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The invention relates to improved nucleic acid reagents comprising an array
of nucleic acid fragments and to combinations of such improved reagents.
The invention also relates to methods for the preparation of nucleic acid
reagents comprised of an array of clones, and combinations of such nucleic
acid reagents, by recombinant-DNA techniques, and to their use for the
identification of nucleic acids by hybridization methods.
Various hybridization methods have commonly been used for the
identification and study of nucleic acids. Some examples are the direct
hybridization methods, in which the sample containing the nucleic acid to
be identified is either in a solution (Brautigam et al., J. Clin.
Microbiol., 1980, 12, 226-234 and the British Patent Publication No.
2,019,408) or affixed to a solid carrier (U.S. Pat. Nos. 4,139,346,
4,302,204, 4,358,535, 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 identified.
Other known hybridization methods include the two-step sandwich
hybridization method presented by Dunn and Hassell in Cell, 12, 23-36,
1977, and the one-step sandwich hybridization methods presented in the
European Patent Publication No. 79,139. For the identification of the
nucleic acids by the sandwich methods two separate nucleic acid reagents
are needed to detect the nucleic acids present in the sample solution. One
of these reagents is affixed to a solid carrier and the other is labeled.
Nucleic acid reagents, both those affixed to a solid carrier and those
which are labeled, are characterized in that their base sequence is
complementary, or nearly complementary, to the nucleic acid to be
identified, i.e. homologous. The nucleic 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 been prepared synthetically or by recombinant-DNA techniques.
Natural plasmids (U.S. Pat. No. 4,358,535), nucleic acids from
bacteriophages (U.S. Pat. No. 4,543,535), ribosomal RNA and messenger RNA
(U.S. Pat. No. 4,302,204), or nucleic acid from different viruses
(Stalhandske et al., Curr. Top. Microbiol. Virol. 104, 1983) have been
used as the nucleic acid reagents. The whole virus genome has been used
for identifying, for example, parts belonging to the different viruses in
the messenger RNA of a hybrid virus (Dunn and Hassell, Cell, 12, 23-36,
1977). Nucleic acid reagents have also been prepared by using
recombinant-DNA techniques (U.S. Pat. Nos. 4,395,486 and 4,359,535, the
European Patent Application No. 79,139 and the British Patent Publication
No. 2,034,323 and the European Patent Application No. 62,286). Nucleic
acid reagents 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 separated clones.
We have developed new, more sensitive nucleic acid reagents, comprising at
least two series of alternating arrays of nucleic acid fragments prepared
from either one or several segments homologous to the nucleic acid to be
identified.
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 reagents. 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 higher sensitivity of the nucleic acid reagents according to the
invention in sandwich hybridization methods 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 (FIGS. 1 and 2). In FIGS. 1 and 2, v
represents vector-derived DNA, x the nucleic acid to be 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 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 FIG.
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 a.sub.1, a.sub.2, and a.sub.3, at one or
several points, depending on the degree of reaction. When the reaction
reaches its final stage, a situation according to FIG. 1 may be produced,
in which the sample strand forms a loop or loops to which the probe or
probes, for example, b.sub.1 and b.sub.2 in FIG. 1, hybridize. At this
time the distance of the vector-derived nucleic acid parts from the
hybridization joining point (1) d creases (FIG. 1), and the hybrid is more
stable than the hybrid formed by one reagent pair (prior art) shown in
FIG. 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.
Our 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 characteristics of the invention are shown in the distinguishing
features of the claims, and the invention is described in greater detail
in the following description and in the a companying drawings, in which
FIG. 1 shows an array of sandwich hybrids,
FIG. 2 depicts a sandwich hybrid of the prior art,
FIG. 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,
FIG. 4 shows the corresponding sites of three alternating series of arrays
of nucleic acid fragments,
FIG. 5 shows an array of nucleic acid fragments according to FIG. 3
separate (a), joined together (b) and both separate and joined together
(c),
FIG. 6 shows an array of sandwich hybrids,
FIG. 6a shows an array of sandwich hybrids which is formed when separate
fragments are used,
FIG. 6b shows an array of sandwich hybrid which is formed when joined
b-fragments are used,
FIG. 6c shows an array of sandwich hybrids which is formed when both
separate and joined b-fragments are used,
FIG. 7 shows an array of nucleic acid reagents which identify different
nucleic acids,
FIG. 8 shows an array of sandwich hybrids which are formed when the array
of nucleic acid reagents according to FIG. 7, identifying different
nucleic acids, are used,
FIG. 9 shows an array of hybrids formed by a direct hybridization method,
FIG. 10 shows the recombinant plasmid pKTH1220,
FIG. 11 shows an array of sandwich hybrids which is formed when an array of
nucleic acid fragments prepared from the recombinant plasmid pKTH1220 are
used,
FIG. 12 shows the recombinant plasmid pKTH1271,
FIG. 13 shows an array of sandwich hybrids which is formed when arrays of
nucleic acid fragments prepared from the recombinant plasmid pKTH1271 are
used.
