LU86238A1 - METHOD FOR THE IDENTIFICATION OF NUCLEIC ACIDS - Google Patents

Description

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The present invention relates to a method - for the identification of nucleic acids by means of solution hybridization. The method according to the present invention is characterized by the use of at least two probes in the hybridization reaction. A mark is attached to the detection probe and to the other probe, the so-called capture probe, is fixed a component having an affinity for another component, by means of which the hybrid probe capture: target nucleic acid: probe Detection resulting from the hybridization can be separated from the other components present in the hybridization mixture.

Various hybridization methods have been used for the identification of nucleic acids. There may be mentioned, for example, direct hybridization methods and sandwich hybridization methods. In hybrid- methods? direct specimen, the nucleic acid specimen is either in solution or attached to a solid support. The nucleic acid to be identified is identified using a labeled probe. In sandwich hybridization methods (US 4,486,539), two separate probes are used by means of which the nucleic acid to be identified is detected in the sample solution. The detection probe is labeled with a labeling substance and the other probe is attached to the solid support.

A new hybridization method has been developed in which two different probes are used and these two probes are in the solution phase. Because the target nucleic acid involved in hybridization and both probes are in the same phase in solution, the hybridization reaction is considerably faster than in a sandwich hybridization, in which a probe is attached to a support solid. In the method according to the present invention, an incubation period of one hour is sufficient. In the single-stage sandwich hybridization (US 4,486,539), sufficient hybridization is not obtained in less than 12 hours. When performing two-stage sandwich hybridization (Dunn "2 & Hassel, Cell. Vol. 12 pp. 23-36, 1977), a hybridization time of at least 24 hours is required.

Advantageously, at least two probes are used in the method according to the present invention. Probes 5 are nucleic acid fragments 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 one another in the nucleic acid to be identified. The probes must not overlap each other.

The probes can be prepared by synthesis, semi-synthesis, by recombinant DNA techniques or from nucleic acids directly isolated from nature. Probes are also commercially available from several sources. A probe can be linked to a suitable vector. It may contain parts of a vector or be completely devoid of these.

The detection probe is labeled with a suitable labeling agent. Various radioactive isotopes or radioactively labeled compounds can be used in this connection. The labeling substance may be fluorescent, luminescent, emit light, be demonstrated by an enzymatic or immunological route, etc. Mention may be made, for example, of marks based on biotin and avidin or streptavi-25 dine, lanthanide chelates, ferritin and heme compounds and haptens which can be demonstrated immunologically, such as acetoxyacetylfluorene derivatives (WO 8302286). Identification can also be done through proteins. The method according to the present invention does not depend on the marking used. All well known labeling substances suitable for nucleic acid hybridization or those which will be further developed can be freely applied in this method.

35 We attach to the other probe, the so-called cap probe! 3 ture, a component with an affinity for another component. Suitable affinity pairs include, for example, biotin-avidin or streptavidin, heavy metal derivatives - thio groups, various homopolynucleotides, such as poly dG - poly dC, poly dA - poly dT, and poly dA - poly U Other affinity pairs can also be used provided that the components have a sufficiently strong affinity for each other. Suitable affinity pairs are found among ligands and conjugates used in immunological methods.

Before performing the hybridization reaction, the specimen is treated to release 8 target nucleic acids in a single stranded 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 have been rendered, if necessary, in a single stranded form. Various suitable buffers can be used as hybridization solutions. Hybridization takes place in a temperature range of 0 to 80 ° C, but it is advantageous to work at 65 ° C. If the hybridization solution contains formamide (40 to 55%), a temperature of 37 ° C can be used. One hour is sufficient for the duration of hybridization.

When the hybridization has taken place, the solution is diluted, if necessary, to make the conditions advantageous for the affinity pair: the mixture is then brought into contact with the other member of the affinity pair. Affinity chromatography columns, filters, plastic surfaces, and glass surfaces, etc. can be used to capture the hybrid capture probe: target nucleic acid: detection probe.

The material serving as support for the affinity column can be for example a cellulose, a latex, a polyacrylamide, a dextran or agarose. These materials can also be used in suspensions in a test tube. It is also advantageous to use test tubes on the inner surface of which the other component of the affinity pair is fixed. That it is possible to fix on this material, a component having an affinity for the component 5 attached to the capture probe, constitutes a prerequisite for the material chosen.

If the specimen contains the nucleic acid to be identified, hybridization will provide a capture probe hybrid: target nucleic acid: detection probe. This hybrid 10 adheres to the support during the fractionation. The substance marking the fraction sticking to the support can be measured by conventional methods, directly from the support or after elution, from the eluted solution.

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

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

In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows.

The invention will be better understood with the aid of the additional description which follows, which refers to examples of implementation of the method which is the subject of the present invention.

