NL8503597A - METHOD FOR IDENTIFYING NUCLEIC ACIDS. - Google Patents

Description

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Short designation: Method for identifying nucleic acids

The invention relates to a method of identifying nucleic acids by hybridization in solution. The method of the invention is characterized in that at least two probes are used in the hybridization reaction. A label is attached to the detectors probe, and to the other probe, the so-called capture probe, a component with affinity for another component is attached, whereby the capture probe / target nucleic acid / detector probe hybrid resulting from the hybridization can be separated from the other components present in the hybridization mixture.

Various hybridization methods have been used to identify nucleic acids. Direct hybridization methods and sandwich hybridization methods can be mentioned as examples. In direct hybridization methods, the nucleic acid specimen is either in solution or bound to a solid support. The nucleic acid to be identified is demonstrated using a labeled probe. Sandwich hybridization methods (U.S. Patent 4,486,539) use two separate probes by which the nucleic acid to be identified is demonstrated in the sample solution. The detector probe is labeled with a label substance and the other probe is bound to solid support.

A new hybridization method that uses two different probes and where both probes are in the solution phase has been developed. Since both the target nucleic acid participating in the hybridization and the two probes are in the same solution phase, the hybridization reaction is considerably faster than in sandwich hybridization, with one probe attached to a solid support. In the method according to the invention, an incubation time of one hour is sufficient. In the single-stage sandwich hybridization (U.S. Patent 4,486,539), sufficient hybridization is not accomplished in less than 12 hours.

When two-stage sandwich hybridization (Dunn and Hassel, Cell, 12, 23-36 (1977)) is performed, a hybridization time of at least 24 hours is required.

At least two probes are advantageously used in the method according to the invention. The probes are nucleic acid fragments that are homologous to the target nucleic acid. It is advantageous but not: * i 7 - * ^ - »Γ - 2 - it is necessary that the probes are homologous to sites relatively close together in the nucleic acid to be identified. The probes must be non-overlapping with each other.

The probes can be prepared synthetically, semi-synthetically, using recombinant DNA techniques or from nucleic acids isolated directly from nature. Probes are also commercially available from a variety of sources. A probe can be bound to a suitable vector.

The probe can contain vector parts or be completely free of vector parts.

The detector probe is labeled with an appropriate label. Various radioactive isotopes or radiolabeled compounds can be used as labels. The label substance can also be fluorescent, luminescent, light-emitting, enzymatic or immunologically demonstrable, etc. Labels based on biotin and avidin or streptavidin, lanthanide chelates, ferritin and heme compounds, and immunologically demonstrable hapten, such as acetoxyacetylfluorene derivatives (WO 8302286) can be mentioned as examples. Identification through proteins is also possible. The method according to the invention does not depend on the label used. All currently known label substances suitable for nucleic acid hybridization, or those which will be developed in the future, can be freely applied to the method.

A component with affinity for another component is attached to the other probe, the so-called capture probe. Biotin / avidin or streptavidin, heavy metal derivatives / thio groups, various homopoly nucleotides such as poly-dG / poly-dC, poly-dA / poly-dT and poly-dA / poly-U are suitable affinity pairs. But other affinity pairs can also be used, provided that the components have sufficiently strong affinity for each other. Suitable affinity pairs are found among ligands and conjugates used in immunological methods.

Before the hybridization reaction is performed, the specimen is treated to release the target nucleic acids in single strand form in the hybridization solution. Hybridization is carried out in a hybridization mixture in which the target nucleic acids, the labeled probe and the capture probe are made single stranded, if necessary. Various suitable buffers can be used as hybridization solutions. Hybridization takes place within the temperature range of 0-80 ° C, but it is advantageous to use a temperature of 65 ° C. If the hybridization solution contains formamide (40-55%), a temperature of 37 ° C can be used. One hour is sufficient as a hybridization time.

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When hybridization has occurred, the solution is diluted as necessary to make the conditions beneficial to the affinity pair. The mixture is then contacted with the other member of the affinity pair. Affinity chromatography columns, filters, plastic surfaces, glass surfaces, etc. can be used to capture the capture probe / target nucleic acid / detector probe hybrid.

The support material of the affinity column can be, for example, cellulose, latex, polyacrylamide, dextran or agarose. These materials can also be used as suspensions in a test tube. It is also advantageous to use test tubes in which the other component of the affinity pair is bound to its inner surface. It is a requirement for the selected material that it is possible to bind to it a component that has affinity for the component attached to the capture probe.

If the specimen contains the nucleic acid to be identified, a capture probe / target nucleic acid / detector probe hybrid results from the hybridization. During fractionation, this hybrid attaches to the support. The label of the fraction adhering to the support can be measured by conventional methods, directly from the support or after elution from the eluted solution.