Our invention 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.
The arrays of nucleic acid reagents can be prepared synthetically. In this
case the fragments from the two alternating series of arrays of nucleic
acid fragments, must not be homologous to each other. But they must be
sufficiently homologous to alternating sites in the nucleic acids to be
identified. These fragments can easily be prepared by fully automatic
machines after characterization of the nucleic acid sequence of the
nucleic acid to be identified.
The nucleic acid reagents according to the invention are composed of
separate, or joined, or both separate and joined array of nucleic acid
fragments.
The arrays of nucleic acid fragments may be joined to a vector, contain
parts of vectors, or be totally devoid of vector parts.
The nuclei 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 nucleotides. The nucleic acid
fragments according to the invention are derived either from the genome to
be identified or from one part of the genome, for example from a
relatively large clone representin a certain part of the genome. The
arrays of nucleic acid fragments according to the inventio 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 identical to the nucleic acid to
be identified but sufficiently homologous, so that a stable hybrid is
formed between the reagent and the nucleic acid to be identified. The
preparation of suitable arrays of nucleic acid fragments: is by no means
limited to the isolation of suitable nucleic acid fragments from the
genome. There are available many equally useful methods to prepare such
arrays 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 series of
alternating nucleic acid fragments a.sub.1, a.sub.2, a.sub.3, etc., and
b.sub.1, b.sub.2, b.sub.3, etc., are obtained. The nucleic acid fragments
belonging to the series a.sub.1, a.sub.2, a.sub.3, etc. are composed of
fragments situataed close to but not adjacent to one another. The nucleic
acid fragments belonging to the series b.sub.1, b.sub.2, b.sub.3, etc. are
also composed of nucleic acid fragments situated close to but not adjacent
to one another. The nucleic acid fragments belonging to the series
a.sub.1, a.sub.2, a.sub.3, etc. and those belonging to the series b.sub.1,
b .sub.2, b.sub.3, etc. must be homologous to each other. It is preferable
that the nucleic acids belonging to the series a.sub.1, a.sub.2, a.sub.3,
etc. and those belonging to the series b.sub.1, b.sub.2, b.sub.3, etc. are
isolated in such a way that every second fragment belongs to the a-series
and every second to the b-series, as shown in FIG. 3. In FIG. 3, a.sub.1,
a.sub.2, a.sub.3, and b.sub.1, b.sub.2, b.sub.3 are arrays of nucleic acid
fragments sufficiently homologous to the nucleic acid to be identified. It
is, of course, possible that even a third nucleic acid fragment series,
c.sub.1, c.sub.2, c.sub.3, etc., is isolated from the same nucleic acid,
as shown in FIG. 4. It is 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
separate fragments a.sub.1, a.sub.2, a.sub.3, etc., and b.sub.1, b.sub.2,
b.sub.3, etc. (FIG. 5a) or joined together into longer strands a.sub.1
-a.sub.2 -a.sub.3, etc., and b.sub.1 -b.sub.2 -b.sub.3, etc. (FIG. 5b). It
is, of course, possible to prepare all kinds of intermediate forms such
as, for example, an a-series in which a.sub.1 is a separate fragment and
a.sub.2 -a.sub.3 are joined together, and in the b-series, for example,
b.sub.1 -b.sub.2 are joined together and b.sub.3 is separate, etc., as
shown in FIG. 5c.
