagent pairs Al and Bl, 15 A2 and B2, A3 and B3, etc., or Bl and Al, B2 and A2, B3 and A3, etc. Reagents containing arrays of nucleic acid fragments which identify different nucleic acids can also be combined so that a probe AX-Ay~Az is obtained, which, for example, comprises an array of nucleic acid fragments (al-a2-a3) -~al-a2-a3) -20 (al-a2-a3)z, as shown in Figure 7, in which alX, a2 and a3x are arrays of nucleic acid fragments Ax which identify nucleic acid x; aly, a2y and a3y are arrays of nucleic acid fragments Ay which identify nucleic acid y; alz, a~z and a3 are arrays of nucleic acid fragments Az which identify nucleic acid z, and v is a vector-derived nucleic acid part. Joined arrays of nucleic acid fragments can, of course, also be used as separate fragments, as suitable mixtures.

The arrays of sandwich hybrids according to Figure 8 are obtained by using the reagents shown in Figure 7. If simul~aneous identification of several different nucleic acids is desired, it is, of course, necessary to use separate filtexs, as shown in Figure 8. Fisure 8a shows a solid carrier identifying the nucleic acid x, Figure 8b a solid carrier identifying the nucIeic acid y, and Figure 8c a solid carrier identifying the nucleic acid z. In Pigures 8a,8b and 8c, blX, b2X

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'' ~24~ 5 and b3x are array6 of nucleie acid fragmerlt6 affixed to a solid carrier and identifying the nucleic acid x; bly, b2y, and b3y arearrays of nucleic acid fragmen~s affixed to a solid carrier and identifying the nucleic acid y; and bl , b2 ~ and b3 are arrays of 05 nucleic acid fragments affixed to a solid carrier and idelltifying the nucleic acid z; and x, y and z are the nucleic acids to be identified. Fx, F and F are the respective solid carriers or filters, A -Ay~Az i~ a probe which iden~ifies all the three nucleic acid~ simultaneously, if separate solid carriers are used.

The above-described nucleic acid fragment series, reagents and reagent combinations can be prepared by recombinant-DNA techni-ques known ~ se. A number of nucleic acid fragments of diffe-lS rent lengths are generated, by using restriction enzymes, fromthe nucleic acid to be identified or from a part representing it. If the restriction map of the genome to be identified is known, it is possible to select from the genome the suitable adjacent ~ragments, generated by using restriction enzymes, and the fragments are isolated and amplified by using recombinant DNA techniques.

When an unknown genome is involved, an intermediate stage can be used in the prepara~ion of the reagents, in such a way that a relatively large restriction fragment is cloned, this fragment is mapped, and the arrays of nucleic acid fragments series al, a2, a3, etc., and bl, b2, b3, etc., are produced on the basis of the information thus obtained.

It is, of course, possible to use combin~tions of the above methods and to use several large separate cloned restriction fragments as starting material, and to prepare several separate series, which are combined to form ~uitable combinations.

It is advantageous to prepare the nucleic acid fragment series al, a2, a3, etc., and bl, ~2' b3, etc~., according to the inven-tion by using recombinant-D~A technigues in such a way that the :
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1 o ~ %~8~5 series a i6 cloned il~to one vector, for example into the plas~
mid pBR322, and whereas the series b i6 cloned into another suitable vector, which does not have sequences ir, common with the previous vector. The bacteriophage M13 is an example of 05 such a second advantageous vector. The fragments belonging to the series a can be joined to one another, and the joined series can be cloned into one vector. For example, al-a2, join~d together, can be cloned a~ a continuous insert into the same pBR322 vector. In a corresponding manner it is possible to prepa~e a reagent ~eries bl-b2. In the cloning it is preferred to use vectors to which very large inserts of foreign DNA can be joined. For example, lambdaphage and cosmid vectors are sui-table for this purpose.

Thus, two reagent pairs comprising arrays of nucleic acid fragments are needed in the sandwich hybridization method according to the invention, a reagent labeled with the label substance to be identified, i.e. a probe, and a so-called filter reagent a~fixed to a solid carrier.
Mos~ commonly, radioactive isotopes are used or labeling the probes. For example in the British Patent Publication No.
2,034,323, the US-Patents Nos 4,358,535 and 4,302,204 the following isotopes are used: 32p 125I 131I d 3 European Patent Publication ~o. 79,139, the isotope 125I is used. Nucleic acid probes have also been modified in differen~
ways and labeled with, e.g. fluorescent labels (French Paten~
Publication ~o. 2,518,755). Also enzymatic or enzymatically measureable labels are used (the British Patent Publication No.
2,019,408, the European Paterlt Publication ~o. 63,879 and the French Patent Publication No. 2,519,005). The European Patent Publications Nos 70,6~5 and 70,687 de cribe a light-emitting label and labeling method, and the French Patent Publication ~o. 2,518,755 describes an immunologically measurable label.
The lanthanide chelates described in US-Patent No. 4,374,120, especially europium, can be used as label subst~rlces. Also the 8~

biotin-avidin label substarlce de6cribed by Leary et al. (PNAS
80, 4045-4049, 1983) is suitable as a label. A few examples of labels which can be used for the labeling of nucleic acid rea-gents according to the invention are mentiorled above, but it i6 05 evident that there will be developed new, improved labPl sub-stances which are also suitable for the labeling of arrays of nucleic acid fragments according to the invention.