It should be understood, however, that these examples of implementation are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

EXAMPLE 1

Identification of adenovirus DNA from a cell lysate with homopolynucleotide acid.

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The detection probe used was the recombinant phage mKTH 1206 labeled with H5jr which contains a Bgl II fragment of the adenovirus genome at positions 42-45.3% on the genetic map of type 2 d1 adenovirus. The recombinant pha-5 ge was deposited in the collection of cultures Deutsche Sammlung von Mikro-Organismen under the number DSM 2827. Its specific activity is 7 x 107 cpm / ^ / g of DNA. The probe is further described by Ranki et al. 1983, Gene 21, 77-85.

The capture probe used was the recombinant plasmid pKTH 1202, which has been deposited in the Collection of Cultures Deutsche Sammlung von Mikroorganismen under the number DSM 2824 and comprises a BamHI D fragment of adenovirus (position on the map: 29-42% ), cloned into plasmid pBR 322. The recombinant plasmid pKTH 1202 (DSM 2824) was cut using the restriction enzyme Hae III and a poly-A tail was linked to the 3 'ends of the fragments with a terminal tranferase enzyme. The tail length was measured by incorporating ^ h-A. The length was on average about 70 residues A. The capture probe was denatured by bringing it to a boil before use. The sample used consisted of A-549 cells infected with the adenovirus. The cells were incubated for 21 hours after infection. The cells were collected and lysed with a 1% solution of sodium dodecyl sulfate.

The solution contained about 10 ^ cells / ml. Its viscosity was lowered by ultrasonic treatment. Uninfected and identically treated A-549 cells were used as a control. Before hybridization, the specimen was boiled for 5 minutes in 0.02 M NaOH, cooled to 0 ° C and neutralized with acetic acid.

To carry out the test, a detection probe (500,000 cpm), 50 ng of capture DNA probe and 10 μl of specimen were combined in a test tube. The vo-35 lume was adjusted to 50 ytil and the buffer solution used comprised 0.6 M

* 6 sodium chloride, 0.06 M sodium citrate, 0.02 M sodium phosphate, (pH = 7.6) and 0.5% sodium dodecyl sulfate. The mixture was incubated for 1 hour at 65 ° C.

After hybridization, the solution was cooled to 5 + 20 ° C and flowed slowly through a chromatography column having 1 ml of oligo dT-cellulose. The solution thus collected was again sent to the column. The column was then washed with 20 ml of a solution which contained 0.15 M sodium chloride, 0.015 M sodium phosphate (pH = 7.6) and 0.5% sodium dodecyl sulfate. Finally, the fixed DNA was detached using 1 ml of 0.02 M NaOH. This solution was recovered and its radioactivity determined.

15 Result: Activity of the 125 d (cpm)

Target nucleic acid: _ .. n ..

Infected 'cells' - Control cells 5230 325 20 EXAMPLE 2

Identification of Chlamydia trachomatis DNA using biotin-streptavidin

The detection probe used was the 125jf-labeled recombinant phage mKTH 1245 which contains two BamHI-SalI DNA fragments from the clone pKTH 1220, which are linked together to the vector M13np8. The clone pKTH 1220 was deposited in the Collection of Cultures Deutsche Sammlung von Mikroorganismen under the number DSM 2825 and it is described in Palva et al., FEMS Microbiology Letters 23 pp. 83-89, 30 1984.

The capture probe used was the recombinant plasmid pKTH 1250. This plasmid comprises a Sall-ClaI fragment of 2.9 kb originating from the plasmid pKTH 1220 (DSM 2825) and the vector pAT153.

Biotin molecules have been covalently linked to the DNA of * 7 pKTH 1250, according to the known nick-translation method (Rigby et al. J. Mol.Biol. 113, pp. 237-251, 1977), with biotin-ll-UTP as a substrate (Bethesda Research Laboratories). The DNA capture probe 5 was brought to the boil in a buffer comprising 10 mM Tris-Cl, pH = 7.6 and 1 mM EDTA, for 5 minutes before using it.

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, linked to the vector pBR 322. The plasmid serves as model DNA, representing the DNA of the genome of the bacterium. Before use, the plasmid was heated to boiling for 5 minutes in 0.02 M NaOH, then the solution was neutralized with acetic acid.

Streptavidin, used as the affinity material, was fixed on Sepharose activated by CNBr (Pharmacia) according to Axen et al. Nature 214 pp. 1302-1304, 1967.

To carry out this test, the probe (500,000 cpm), 50 ng of capture DNA probe and 10 ng of target DNA were combined in a test tube. The volume is adjusted to 20 μΐ and the buffer solution was the same as that used in Example 1. The control DNA was calf thymus DNA.