FIG. 6 depicts various arrays of sandwich hybrids. FIG. 6a shows an array
of sandwich hybrids in which the arrays of nucleic acid fragments are
separate. FIG. 6b shows an array of hybrids in which the labeled array of
nucleic acid fragments are joined together. FIG. 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 FIG. 6, x represents the
nucleic acid to be identified; b.sub.1, b.sub.2, and b.sub.3 represent the
labeled probe, and a.sub.1, a.sub.2, and a.sub.3 represent arrays of
nucleic acid fragments affixed to a solid carrier.
Nucleic 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 reagents which belong to the a-series
can be affixed to a solid carrier in such a way that a nucleic acid
reagent A bound to a solid carrier is obtained. It is, of course,
alternatively possible to prepare a labeled nucleic acid reagent A, and a
corresponding nucleic acid reagent B bound to a solid carrier.
Such nucleic acid pairs A and B, or B and A, labeled and respectively
affixed to a solid carrier can be prepared for several different nucleic
acids to be identified. They can be combined into suitable nucleic acid
reagent combinations, which are composed of different nucleic acid reagent
pairs A.sub.1 and B.sub.1, A.sub.2 and B.sub.2, A.sub.3 and B.sub.3, etc.,
or B.sub.1 and A.sub.1, B.sub.2 and A.sub.1, B.sub.2 and A.sub.3, etc.
Reagents containing arrays of nucleic acid fragments which identify
different nucleic acids can also be combined so that a probe A.sub.x
-A.sub.y -A.sub.z is obtained, which, for example, comprises an array of
nucleic acid fragments (a.sub.1 -a.sub.2 -a.sub.3).sub.x -(a.sub.1
-a.sub.2 -a.sub.3).sub.y-(a.sub.1 -a.sub.2 -a.sub.3).sub.z, as shown in
FIG. 7, in which a.sub.1x, a.sub.2x and a.sub.3x are arrays of nucleic
acid fragments A.sub.x which identify nucleic acid x; a.sub. 1y, a.sub.2y
and a.sub.3y are arrays of nucleic acid fragments A.sub.y which identify
nucleic acid y; a.sub.1z, a.sub.2z and a.sub.3z are arrays of nucleic acid
fragments A.sub.z 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 FIG. 8 are obtained by using
the reagents shown in FIG. 7. If simultaneous identification of several
different nucleic acids is desired, it is, of course, necessary to use
separate filters, as shown in FIG. 8. FIG. 8a shows a solid carrier
identifying the nucleic acid x, FIG. 8b a solid carrier identifying the
nucleic acid y, and FIG. 8c a solid carrier identifying the nucleic acid
z. In FIGS. 8a, 8b and 8c, b.sub.1x and b.sub.2x are arrays of nucleic
acid fragments affixed to a solid carrier and identifying the nucleic acid
x; b.sub.1y and b.sub.2y are arrays of nucleic acid fragments affixed to a
solid carrier and identifying the nucliec acid y; and b.sub.1z and b.sub.2
are arrays of nucleic acid fragments affixed to a solid carrier and
identifying the nucleic acid z; and x, y and z are the nucleic acids to be
identified. F.sub.x, F.sub.y and F.sub.z are the respective solid carriers
or filters, A.sub.x -A.sub.y -A.sub.z is a probe which identifies all the
three nucleic acids simultaneously, if separate solid carriers are used.
The above-described nucleic acid fragment series, reagents and reagent
combinations can be prepared by recombinant-DNA techniques known per se. A
number of nucleic acid fragments of different lengths are generated, by
using restriction enzymes, from the 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 fragments, 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 preparation 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 a.sub.1, a.sub.2, a.sub.3, etc., and
b.sub.1, b.sub.2, b.sub.3, etc., are produced on the basis of the
information thus obtained.
It is, of course, possible to use combinations 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 suitable combinations.
It is advantageous to prepare the nucleic acid fragment series a.sub.1,
a.sub.2, a.sub.3, etc., and b.sub.1, b.sub.2, b.sub.3, etc., according to
the invention by using recombinant-DNA techniques in such a way that the
series a is cloned into one vector, for example into the plasmid pBR322,
and whereas the series b is cloned into another suitable vector, which
does not have sequences in common with the previous vector. The
bacteriophage M13 is an example of 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, a.sub.1
-a.sub.2, joined together, can be cloned as a continuous insert into the
same pBR322 vector. In a corresponding manner it is possible to prepare a
reagent series b.sub.1 -b.sub.2. 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 suitable 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 affixed to a solid carrier.