The carriers suitable for filter reagents include various nit-rocellulose filters ~US-Patent ~o. 4,358,535 and the British Patent Publication No. 2,095,833). The DDR-Patent Publication ~o. 148,955 desribes a method of binding nucleic acids chemi-cally to the carrier (paper). US-Patents Nos 4,359,535 and 4,302,204 describe chemically modified papers which can be used as solid carriers. Other alternatives include nylon membranes and modified nitrocellulose filters. But it is evident that there will be developed new materials which will be even more suitable for use as solid carriers according to the invention.
It is, of course, possible to use also other solid carriers, such as various chromatography matrices such as triazine- or epoxy-activated cellulose, latex, etc. In principle, there are no other limitations to the selection of the solid carrier than those to be described below. It has to be possible to affix nucleic acid in a single-stranded form to the solid carrier so that these single-stranded nucleic acids can hybridize with the complementary nucleic acid. The solid carrier must also be easy to remove from the hybridization solution, or the hybridization solution must be easy to remove from the solid carrier. Also, the probe must not adhere to the carrier material itself so that it cannot be washed off.

The abo~Te-described combinations~of the arrays of nucleic acid reagent pairs A and B, or B and A, labeled and affixed to a ~o-lid carrier respectively, and from such nucleic acid pairs made for the identification of different nucleic a~ids it is possib-le to as6emble a combination Ax and Bx, Ay and By, A and B .

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12 ~Z~81395 These combinations can be used for the simultaneous identifica-tion of the nucleic acids x, y arld z by sandwich hybridization methods.

05 The sample is treated in such a way that the nucleic acids are released into the hybridization solution, and they are rendered single-stranded. The hybridization is carried out in a hybridi-~ation solution, to which both the nucleic acid reagent~ affi-~ed to a solid carrier and the labeled ones are added. When hybridization has taken place, the filters are lifted from the hybridization solution, if filters have been used as solid car-riers. If chromatography matrices, latex, or the like have been used, the hybridization solution is removed. The solid carriers are rinsed with ~ suitable washing solution. The arrays of sandwlch hybrids formed (Figures 8a, 8b, 8c) are detected by methods known per se. The radioactive label is measured, for example, by autoradiography, by a scintillation counter or by a gamma-counter. For example, an enzymatic label is identified after, for example, a color reaction, by photometry or on the basis of a precipitate. Lanthanide chelates can be detec~ed by a ~o-called "time resolved fluorescence" method. An immunological label is detected by immunological methods suitable for the purpose.

Several different mixtures can be used as the hybridization solution; the alternatives presented in the European Patent Publication No. 79,139 and US-Patent 4,302,204 are mentioned as examples. It is, of course, also possible to use other hybridization mixtures. The hybridization takes place at a temperature of 0-80 C, but is advantageous to use, for example, a temperature of 65C. Sufficient hybridization may occur in a very short period, but it is advantageous to use hybridization periods of,~for example, 12-20 hours.
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The two-~tep sandwich hybridization method i~s carried out in principle in the same manner, but in this case the nucleic acid :

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13 ~ 3895 reagent affixed ~o a solid carrier is fir~t added to the hybri-di~ation solution. When the hybridization has taken place, the solid carrier is washed and a second hybridization is carried out in which the labeled nucleic acid reagent i6 pre ent.

The above-described labeled nucleic acid reagents or reagent combinations Ax, A , Az, etc., and Bx, By, Bz, etc., can, of course, be used in direct hybridization methods. In such a case the nucleic acid sample in a solution must be divided for each nucleic acid x, y and z to be identified or, if the sample i8 affixed to a solid carrier, a 6eparate sample affixed to a car-rier must be prepared for each sample. The formed array of hybrids ~Figure 9) is detected by methods Xnown ~er se. In Figures 9, F represents the solid carrier, i.e. the filter, x the nucleic acid to be identified, and v the vector-derived parts. The labeled probes used are al, a2 and a3 (Figure 9a), b and b (Figure 9b), and al, bl, a2~ b2; 3 As already described above, various combinations of nucleic acid reagents can be made up from ~he arrays of nucleic acid fragments according to the invention. It is possible by using these combinations to identify several different nucleic acids simultaneously. Arrays of nucleic acid frayments homologous to the different nucleic acids to be identified can be used as separate fragments in the mixtures or joined together in such a manner that one probe identifying several different nucleic acids is obtained. Nucleic acid reagents affixed to a solid carrier must, of course, be Xept separate in order for the identification to be successful.
Hybridization using arrays of nucleic acid fragments can be used for identifying various human, animal and plant pathogenic microorganisms. By the method it is possible to identify microorganisms present in foodstuffs, such as clostridia, salmonellae, staphylococci, which cause food poisoning The method is suitable for the ident1fication of contaminants present in water, such as enterobacteria and enteroviruses.

