DK164932B - PROCEDURE FOR IDENTIFICATION OF NUCLEIC ACIDS BY A METHOD OF HYBRIDIZATION, WHICH AT LEAST TWO SUNS ARE IN THE SAME SOLUTION PHASE, SINCE THE ONE, THE DETECTOR SENSOR, IS MARKED WITH A DETECTABLE MARKET - Google Patents

Description

in

DK 164932B

The present invention relates to a method for identifying nucleic acids by hybridization in solution, wherein at least two probes are in the same dissolution phase of the hybridization reaction. To the detector-5 probe is attached a marker and to the second probe, a so-called capture probe, there is attached a component with affinity to another component by which the capture probe / target nucleic acid / detector probe hybrid resulting from the hybridization can be separated from the other 10 components present in the hybridization mixture.

Various hybridization methods have been used to identify nucleic acids. Direct hybridization methods and so-called sandwich hybridization methods can be cited as examples. By direct hybridization methods, the nucleic acid sample is either in solution or attached to a solid support. The nucleic acid to be identified is identified by one labeled probe. In sandwich hybridization methods (U.S. Patent No. 4,486,539), two separate probes are used by which the nucleic acid for identification is detected in the sample solution. The detector probe is labeled with a labeling substance and the other probe is attached to a solid support.

A new hybridization method has now been developed using two different probes and where both probes are in the solution phase. Since both the target nucleic acid participating in the hybridization and the two probes are in the same dissolution phase, the hybridization reaction is substantially faster than that of sandwich hybridization at which one probe is bound to a solid support. In the method of the invention, an incubation time of one hour is sufficient. In the one-step sandwich hybridization (U.S. Patent No. 4,486,539), sufficient hybridization in less than 12 hours cannot be achieved. When performing two-step sandwich hybridization (Dunn & Hassell, Cell. Vol. 12, pages 23-36, 1977), a hybridization time of at least 24 hours is required.

35 WO 84/03520 discloses the detection of a nucleic acid by means of two nucleic acid probes, one of which is labeled. However, the labeled nucleic probe is not directly attached to the nucleic acid to be detected, and by

DK 164932 B

In a described practical embodiment, the nucleic acid to be detected is immobilized.

Advantageously, at least two probes are used in the method according to the invention. The probes are nucleic acid fragments which are sufficiently homologous to the target nucleic acid. It is advantageous, but not necessary, for the probes to be homologous to sites located relatively close to each other in the nucleic acid for identification.

The probes can be made synthetically, semi-synthetically, by 10 recombinant DNA techniques or from nucleic acid isolated directly from nature. There is also. probes commercially available from several different sources. A probe may be bound to an appropriate vector. It may contain vector parts or be completely free of vector parts.

15 The detector probe is labeled with an appropriate marker. Various radioactive isotopes or radiolabelled compounds can be used as a marker. For example, the labeling substance may also be fluorescent, luminescent, light emitting enzymatic or immunologically detectable. Markers based on 20 biotin and avidin or streptavidin, lanthanide chelates, ferritin and heme compounds as well as immunologically detectable haptenes such as acetoxyacetylfluorene derivatives (WO '8302286) are examples. Identification with the involvement of proteins is also possible. The method according to the invention does not depend on the marker used. All currently known labeling substances suitable for nucleic acid hybridization or those which may be developed in the future may be freely used in the present process.

To the second probe, the so-called capture probe, there is attached one component with affinity to another component. Biotin, avidin or streptavidin, various homopolynucleotides such as poly dG-poly dC, poly dA-poly dT, and poly dA-poly U are suitable affinity pairs. However, other affinity pairs may also be used provided the components have sufficiently strong affinity 35 for each other. Suitable affinity pairs are found among ligands and conjugates used in immunological methods.

DK 164932B

3 Prior to the hybridization reaction, the gel release of the sample is treated by the target nucleic acids in single stranded form in the hybridization solution. The hybridization is carried out in a hybridization mixture in which the target nucleic acids, of the labeled probe and the capture probe, if necessary, have been single-stranded. Various suitable buffers can be used as hybridization solutions. The hybridization takes place in the temperature range of 0-80 ° C, but it is advantageous to use a temperature of 65 ° C. If the hybridization solution contains formamide 10 (40-55%) a temperature of 37 ° C can be used. One hour is sufficient as hybridization time.