Most commonly, radioactive isotopes are used for labeling the probes. For
example in the British Patent Publication No. 2,034,323, the U.S. Pat.
Nos. 4,358,535 and 4,302,204 the following isotopes are used: .sup.32 P,
.sup.125 I, .sup.131 I and .sup.3 H. In the European Patent Publication
No. 79,139, the isotope .sup.125 I is used. Nucleic acid probes have also
been modified in different ways and labeled with, e.g. fluorescent labels
(French Patent Publication No. 2,518,755). Also enzymatic or enzymatically
measureable labels are used (the British Patent Publication No. 2,019,408,
the European Patent Publication No. 63,879 and the French Patent
Publication No. 2,519,005). The European Patent Publications Nos. 70,685
and 70,687 describe a light-emitting label and labeling method, and the
French Patent Publication No. 2,518,755 describes an immunologically
measurable label.
The lanthanide chelates described in U.S. Pat. No. 4,374,120, especially
europium, can be used as label substances. Also the biotin-avidin label
substance described 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 reagents according to the invention are mentioned above, but
it is evident that there will be developed new, improved label substances
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 nitrocellulose
filters (U.S. Pat. No. 4,358,535 and the British Patent Publication No.
2,095,833). The DDR-Patent Publication No. 148,955 desribes a method of
binding nucleic acids chemically to the carrier (paper). U.S. Pat. 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 above-described combinations of the arrays of nucleic acid reagent
pairs A and B, or B and A, labeled and affixed to a solid carrier
respectively, and from such nucleic acid pairs made for the identification
of different nucleic acids it is possible to assemble a combination
A.sub.x and B.sub.x, A.sub.y and B.sub.y, A.sub.z and B.sub.z.
These combinations can be used for the simultaneous identification of the
nucleic acids x, y and z by sandwich hybridization methods.
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 hybridization solution, to which
both the nucleic acid reagents affixed 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
carriers. If chromatography matrices, latex, or the like have been used,
the hybridization solution is removed. The solid carriers are rinsed with
a suitable washing solution. The arrays of sandwich hybrids formed (FIGS.
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 detected by a so-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
U.S. Pat. No. 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.degree.-80.degree. C., but is advantageous to
use, for example, a temperature of 65.degree. C. Sufficient hybridiztion
may occur in a very short period, but it is advantageous to use
hybridization periods of, for example, 12-20 hours.
The two-step sandwich hybridization method is carried out in principle in
the same manner, but in this case the nucleic acid reagent affixed to a
solid carrier is first added to the hybridization 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 is
present.
The above-described labeled nucleic acid reagents or reagent combinations
A.sub.x, A.sub.y, A.sub.z, etc., and B.sub.x, B.sub.y, B.sub.z, 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 is affixed to a solid carrier,
a separate sample affixed to a carrier must be prepared for each sample.
The formed array of hybrids (FIG. 9) is detected by methods known per se.
In FIG. 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 a.sub.1, a.sub.2 and a.sub.3 (FIG. 9a), b.sub.1 and b.sub.2 (FIG.
9b), and a.sub.1, b.sub.1, a.sub.2, b.sub.2 ; a.sub.3 (FIG. 9c).
As already described above, various combinations of nucleic acid reagents
can be made up from the 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 fragments
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 kept
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 poisonings. The method is suitable for the identification of
contaminants present in water, such as enterobacteria and enteroviruses.
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 amplification. This characteristic is
significant in, for example, the 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 strand. If changes with respect to the distance between
these two areas do occur, the change is cleary observable by this method.
Therefore the method is also suitable for the detection of changed mRNA,
chromosomal rearrangements, the rearrangement of immunoglobulin genes for
expression, and hereditary diseases. It is thus 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 containing the recombinant plasmids
pKTH1220 and pKTH1271 have been deposited at the culture collection
Deutsche Sammlung von Mikroorganismen (DSM), Griesebachstrasse 8, D-3400
Gottingen, West Germany. The number of the deposit containing the
recombinant plasmid pKTH1220 is DSM2825 and the number of the deposit
containing the recombinant plasmid pKTH1271 is DMS2826. The deposits will
be freely available once the patent application has been made public.
The invention is described in greater detail in the following examples.