Other systems, for example phase extraction or magnetic fields, can also be used in fractionation instead of affinity chromatography.

The method of the invention is described in more detail in the following examples. The method according to the invention does not depend on the nucleic acid fragments used in the examples.

Example 1

Identification of adenovirus DNA from a cell lysate by the acid of homopolynucleotides. The detector probe used is 12% labeled recombinant phage mKTH 1206 containing a Bgl II fragment of the adenovirus genome from position 42-45.3% at contains the gene map of the adenovirus type 2. The recombinant phage has been deposited under number DSM 2827 in the culture collection Deutsche Sammlung von Mikroorganismen. Its specific activity is 7x10x10 cpm / µg DNA. The probe is described in more detail in Ranki et al., Gene 21, 77-85 Π983).

The capture probe used is the recombinant plasmid pKTH 1202, which has been deposited under number DSM 2824 in the culture collection Deutsche Sammlung von Mikroorganismen. This includes a BamHI D fragment of the a4 adenovirus (map position 29-42%) cloned into the plasmid pBR322. The recorabinant plasmid pKTH 1202 (DSM 2824) was fragmented using the restriction enzyme Hae III and a poly-A tail was linked to the 3 'ends of the fragments using 5 term transferase enzyme. Tail length was measured by 2H-A incorporation. The length averaged about 70 A residues. The capture probe was denatured by boiling before use.

The sample used consisted of adenovirus infected A-549 cells. The cells were incubated 21 hours after the infection. The 10 cells were collected and lysed using a 1% sodium dodecyl sulfate solution. The solution contained about 10 ° cells / ml. Its viscosity was reduced by sonication. Identically treated uninfected A-549 cells were used as a control. Before hybridization, the specimen was boiled in 0.02 M 15 NaOH, cooled to 0 ° C, and neutralized with acetic acid.

For the test, 500,000 cpm detector probe, 50 ng capture probe DNA and 10 µl specimen were combined in a test tube. The volume was adjusted to 50 µl and the buffer solution used was 0.6 M sodium chloride, 0.06 M sodium citrate, 0.02 M sodium phosphate (pH 7.6) and 0.5% sodium 20 dodecyl sulfate. The mixture was incubated at 65 ° C for 1 hour.

After hybridization, the solution was cooled to + 20 ° C and slowly passed through a chromatography column with 1 ml oligo-dT cellulose. The solution that came through was collected and passed through the column again. The column was then washed with 20 ml of a solution containing 0.15 M sodium chloride, 0.015 M sodium phosphate (pH 7.6) and 0.5% sodium dodecyl sulfate. The captured DNA was finally released using 1 ml 0.02 M NaOH. This solution was collected and its radioactivity determined.

Result; 12 ^ I activity (cpm). Target nucleic acid: Infected cells. Control cells 5230 325

Example 2

Identification of the DNA of Chlamydia trachomatis using biotin / streptavidin. The detector probe used was 12 µl-labeled recombinant phage mKTH 1245 containing two BamHI-Sall DNA fragments from the clone pKTH 1220, which together are attached to the M13mp8 vector are cross-linked. The clone pKTH 1220 has been deposited under number DSM 2825 in the culture collection Deutsche? '*. '' j 7 * <- - 5 -

Sammlung von Microorganisms and is described in Palva et al., FEMS Microbiology Letters 23, 33-39 (1934).

The capture probe used was the recombinant plasmid pKTH 1250.

This plasmid consists of a 2.9 kb Sall-Clal fragment from the plasmid pKTH 1220 (DSM 2825) and from the vector pAT 153. Biotin molecules were covalently cross-linked with the pKTH 1250 DNA using the known nick translation method (Rigby et al., J. Mol. Biol. 113, 237-251 (1977)) and the biotin-11-UTP as substrate (Bethesda Research Laboratories). The capture probe DNA was boiled for 5 min in a buffer containing 10 mM Tris-Cl pH 7.6, 1 mM EDTA before use.

The target nucleic acid was the recombinant plasmid pKTH 1220 (DSM 2825). This plasmid contains about 10 kb of the DNA characteristic of Chlamydia trachomatis cross-linked with the vector pBR322. The plasmid functions as model DNA, representing the genomic DNA of the bacterium. Before use, the plasmid was boiled in 0.02 M NaOH for 5 min, after which the solution was neutralized with acetic acid.

Streptavidin, which was used as an affinity material, was attached to CNBr-activated Sepharose (Pharmacia) according to Axen et al., Nature 214, 1302-1304 (1967).