Once hybridization has taken place, the solution is diluted, if necessary, to make the conditions favorable for affinity pairs. Then, the mixture is contacted with the other member of the affinity pair. Finite 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 in the affinity column may 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 with the second component of the affinity pair attached to its inner surface. It is a prerequisite for the material selected that it is possible to attach a component to that which has affinity to the component attached to the captive probe.

If the sample contains the nucleic acid for identification, the hybridization results in a capture probe / target nucleic acid / detector probe hybrid. During fractionation, this hybrid will hang at the carrier. The marker on 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, such as phase extraction or magnetic fields, may also be used in place of affinity chromatography by fractionation.

The method according to the invention will be described in more detail in some examples in the following. The process

DK 164932 B

4 of the invention does not depend on the nucleic acid fragments used in the examples.

Example 1 5

Identification of adenovirus DNA from a cell lysate by homopolynucleotides_ 125

The detector probe used is the I-labeled recombinant phage mKTH 1206, which contains a Bgl II fragment of the adenovirus 10 genome from position 42-45.3% on the gene map of adenovirus type 2. The recombinant phage is deposited in the culture collection Deutsche Sammlung von Microorganism under the number DSM 2827 on December 15, 1983. Its specific activity of 7 x 10 6 cpm / g g DNA. The probe is described in more detail in Ranki et al Gene 21, pages 77-85, 1983.

The capture probe used is the recombinant plasmid pKTH 1202, which has been deposited in the culture collection Deutsche Sammlung von Microorganism under the number DSM 2824 on December 15, 1983, and it comprises a BamHI D fragment of the adenoviruses (short position 29-42%) cloned into the plasmid pBR322. The recombinant plasmid pKTH 1202 (DSM 2824) was fragmented using the restriction enzyme Hae III and a poly A tail was coupled to the 3 'ends of the fragments by terminal transferase enzyme. The length of the tail was measured by H-A in-25 corporation. The average length was approx. 70 A residues. Prior to use, the capture probe was denatured by boiling.

The sample used consisted of adenovirus-infected A-549 cells. The cells were incubated for 21 hours after infection.

The cells were collected and lysed by means of a 1% sodium dodecyl sulfate solution. The solution contained approx. 10 cells / ml. Its viscosity was decreased by ultrasound treatment. Identically treated uninfected A-549 cells were used as controls. Prior to hybridization, the sample was boiled for 5 minutes in 0.02 M NaOH, cooled to 0 ° C and neutralized with acetic acid.

For the sample, 500,000 cpm detector probe, 50 ng of capture probe DNA, and 10 μΐ sample were pooled into a test tube. The volume was adjusted to 50 μΐ and the buffer solution used was 0.6 M sodium chloride, 0.06 M sodium citrate, 0.02 M sodium phosphate

DK 164932 B

5 (pH 7.6) and 0.5% sodium dodecyl sulfate. The mixture was incubated for one hour at 65 ° C.

After the hybridization, the solution was cooled to + 20 ° C and slowly passed through a chromatography column with 1 ml of oily oil dT cellulose. The solution that ran through was collected and redirected through the column. Then the column was 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 associated DNA was finally separated using 10 1 ml of 0.02 M NaOH. The solution was collected and its radioactivity determined.

results:

12S

I activity (cpm) 15- Target nucleic acid: infected cells Control cells 5230 325

Example 2 20

Identification of DNA from Chlamydia trachomatis by biotin-streptavidin_ 125

The detector probe used was cI-labeled recombinant phage mKTH 1245 which contains two Bam HI-SalI DNA fragments from the clone pKTH 1220, which are linked together to the M13np8 vector. The clone pKTH 1220 is deposited in the culture collection Deutsche Samm-lung von Microorganism under the number DSM 2825 on December 15, 1983 and is described in Palva et al. FEMS Microbiology Letters, 23, pages 83-89, 1984.

The capture probe used was the recombinant plasmid pKTH 1250.

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

DK 164932 B

6 The target nucleic acid was recombinant plasmid pKTH 1220 (DSM 2825). This plasmid contains approx. 10 kb of the DNA characteristic of Chlamydia trachomatis, bound to the vector pBR322. The plasmid serves as model DNA and represents the bacterial genome DNA. Prior to use, the plasmid was boiled for 5 min in 0.02 M NaOH and the solution neutralized with acetic acid.