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14 3~ 5 Since the sandwich hybridization test using arrays of nucleic acid fragments is a quantitative method, it is applicable to, for example, the detection and measurement of gene amplifi-cation. Thi8 characteristic is significant in, for example, the 05 detection and treatment of cancer. The formation of a stable array of hybrids requires that the homologous sequences of the probe reagent and the filter reagent are located within a moderate, preferably less than 5 kilobase (kb), distance from each other in the sample ~trand. If changes with respect to the ~istance between these two areas do occur, the change is cleary obs~rvable by this method. Therefore the method is also suitab-le for the detection of changed mRNA, chromosomal rearrange-ments, the rearrangement of immunoglobulin genes for expression, and hereditary diseases. It is thu~ possible to construct various reagent combinations from the arrays of nucleic acid fragments. For example, for the identification of the causative agents of venereal diseases it is possible to prepare kits which include a probe which contains arrays of nucleic acid fragments which identify gonorrhea, syphilis, herpes and chlamydiae. The identification is in this case possible by using separate filters for gonorrhea, syphilis, herpes and chlamydiae.

The invention relates in particular to arrays of nucleic acid fragments comprising the recombinant plasmids pKTH1220 and pKTH1271. The recombinant plasmid pKTH1220 comprises, in the plasmid vector pBR322, DNA of Chlamydia trachomatis L2 which is specific to the Chlamydiae. This recombinant plasmid is cloned into the host Escherichia coli K12 HB101. The recombinant plasmid 1271 comprises, in the plasmid vector pBR325, DNA from the cytomegalovirus AD169. This recombinant plasmid is cloned into host Escherichia coli K12 HB101. The hosts containîng the recombinar~t plasmids pRTH1220 and pKTH1271 have been deposited at the culture collection Deutsche Sammlung von Mikroorganismen

3~ (DSM), Griesebachstrasse 8, D-3400 G~ttingen, West Germany. The number of the deposit containlng the recomblnant plasmid : . ...................... .
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~ 19~i pKTH1220 is DSM2825 and the number of the deposit containing the recombinant plasmid pKTH1271 i8 DMS2826. The deposits will be freely available once ~he pater~t application has been made public.

The invention iB described in greater detail in the following examples. These examples must not, however, be underRtood as limiting the protective scope of the invention. The ~tructure of the nucleic acid (DNA and RNA) i~ similar whether the ~ues-tion is of a nucleic acid derived from a eucaryotic or a pro-caryotic cell. For this reason the principles presented in the examples are equally well applicable to the nucleic acid~ of animals (man included), plants and microbes or viruses. Thus the reagents according to the invention can be used to detect the nucleic acids of man, animals, plants, microbes and viru-ses. The arrays of nucleic acid fragment~ can be prepared synthetically, too. The sequence of nucleic acids to be iden-tified can be characterized and homologous arrays of fragments prepared by automatic nucleic acid preparing machines.
Example_l.

a) Arrays of nucleic acid reagents from Chlam dia trachomatis and their preparation DNA fragments suitable for the diagnostics of the Chlamydia trachomatis group were prepared from the DNA of Chlamydia trachomatis serotype L2. The DNA was isolated and fragmented by known methods, and the resulting DNA fragments were cloned into the plasmid pBR322 and transferred to the host oxganism Esche~
30 richia coli K12 HB101, by known methods. A gene bank of the Chlamydia trachomatis L2 bacterium was obtained as a result of the cloning, i.e. a large number of recombinant plasmids, each having a separate BamHI restriction fragment of DNA derived from chlamydiae. For reagent production, recombinant plasmids containing maximally large DNA inserts derived fFom chlamydial .:

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16 i~hg~8~95 DNA were 6elected from the gene bank. One such plasmid is the one designed pKTH1220, which has been deposited at the culture collection Deutsche Sammlung von Microorganismeh under the rlum-ber (DSM 2825) and the ~uitability of which for use as a rea-05 ge~t was demonstrated by a direct hybridizatiorl test. The testshowed that pKTH1220 identified all of the nucleic acids deri-ved from differe~t Chlamydia trachomati6 serotypes, but no other nucleic acids.