For the test, 500,000 cpm probe, 50 ng capture probe DNA and 10 ng target DNA were combined in a test tube. The volume was adjusted to 20 µl and the buffer solution was the same as in Example 1. The control DNA was calf thymus DNA.

The mixture was incubated at 65 ° C for 60 min. Then 500 µl of a buffer solution of the composition 0.1 M Tris-Cl pH 7.5, 0.1 M NaCl, 2 mM MgCl 2, 0.05% Triton X-100 was added. Finally, the solution was fractionated in a 0.2 ml Streptavidin Sepharose column. The column was washed with 10 ml of the above buffer and 10 ml of 0.015 M sodium chloride, 0.015 M sodium phosphate (pH 7.6), 0.5% sodium dodecyl-30 sulfate (50 ° C). Thus, the biotinylated DNA attached to the Streptavidin while the other DMA passed through the column. The radioactive probe adhered only as a result of hybrid formation. The captured radioactivity was determined by transferring the entire column into the counter of a gamma counter.

35 Result: 12 ^ I activity (cpm)

Target nucleic acid: 10 ng pkTH 1220 10 ng Control DNA

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Example 3

Identification of piasmid pBR322 DNA using an antigen-antibody pair

The detector probe used is a derivative of the plasmid pBR322 (commercially available from various sources) from which the Pstl-Sall (3613-651) fragment has been removed. The plasmid was labeled with photobiotin using a known method (Forster et al., Nucleic Acids Res. 13, 745-761 (1985)) and commercial reagents (Bresa, Adelaide, Aus gralie).

The capture probe used was DNA from a recombinant phage M13mp11 into which the pBR322 Pst1-Sall fragment had been introduced. The DNA was sulfonated by a known method (Orgenics Ltd, Yavne, Israel).

The specimen was E. coli HB101 carrying the plasmid pBR322, the amount of which was increased using chloramphenicol amplification (Maniatis et al., Molecular cloning, A laboratory manual, Cold Spring Harbor Laboratory 1982). The bacterial cells were lysed with lysozyme followed by boiling in NaOH as described in the publication Palva, J. Clin. Microbiol. 18, 92-100 (1983).

Antisulfone monoclonal antibodies were used to coat polystyrene microtiter plates by standard methods (McKearn, in Hybri-domas: A New Dimension in Biological Analyzes, ed. Kennett et al., Plenum Press 1980).

For the test, 5x10 ^ were lysed Ξ. coli cells (both with and without pBR322) combined with 100 ng capture DNA and 100 hg detector probe in a 50 µl hybridization mixture. The conditions were as in Example 1 except that 5% polyethylene glycol (PEG 6000) was added to the mixture and the sodium dodecyl sulfate concentration was 0.1%. After hybridization, the solution was diluted to 250 µl by adding 0.02 M sodium phosphate (pH 7.6) and the solution was transferred to the antibody-coated microtiter plate. This was followed by incubation at 37 ° C for two hours. The plate was then washed with a solution containing 0.015 M sodium chloride, 0.02 M sodium phosphate pH 7.6 and 0.05% Triton X-100. The presence of detector probe was visualized by adding streptavidin (Bethesda Research Laboratories BRL), washing, adding biotinylated alkaline phosphatase (BRL), and washing as described by Leary et al., Proc. Natl. Acad. Sci. USA 80, 4045-4049 (1983). Finally, 250 μl of a solution of 35 mg / l paranitrophenylphosphate (Sigma) in diethanolamine buffer (pH 10) was added: -. - V7 ^ v * * .....

t - 7 - added. After 60 min, the reaction was stopped and the absorbance was measured at 410 nm.

Ssult: A41Q nm

Di-nucleic acid: Horns with pBR322 Cells without pBR322 5> 2 0.15 o 2 'l j 7

Claims (8)

Method for identifying nucleic acids, characterized in that at least two probes present in the same solution phase are used in a hybridization method, wherein the detector probe is labeled with a detectable label and in which the capture probe is a member of an affinity pair and wherein the capture probe / target nucleic acid / detector probe hybrid formed in the hybridization reaction is isolated by the other member of the affinity pair. A method according to claim 1, characterized in that the affinity pair is biotin / streptavidin or biotin / avidin. The method according to claim 1, characterized in that the affinity pair is a homopolynucleotide pair. 4. A method according to claims 1 and 3, characterized in that the affinity pair is poly-dC / poly-dG. A method according to claims 1 and 3. characterized in that the affinity pair is poly-dA / poly-dT. A method according to claims 1 and 3, characterized in that the affinity pair is poly-dA / poly-U. 20 A method according to claim 1, characterized in that the affinity pair is a heavy metal derivative / a thio group. A method according to claim 1, characterized in that the affinity pair is an antigen / an antibody. "Y .. · _} :) J ƒ