The mixture was incubated for 60 minutes at 65 ° C.

Then, 500 µl of a buffer solution having the following composition was added: 0.1 M Tris-Cl pH 7.5, 0.1 M NaCl, 2 mM MgCl2r 0.05% Triton x- 100. Finally, the solution was fractionated in a 0.2 ml Streptavidin-Sepharose column. The column was washed with 10 ml of the aforementioned tam-30 pon and 10 ml comprising 0.015 M sodium chloride, 0.015 M sodium phosphate (pH = 7.6), 0.5% sodium dodecyl sulfate (50 ° C). The biotinylated DNA therefore adhered to streptavidin, while the other DNA crossed the column. The radioactive probe only adhered after hybrid formation. The radio has been determined.

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8 activity captured by transferring the entire column to the counting tube of the gamma counter.

Activity of 125 d (Cpm) 5 - Result: Target nucleic acid: .n Ί,, - 10 ng of pKTH 1220 10 n9 of control DNA 1350 115 10 EXAMPLE 3

Identification of the DNA of the plasmid pBR322 using an antigen antibody pair

The detection probe used was a derivative of plasmid pBR322 (commercially available from several sources), from which the Pstl-SalI fragment (3613-651) was removed. The plasmid was labeled with photobiotin using a known method (Förster and Coll. Nucleic Acids Res. 13, pp. 745-761, 1985) and industrial reagents (Bresa,

Adelaide, Australia).

The capture probe used was DNA from a recombinant phage Ml3mpl1, into which the Pstl-SalI fragment of pBR322 had been introduced. The DNA was sulfonated according to a known method (Organics Ltd. Yavne, Israel).

The specimen was 1Έ. coli HB101 carrying the plas-25 mide pBR322, the quantity of which had been increased by amplification with chloramphenicol (Maniatis and Coll. Molecular cloning, A laboratory manual, Cold Spring Harbor Laboratory 1982). Bacterial cells were lyzed with lysozyme and then boiled in NaOH, as described in the publication by Palva, J. Clin. Microbiol. 18, pp. 92-100, 1983).

Antisulfone monoclonal antibodies have been used to coat polystyrene microtiter wells, according to conventional methods (McKearn, in Hybridomas: A 35 New Dimension in Biological Analyzes, ed. Kennett et al., 9 *

Plenum Press 1980).

. To carry out this test, 5.10 ^ E cells were combined. coli lysed (both with and without pBR322) with 100 ng of each of the capture and detection probes in 50 μΐ 5 of hybridization mixture. The conditions were those of Example 1, except that 5% of polyethylene glycol (PEG 6000) was added to the mixture and that the concentration of sodium dodecyl sulfate was 0.1%. After hybridization, the solution was diluted to 250 µl by adding 0.02 M sodium phosphate (pH = 7.6), then the solution was transferred to the antibody coated microtiter well. An incubation was then carried out for 2 hours at 37 ° C. The well 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. The presence of the detection probe was made visible by adding streptavidin (Bethesda Research Laboratories BRL) by washing, adding biotinylated alkaline phosphatase (BRL) and washing as described by Leary et al. Proc. Natl. Acad. Sei. USA 80, 20 pp. 4045-4049, 1983. Finally, 250 µl of a 36 mg / ml solution of paranitrophenyl phosphate (Sigma) in diethanolamine buffer (pH 10) was added. Sixty minutes later, the reaction was stopped and the absorption rate measured at 410 nm.

25 _ Result: A41Q nm

Target nucleic acid: _ ...

,,. ^ Cells without pBR322 cells with pBR322 ^ 30> 2 0.15

As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the scope or the scope of the present invention.

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Claims (4)

VP ia, 10 1) Method for the identification of nucleic acids, characterized in that at least two probes which are in the same phase in solution are used in a hybridization method, the detection probe being labeled with a detectable labeling substance and a member of an affinity pair attached to the capture probe, and the capture probe hybrid is isolated: target nucleic acid: detection probe formed in the hybridization reaction by means of the other member of the affinity pair. 2 °) Method according to claim 1, characterized in that the affinity pair comprises biotin and streptavidin or biotin and avidin. 3) Method according to claim 1, characterized in that the affinity pair is a pair of homopoly-nucleotides. 4 °) Method according to claims 1 and 3, characterized in that the affinity pair is poly dC - poly dG. 5) method according to claims 1 and 3, characterized in that the affinity pair is poly dA - poly dT. 6 °) Method according to Claims 1 and 3, characterized in that the affinity pair is poly dA - poly U. 25 7 °) Method according to Claim 1, characterized in that the affinity pair is a derivative of heavy metal - a thio group. 8 °) Method according to claim 1, characterized in that the affinity pair comprises an antigen - an antibody.