The applicable fragments, obtainable by using different re-striction enzymes, were selected from the pKTH1220-plasmid DNA, and some of these fragment~ were transferred by further cloning into pAT153 plasmid (Maniatis et al., Molecular Cloning. A La-boratory Manual, Cold String Harbor Laboratory, p.6, 1982) and some to M13 phage. Figure 10 shows ~he recombinant plasmid pKTH1220, havin~ a molecular length of 14 kb. In Figure 10, BamHI, SalI and ClaI represent the restriction enzymes used, and al, a2, bl, b2 and b3 illustrate the size and mutual lo-cations of the fragments produced with the aid of these re-striction enzymes. The fragments belonging to the series b aslabeled probes. Table 1 lists the ~izes of the fragments and the vectors used for further cloning, the names of the recom-binant plasmids, and their use.

Table 1.

.
Fragment Size Vector Recombinant Use plasmid _ al ClaI-SalI 3.Okb pAT153 pKTH1252 Filter a2 SalI-ClaI 2.9kb pAT153 pKTH1250 Filter bl SalI-BamHI 0.7kb M13mp8 mKTH1242 Labeled probe b2 BamHI-SalI 1.4kb M13mp8 mKTH1239 Labeled probe b3 ClaI-ClaI 1.7kb M13mp8 mKTH1248 Labeled probe bl-b2 BamHI-BamHI 2.lkb M13mp8 mKTH1245 L2beled probe ., ' ~;
, ..

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17 ~Z~8~9~D

The fragmel~ts listed in Table 1 were isolated from an agarose ~el by electroelution and were cloned into the appropriate re-stric~ion enzyme identification sites of the vectQrs listed in Table 1, by using known methods.

The fragment BamHI-BamHI 2.lkb was produced as follows: t~e fragments BamHI-SalI 1.4kb and SalI-BamHI 0.7kb of the plasmid pKTH1220 were separated by gel electrophoresis in agarose gel, from which they were isolated. The purified fragmen~s were joined to each other with the aid of T4 ligase enzyme, and of the 2.lkb D~A fragments produced in the reaction, those which had free ends which were identified by the BamHI enzyme were fur~her joined to the BamHI restriction site of the double-stranded form of the M13mp8 phage DNA. Thus there was made a recombinant phage-DNA (mKTH1245) which contains Chlamydia trachomatis D~A comprising two separate DNA fragments which are not located adjacently in the genome. However, in the genome they are located adjacent to the DNA reagents pKTH1250 and pKTH1252 to be affixed to the filter (Figure 11). Figure 11 shows an array of sandwich hybrids which is formed when the recombinant plasmids and recombinant phages listed in Table 1 are used as arrays of nucleic acid reagents.

b~ Demonstration of ~he sensitivity of an array of nucleic acid rea ents from Chlamydia trachomatis by using the sandwich g _ ~
hybridization method The sensitivity of an array of nucleic acid reagents as compared with a single continuous reagent pair was studied by the sandwich hybxidization method. The test was carried out using filters which all contained 1011 molecules of both pKTH1250 ~a2) and pKTH1252 (al) ~NA rendered single-stranded.
The sample to be studied was the plasmid pKTH1220, which for the test was rendered single-stranded by bviling for 5 min in 0.17 M NaOH, whereafter it was transferred to oC and neutralized with an equimolar amount of acetic acid. The -18 ~Z~1~38~5 following probes labeled with 125J~ ted in Table 1, were used in the tests: mKTH1242(bl), mKTH1239(b2), mKTH1248(b3) and mKTH1245(bl-b2)-05 The hybridization was performed at +65C for 17 hour6 in a hyb-ridization solution having the following composition: 4 x SSC, 0.02 % Ficoll, 0.02 % polyvinyl pyrrolidone, 0.2 % SDS, and 200 ~g/ml herring ~perm D~A. The filters were wa~hed for 2 h at 50C with a washing ~olution, having khe following comp~sition:
0.1 x SSC, 0.2 % SDS, and were counted using a gamma-counter.
The results are ~hown in Table 2 and are the means of five parallel tests.

Table 2 -.
Hybridized radioactivity, 20 Specimen with (b) a~ the probe_ _ _ molecules/test bl b2 b3 bl,b2 (b -b ) (bl-b2),b3 bl 380,000 cpm/test; 5 x 10 cpm/~gDNA
b2 340,000 cpm/test, 4 x 107 cpm/~gDNA
b 350,000 cpm/test; 5 x 107 cpm/~gDNA

b -b2 310,000 cpm/test; 7 x 10 cpm/~gDNA
bl,b2 700,000 cpm/test;
(bl_b2)~b3 700,000 cpm/tes~;

:
.