These examples must not, however, be understood as limiting the protective
scope of the invention. The structure of the nucleic acid (DNA and RNA) is
similar whether the question is of a nucleic acid derived from a
eucaryotic or a procaryotic cell. For this reason the principles presented
in the examples are equally well applicable to the nucleic acids 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 viruses. The arrays of nucleic acid
fragments can be prepared synthetically, too. The sequence of nucleic
acids to be identified can be characterized and homologous arrays of
fragments prepared by automatic nucleic acid preparing machines.
EXAMPLE 1.
(a) Arrays of nucleic acid reagents from Chlamydia 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
organism Escherichia 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 from chlamydial DNA were selected from the gene bank. One
such plasmid is the one designed pKTH1220, which has been deposited at the
culture collection Deutsche Sammlung von Microorganismen under the number
(DSM 2825) and the suitability of which for use as a reagent was
demonstrated by a direct hybridization test. The test showed that pKTH1220
identified all of the nucleic acids derived from different Chlamydia
trachomatis serotypes, but no other nucleic acids.
The applicable fragments, obtainable by using different restriction
enzymes, were selected from the pKTH1220-plasmid DNA, and some of these
fragments were transferred by further cloning into pAT153 plasmid
(Maniatis et al., Molecular Cloning. A Laboratory Manual, Cold String
Harbor Laboratory, p. 6, 1982) and come to M13 phage. FIG. 10 shows the
recombinant plasmid pKTH1220, having a molecular length of 14 kb. In FIG.
10, BamHI, SalI and ClaI represent the restriction enzymes used, and
a.sub.1, a.sub.2, b.sub.1, b.sub.2 and b.sub.3 illustrate the size and
mutual locations of the fragments produced with the aid of these
restriction enzymes. The fragments belonging to the series b as labeled
probes. Table 1 lists the sizes of the fragments and the vectors used for
further cloning, the names of the recombinant plasmids, and their use.
TABLE 1
__________________________________________________________________________
Recombinant
Fragment Size Vector
plasmid
Use
__________________________________________________________________________
a.sub.1
ClaI-SalI
3.0 kb
pAT153
pKTH1252
Filter
a.sub.2
SalI-ClaI
2.9 kb
pAT153
pKTH1250
Filter
b.sub.1
SalI-BamHI
0.7 kb
M13mp8
mKTH1242
Labeled probe
b.sub.2
BamHI-SalI
1.4 kb
M13mp8
mKTH1239
Labeled probe
b.sub.3
ClaI-ClaI
1.7 kb
M13mp8
mKTH1248
Labeled probe
b.sub.1 -b.sub.2
BamHI-BamHI
2.1 kb
M13mp8
mKTH1245
Labeled probe
__________________________________________________________________________
The fragments listed in Table 1 were isolated from an agarose gel by
electroelution and were cloned into the appropriate restriction enzyme
identification sites of the vectors listed in Table 1, by using known
methods.
The fragment BamHI-BamHI 2.1 kb was produced as follows: the 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 fragments were joined to each other with the aid of
T4 ligase enzyme, and of the 2.1 kb DNA fragments produced in the
reaction, those which had free ends which were identified by the BamHI
enzyme were further joined to the BamHI restriction site of the
doublestranded form of the M13mp8 phage DNA. Thus there was made a
recombinant phage-DNA (mKTHl245) which contains Chlamydia trachomatis DNA
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 (FIG. 11). FIG.
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 the sensitivity of an array of nucleic acid reagents
from Chlamydia trachomatis by using the sandwich hybridization method
The sensitivity of an array of nucleic acid reagents as compared with a
single continuous reagent pair was studied by the sandwich hybridization
method. The test was carried out using filters which all contained
10.sup.11 molecules of both pKTH1250 (a2) and pKTH1252 (a.sub.1) DNA
rendered single-stranded. The sample to be studied was the plasmid
pKTH1220, which for the test was rendered single-stranded by boiling for 5
min in 0.17 M NaOH, whereafter it was transferred to 0.degree. C. and
neutralized with an equimolar amount of acetic acid. The following probes
labeled with .sup.125 I, listed in Table 1, were used in the tests:
mKTH1242 (b.sub.1), MKTH1239 (b.sub.2), MKTH1248 (b.sub.3) and
mKTH1245(b.sub.1 -b.sub.2).
The hybridization was performe | | |