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

For the test, 500,000 cmp probe, 50 ng of capture probe DNA and 10 ng of target DNA in test tubes were combined. The volume was adjusted to 20 μΐ and the buffer solution was the same as in Example 1. Control DNA was calf thymus DNA.

The mixture was incubated for 60 minutes at 65 ° C. Then, 500 μΐ of a buffer solution of composition 0.1 M Tris-Cl pH 7.5, 0.1 M NaCl, 2 mM MgCl 2, 0.05% Triton® x-100 was added. , 2 ml of strep-tavidin "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 sulfate (50 ° C). The biotinylated DNA adhered to the streptavidin while the other DNA passed through the column, the radioactive probe adhering only as a result of hybrid formation, and the captured radioactivity was determined by transferring the entire column to the counter tube in a gamma counter.

Results: 125 I activity (cpm) 30 - Target nucleic acid:

10 ng pKTH 1220 10 ng control DNA

1350 115

DK 164932 B

7

Example 3

Identification of the plasmid pBR322 DNA by an antigen-antibody pair

The detector probe used is a derivative of the plasmid pBR 5,322 (commercially available for multiple sources) from which the PstI-SalI fragment (3613-651) has been removed. The plasmid was labeled with photobiotin using a known method (Forster et al. Nucleic Acids Res. 13, pages 745-761, 1985) and commercial reagents (Bresa, Adelaide, Australia).

10 The probe used was DNA. from a recombinant phage M13mpII into which the fragment pBR322 PstI-SalI had been introduced. DNA had been sulfonated by a known method (Orgenics Ltd. Yavne, Israel).

The sample was Escherichia coli HB101 with the plasmid pBR322, the amount of which was increased by chloramphenicol enhancement (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, pages 92-20 100, 1983).

Antisulfone monoclone antibodies were used to coat polystyrene microtiter chambers by standard method (McKaern in Hybridomas: A New Dimension in Biological Analysis, ed. Ken-nett et al., Plenum Press 1980).

g 25 For the sample, 5 x 10 5 lysed Escherichia coli cells (both with and without pBR322) were combined with each 100 ng of capture DNA and detector probe in a 50 μΐ 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 μΐ with the addition of 0.02 M sodium phosphate (pH 7.6) and the solution transferred to the antibody coated microtiter well (microtiter well). This was followed by incubation for 2 hours at 37 ° C. The chamber was then washed with a solution containing 0.15 M sodium chloride, 0.02 M sodium phosphate pH 7.6 and 0.05% Triton® X-100.

DK 164932 B

8 of the detector probe was visualized by the addition of streptavidin (Bethesda Research Laboratories BRL), washing, addition of biotinylated alkaline phosphatase (BRL), and washing as described by Leary et al. in Proc. Natl. Acad. Sci. USA 80, pages 4045-4049, 1983.

Finally, 250 μΐ of a 35 mg / ml solution of paranotrophenyl phosphate (Sigma) in diethanolamine buffer (pH 10) was added. After 60 minutes, the reaction was stopped and absorption was measured at 410 nm.

Result: A410 nm Target nucleic acid:

Cells with pBR 322 Cells without pBR 322 15> 2 0.15

Claims (7)

A method for identifying nucleic acids by a hybridization method wherein at least two probes are in the same solution phase, one detector probe being labeled with a suitable detectable marker, characterized in that the other detector probe is attached to it. one member of an affinity pair upon which the capture probe / target nucleic acid / detector probe hybrid formed by the hybridization reaction is isolated from the other components present in the hybridization solution by the second member of the affinity pair associated with a solid support. · Process according to claim 1, characterized in that the affinity pair is biotin-streptavidin or biotin-avidin. Method according to claim 1, characterized in that the affinity pair is a homopolynucleotide pair. Method according to claims 1 and 3, characterized in that the affinity pair is poly cD-dG. Process according to claims 1 and 3, characterized in that the affinity pair is poly dA-poly dT. Process according to claims 1 and 3, characterized in that the affinity pair is poly dA-poly U. The method of claim 1, characterized in that the affinity pair is an antigenic antibody.