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Statistically calculated, th~ 95 % confidence limit of the tests performed without a sample (= negative controls) was regarded as the lower limit for positivity. These values were 52-54 cpm when the probe was bl, b2 or b3, 58 cpm when the 05 probe was bl, b2, 56 cpm when the probe was bl-b2, and 65 cpm when the probe was bl-b2, b3.

c) Chlamydia diagnostics by usin~Lsandwich hybridization with arrays of nucleic acid fragments Specimens taken from three men suffering from urethritis and three women suffering from cervicitis were selected for the test. Chlamydia trachomatis had been isolated from the male urethral specimens and the female specimens taken from the cervix. In addition, a corresponding number of similar patient specimens, from which chlamydia had not been isolated, were studied. The specimens to be examined were taken with cotton-tipped swabs which were immersed in a chlamydia sample-takiny buffer containing 0.2 M saccharose, 20 mM phosphate buffer, 3 %
fetal calf serum, 10 ~g/ml gentamicin, lOOJug/ml vancomycin, and 25 IU/ml nystatin.

Chlamydia was cultivated from the specimens. The original specimens were also assayed by sandwich hybridization using an array of nucleic acid fragments. The specimens were concentrated by using 2-butanol to remove liquid from them in such a way that the final volume was about 80 ~1, their concentration for the testing thus being about 3-7 fold.
Thereafter 70 mM EDTA, 0.7 % SDS, 200 ug/ml proteinase X enzyme were added to the specimen, and it was treated for 15 min at 55C and for 45 min at 37C. Thereafter the specimen was boiled for 5 min in 0.175 M NaOH. The boiled specimen was transferred to 0 C and nèutrali2ed with an equimolar amount of acetic acid and tested. The filters and hybridization conditions described in Example lb were used in the test. The probe used was mKTH1245 (bl-b2)~ 300,000 cpm/400 ~l~hybridization reaction.
The results are shown in Table 3.

.~ . . . .

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Table 3.

Specimen Hybridized Result of 05 radioactivity chlamydia culture Man 1. 151 Man 2. 164 10 Man 3. 154 Man 4. 61 Man 5. 76 Man 6. 55 15 Woman 1. 343 +
Woman 2. 509 +
Woman 3. 362 Woman 4. 57 Woman 5. 58 20 Wo~an 6. 81 Buffer, X4 30-55 Chl. trachomatis 25 L2 bacterium, 1o6 419 +

The limit for positivity iD the tests was 104 cpm.

The result in Table 3 shows that sandwich hybridization using an array of nucleic acid fragments is suitable for diagnosis venereal diseases. The samples which were negative in the culture tests were negative al80 in the sandwich hybridization test.

, 2~ 38~3~

Example 2.

a) An array of r~ucleic acid reagents from Cytomegalovirus and their_preparatio_ ~5 DNA fragments suitable for the diagnostics of Cytomegalovirus were prepared from Cytomegalovirus (AD 169, ATCC VR-538)-(CMV).
DNA was isolated and fragmented by known methods. EcoRl frag-ment I of about 9 kb, defined in Spector et al., J. Virol. 42, 558-582, 1982, was isolated from agarose gel by electroelution after the EcoRI restriction fragme~lts had been ~eparated on the basis of their size. The eluted DNA was extracted with phenol, whereafter it was precipitated with ethanol. The DNA thus puri-fied was joined by means-of T4-ligase to the pBR325 plasmid vector opened by using the EcoRI enzyme, and the produced re-combinant-D~A ware transferred to E.coli K12 HB101 host bac~
teria. From among ampicillin and tetracyclin resistent but chloramphenicol sensitive clones there was selected one which contained a cytomegalovirus-specific DNA insert of the correct si7e. The character of the cloned cytomegalovirus D~A was as-certained by the Southern blot method. This test ensured that the described 9 kb EcoRI-DNA fragment was derived from the DNA
of Cytomegalovirus and, more specifically, was included in its HindIII-D fragment (Oram et al., J.Gen. Virol., 59, 111-129, 1982). The recombinant plasmid thus described was designated pKTH1271, and it was deposited at the culture collection Deut-sche Sammlung von Microorganismen under number DSM 2826. The recombinant plasmid was grown and puriied by known techniques.

The further clonings were carried out by known techniques by using as vectors the pBR322 plasmid and the M13mp7 and M13mp8 phages. Figures 12 shows the hybrid plasmid pKTH1271 having a molecular length of about 9 kb. The array of nucleic acid fragments shown in Figure 12 were prepared by using the re-striction enzymes EcoRI, BamHI, ClaI and PstI. Figure 12 showsthe fragments obtained by using tbe restriction enzymes as well ~Z~L8~3~5 as their relative size and location. Table 4 lists the 6izes of the fragments in question ar~d the vectors used for the further cloning, ~he names of the thus obtained recombinant plasmids, and their use either as filter reagente or as labeled probes.
05 Figure 13 shows an array of sandwich hybrid~ which i6 formed w~en the array of nucleic acid fragme2lts li~ted in Table 4 are used.

Table 4.

_ Restriction fragment Vector Designation U~e _ . _ al EcoRI-PstI (3.3Xb) pBR322 pKTH1273 Filter a2 ClaI-BamHI (3.Okb) pBR322 pKTF1274 Filter 15 bl PstI-PstI (0.6kb) M13mp7 mKTH1277 Labeled probe b2 PstI-ClaI (l.Okb) M13mp8 mKTH1278 Labeled probe b3 BamHO-EcoRI (1.Okb) M13mp8 m~TH1279 Labeled probe b) Demonstration of the sensitivity of an array nucleic acid reagents from cvtomeqalovirus by the sandwich hybridization method The sensitivity of an array of nucleic acid reagent~ as compared with one continuous reagent pair was assayed by the sandwich hybridization method. The specimen in the tests was CMV DNA, which was boild in 0.17 M NaOH for 5 min. and was thereafter neutralized as in example lb. Filters which all 30 contained 10 molecules of both p~TH1273(alj DNA and pKTH1274~a2) DNA, rendered single-stranded, and the following probes labeled with J listed in Table 4: mXTH1277(bl), mKTH1278(b2) and mKTH1279(b3j were used in the test. The probes each contained 108 cpm/~g DNA. The hybridization was carried out as described in Example lb. The results are shown in Table 5.

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23 ~8~5 Table 5.
_ .
Hybridized radioactivity, 05 Specimen with (b) as the probe molecules/test bl b2 b3 bl,b2 bl'b2'b3 .

0 35 33 38 45 ~3 106 38 44 ~6 1 95 125 1.6x107 203 254 265 415 645 _ bl 310.000 cpm/test b2 320.000 cpm/test b3 300.000 cpm/test bl,b2 300.000 cpm of each/test bl,b2,b3 300.000 cpm of each/test In the test of value of the l~wer limit for positive was 51-55 cpm when the probe was bl, b2 or b3, S9 cpm when the probe was bl, b2, and 63 cpm when the probe was bl, b2, b3.
The results in Table 5 show ~hat sandwich hybridization in which an individual probe reagent in used (bl, b~ or b3~
detects in each case 4 x 10 CMV-D~A molecules. On the other hand, hybridization with a reagent of bl,b2 or bl,b2,b3 detects as few as 10 molecules of CMV-DNA. ~he results show that the array of nucleic acid reagents are four times as sensitive as individual nucleic acid reage~ts.

c) CMV diagnostics by using sandwich hy~r d zation wlth an array of nucleic aci~ S

Clinical spcimens were assayed by using sandwich hybridization with an array of reagents. These samples included two urine specimens from children under 1 year. These children were suspected of suffering from a congenital cytomegalo di~ease. A
lung biopsy specimerl from a patient with CMV pulmonary 05 infection was also a6sayed by thP pre~ent ~andwich hybridization. Both cytomegalovirus-infected and uninfected human fetal cells were also used as specimens in the test.

A solution which contained 1 ~ sarcosyl and 5 mM EDTA and 200 ~Ig calf thymus DNA was added to a 10 ml urine ~pecimen, whereafter the DNA released from the virus, together with the carrier, was precipitated u~ing 10 ml isopropanol at room temperature. The DNA precipitate was dissolved in 200 ~1 of TE
buffer and was brought to a single-stranded form by boiling it for 5 min, whereafter the DNA solution was cooled to 0 C and added to the hybridization ~olution.

The lung biopsy specimen (a few mm ) was minced mechanically, with a knife, 200 ~1 of TE buffer containing 1 ~ SDS solution and 1 mg/ml of proteinase K-enzyme was added to it. A digestion was carried out at +37 C for 1 h whereafter the specimen was drawn into an injection syringe twice through a thin hypodermical needle. The specimen thus homogenized was boiled, whereafter it was added to the test solution.
The cells infected with cytomegalovirus and the uninfected cells were broken up by an SDS, proteinase K treatment, homogenized and boiled, as above.

The reagents in the hybridization test were pKTH1273(al) and pKTH1274(a2) on filters and mKTH1277(bl), mKTH1278(b2), mKTH1279(b3) as probes, each 200.000 cpmlreaction. In other respects the hybridization, the washing of the filters and the coun~ing of the results were carried out as~described in 35 Bxample lb. ~ ~;

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~8~5 The results of the pre~ent hybridization are shown in Table 6.

Table 6.

05 ~ybridi~ed Virus i~olation Specimenradioactivity Infected cells (105) ~521 Not done Urine 1(10 ml) 243 CMV

Urine 2(10 ml) 3215 CMV

Urine from a healthy 15 person ~10 ml) 52 ~ot done Lung biopsy specimen 535 CMV

Control cells 105 68 Not done No specimen 65 Not done The results in Table 6 show that it is possible, by using an array of nucleic acid reagents, to demonstrate CMV in different clinical specimens such as urine, lung biopsy specimens and cells.

The test is specific to cytome~galovirus; it;does~not identify . human DNA, i.e. the test is not interfered~by the human DNA
pr~sent in the sample. In fact the type of~specimen does not interfere with the ~pecifity of test in any way.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Nucleic acid reagent comprising at least two series of at least two arrays of alternating nucleic acid fragments homologous to a nucleic acid which is to be identified but not homologous to one another.

2. Nucleic acid reagent according to claim 1, comprising separate arrays of alternating nucleic acid fragments.

3. Nucleic acid reagent according to claim 1, comprising joined arrays of alternating nucleic acid fragments.

4. Nucleic acid reagent according to claim 1, 2 or 3, comprising arrays of nucleic acid fragments having vector-derived parts.

5. Nucleic acid reagent according to claim 1, 2 or 3, comprising arrays of nucleic acid fragments having no vector-derived parts.

6. Nucleic acid reagent according to claim 1, 2 or 3, comprising labeled arrays of nucleic acid fragments.

7. Nucleic acid reagent according to claim 1, 2 or 3, comprising arrays of nucleic acid fragments affixed to a solid carrier.

8. Nucleic acid reagent according to claim 1, 2 or 3, comprising the recombinant plasmid pKTH1220 or its derivatives, said recombinant plasmid containing the DNA of Chlamydia trachomatis L2 bacterium and being cloned into the host Escherichia coli K12, HB101, wherein the deposit number of this host comprising the recombinant plasmid pKTH1220 is DSM 2825.

9. Nucleic acid reagent according to claim 1, 2 or 3, comprising the recombinant plasmid pKTH1271 or its derivatives, said recombinant plasmid containing the DNA of Cytomegalovirus AD 169 and being cloned into the host Escherichia coli K12 HB101, wherein the deposit number of this host comprising the recombinant plasmid pKTH1271 is DSM
2826 .

10. A method for identifying several different nucleic acids with a set of nucleic acid reagents according to claim 1, 2 or 3, comprising the steps of assembling suitable combinations of nucleic acid reagents from arrays of nucleic acid fragments homologous to the different nucleic acids to be identified and comparing the so assembled combinations to the nucleic acids to be identified.

11. A method for the preparation of nucleic acid reagents according to claim 1, comprising the step of producing arrays of nucleic acid fragments by recombinant-DNA techniques, synthetically or semisynthetically.

12. A method according to claim 11, for the preparation of arrays of nucleic acid fragments, said method comprising:
a) isolating selected nucleic acids of suitable length;
b) cloning the isolated nucleic acids into suitable vectors;
c) fragmenting the selected nucleic acids by the aid of restriction enzymes;
d) combining the suitable fragments into arrays of nucleic acid fragment series by means of suitable ligases;
e) cloning the arrays of fragments into suitable vector;
f) labeling the nucleic acid fragments belonging to one series; and g) affixing to a solid carrier the nucleic acid fragments belonging to the other series.

13. The method according to claim 12, wherein in step (e), fragments belonging to different series are cloned into different vectors.

14. The method according to claim 12, wherein in step (f) and (g), the nucleic acid fragments belonging to one and the other series are separate.

15. The method according to claim 12, wherein in step (f) and (g), the nucleic acid fragments belonging to one and the other series are joined.

16. Nucleic acid reagents comprising arrays of alternating nucleic acid fragments comprising the recombinant plasmid pKTH1220 or its derivatives and which recombinant plasmid contains the DNA of Chlamydia trachomatis L2 bacterium and is cloned into the host Escherichia coli K12, HB101, and the deposit number of this host comprising the recombinant plasmid pKTH1220 is DSM
2825.

17. Nucleic acid reagents comprising two or more series of at least two alternating arrays of nucleic acid fragments sufficiently homologous to the nucleic acid which is to be identified but not homologous to one another, comprising the recombinant plasmid pKTH1220 or its derivatives and which recombinant plasmid contains the DNA
of Chlamydia trachomatis L2 bacterium and is cloned into the host Escherichia coli K12, HB101, and the deposit number of this host comprising the recombinant plasmid pKTH1220 is DSM 2825.

18. A nucleic acid reagent for use in a sandwich hybridization assay to detect the identity of a nucleic acid, said nucleic acid reagent comprising at least two sets of at least two alternating nucleic acid fragments which are homologous to the nucleic acid which is to be identified, said alternating nucleic acid fragments being not homologous to one another, wherein at least one of the sets of nucleic acid fragments are labeled and at least one set of nucleic acid fragments are affixed to a solid carrier.

19. The nucleic acid reagent according to claim 18, wherein some or all of the nucleic acid fragments on one or more of the sets of nucleic acid fragments are joined to form an array of nucleic acid fragments.

20. The nucleic acid reagent according to claim 19, comprising nucleic acid fragments which have vector-derived parts.

21. The nucleic acid reagent according to claim 19, comprising the recombinant plasmid pKTH1271 or derivatives thereof, which recombinant plasmid contains the DNA of Cytomegalovirus AD169 and is cloned into the host Escherichia coli K12 HB101, and the deposit number of said host containing the recombinant plasmid pKTH1271 is DSM
2826.

22. A method for identifying more than one nucleic acid comprising exposing the nucleic acids to the nucleic acid reagent according to claim 19, wherein combinations of nucleic acid reagents are assembled from nucleic acid fragments which are homologous to the nucleic acids to be identified.

23. The method for the preparation of a nucleic acid reagent according to claim 19, wherein the nucleic acid reagent is prepared synthetically, semi-synthetically or by using recombinant-DNA techniques.

24. The method according to claim 23, wherein the preparation of the nucleic acid reagent comprises:
a) isolating nucleic acids which are homologous to the nucleic acids to be identified;
b) cloning said nucleic acids into vectors;
c) fragmenting said nucleic acids using restriction enzymes into at least two sets of at least two alternating nucleic acid fragments;
d) cloning said nucleic acid fragments into vectors;
e) labeling one set of nucleic acid fragments;
and f) fixing said nucleic acid fragments belonging to a second set of nucleic acid fragments to a solid carrier.

25. The method according to claim 24, wherein after step c), some or all of the nucleic acid fragments are combined to form arrays of nucleic acids by using suitable ligases.

26. The method according to claim 24, wherein in step d) the fragments belonging to different sets are cloned into different vectors.

27. The nucleic acid reagent according to claim 18, comprising nucleic acid fragments which have vector derived parts.

28. The nucleic acid reagent according to claim 18, comprising the recombinant plasmid pKTH1271 or derivatives thereof, which recombinant plasmid contains the DNA of Cytomegalovirus AD169 and is cloned into the host Escherichia coli K12 HB101, and the deposit number of said host containing the recombinant plasmid pKTH1271 is DSM
2826.

29. A method for identifying more than one nucleic acid comprising exposing the nucleic acids to the nucleic acid reagent according to claim 18, wherein combinations of nucleic acid reagents are assembled from nucleic acid fragments which are homologous to the nucleic acids to be identified.

30. A method for the preparation of the nucleic acid reagent according to claim 18, wherein the nucleic acid reagent is prepared synthetically, semi-synthetically or by using recombinant-DNA techniques.

31. The method according to claim 30, wherein the preparation of the nucleic acid reagent comprises:
a) isolating nucleic acids which are homologous to the nucleic acids to be identified;

b) cloning said nucleic acids into vectors;
c) fragmenting said nucleic acids using restriction enzymes into at least two sets of at least two alternating nucleic acid fragments;
d) cloning said nucleic acid fragments into vectors;
e) labeling one set of nucleic acid fragments;
and f) fixing said nucleic acid fragments belonging to a second set of nucleic acid fragments to a solid carrier.

32. The method according to claim 31, wherein in step d) the fragments belonging to different sets are cloned into different vectors.

33. A method for conducting a sandwich hybridization assay using a nucleic acid reagent to detect the identity of a nucleic acid, said method comprising exposing said nucleic acid to said nucleic acid reagent, said nucleic acid reagent comprising at least two sets of at least two alternating nucleic acid fragments which are homologous to the nucleic acid which is to be identified, said nucleic acid fragments being not homologous to one another, wherein the nucleic acid fragments from each of said sets alternate in said nucleic acid reagent and at least one of said sets of nucleic acid fragments are labeled and at least one set of nucleic fragments are affixed to a solid carrier.

34. The method according to claim 33, wherein some or all of the nucleic acid fragments on one or more of the sets of nucleic acid fragments are joined to form an array of nucleic acid fragments.

35. The method according to claim 33, comprising nucleic acid fragments which have vector-derived parts.

36. The method according to claim 33, comprising the recombinant plasmid pKTH1220 or derivatives thereof, which recombinant plasmid contains the DNA of Chlamydia trachomatis L2 bacterium and is cloned into the host Escherichia coli K12 HB101, and the deposit number of said host containing the recombinant plasmid pKTH1220 is DSM
2825.

37. The method according to claim 33, comprising the recombinant plasmid pKTH1271 or derivatives thereof, which recombinant plasmid contains the DNA of Cytomegalo-virus AD169 and is cloned into the host Escherichia coli K12 HB101, and the deposit number of said host containing the recombinant plasmid pKTH1271 is DSM 2826.