FI72146C - Procedure for Identifying Nucleic Acids. - Google Patents

Description

721 46

Method for identification of nucleic acids

The invention relates to a method for the identification of nucleic acids by hybridization in solution.

The method according to the invention is characterized in that two probes in the same solution are used in the hybridization reaction. The detector probe is labeled with a marker and a so-called clamp the probe is attached to a component that can act as a party affiniteetiparissa, the hybrid-formed foundation fastener 10 probe: the sample. Detector probe - the hybrid may be separated from other components of the hybridization mixture for the second party by means of an affinity pair.

Various hybridization methods have been used to identify nucleic acids. Examples include direct hybridization methods and layer hybridization methods. In direct hybridization methods, the nucleic acid sample is either in solution or attached to a solid support.

The nucleic acid to be identified is detected using a single labeled probe. Layer hybridization methods (ΓΙ 63596) use two separate probes to detect the identifiable nucleic acid from the sample solution. One of the probes used is labeled with a marker and the other is attached to a solid support.

We have developed a new hybridization method using two different probes but both probes are in the same solution phase. Because the sample involved in the hybridization and both probes are in the same solution phase, the hybridization reaction proceeds significantly faster than in layer hybridization, where one probe is attached to a solid support. In the method according to the invention, an incubation time of one hour is sufficient. In a single-stage layered hybrid-35 foundation (FI 63596), sufficient hybridization occurs only in 12 2 721 46 hours. When performing a two-step layer hybridization, a hybridization time of at least 24 hours is required (Dunn and Hassell, Cell 12, 23-36, 1977).

In the method according to the invention, two probes are preferably used. The probes are nucleic acid fragments sufficiently homologous to the nucleic acid to be identified. It is preferred, but not essential, that the probes be homologous to relatively close to 10 sites in the nucleic acid to be identified. The probes must not be homologous to each other.

Probes can be prepared synthetically, semi-synthetically, by recombinant DNA technology, or directly from nucleic acids isolated from nature. The probe may be bound to a suitable vector. It may contain vector elements or be completely free of vector elements.

The detector probe is labeled with suitable markers.

Various radioactive isotopes or radiolabeled compounds can be used as tracers. The label may also be fluorescent, luminescent, light emitting, enzymatically or immunologically detectable, etc. Examples include labels based on the affinity of biotin and avidin or streptavidin (Leary et al., PNAS 80, 4045-4049, 1983), lanthanide chelates (US lanthanide chelates). and U.S. 4,374,120), ferritin and hemic compounds, immunologically detectable haptens such as AAF and AIF (various acetoxyacetylfluorene derivative requirements (WO 8302286)). Protein-mediated identification is also possible. The method according to the invention is not dependent on the tracer used. All known markers suitable for nucleic acid hybridization are freely applicable to the method.

Il 72146 3

Attached to the fastener probe is a component that can act as a party in the affinity pair. Such affinity droplets are, for example, biotin-avidin or biotin-streptavidin, a heavy metal derivative thio group (Dale and 05 Ward, Biochemistry 14, No. 11, 2458-2469, 1975), various homopolynucleotides, for example poly dG - poly dc, poly dA - poly dT or poly dA - poly U. But other affinity pairs can also be used as long as the components have a sufficiently strong affinity for each other. Among the ligands and conjugates used in immunological methods, suitable affinity pairs can be found.

Prior to performing the hybridization reaction, the sample is treated so that the nucleic acids are released into the hybridization solution in a single-stranded form. The hybridization is performed in a hybridization mixture in which the nucleic acids, the detector probe and the attachment probe have been brought into a single-stranded form, if necessary. Various suitable buffers can be used as the hybridization solution. Hybridization occurs at a temperature in the range of 0-80 ° C, but it is preferred to use a temperature of 65 ° C. If the hybridization solution contains formamide (40-55%), a temperature of 37 ° C can be used. An hour is enough for the hybridization time.

Once hybridization has taken place, the solution is diluted, if necessary, so that the conditions are favorable for the affinity pair. The mixture is then brought into contact with the other party of an affinity pair which is coupled to a suitable carrier. The support can be, for example, an affinity chromatography column, a filter, a plastic or glass surface.

The material of the affinity carrier may be, for example, cellulose, latex, polyacrylamide, dextran or agarose. These materials can also be used as suspensions in 35 test tubes. It is also preferred to use test tubes with the other side of the affinity pair attached to the inner surface of the 4 72146. The choice of materials requires that a component with affinity for the component attached to the attachment probe be attached.

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If the sample contains nucleic acid for identification, hybridization results in an attachment probe: sample: detector probe hybrid. This hybrid adheres to the solid support during fractionation. The label 10 of the fraction adhering to the support can be measured by conventional methods directly from the support or from the solution eluted after elution.

Other systems, such as phase extract or magnetic fields, can be used in the fractionation instead of affinity chromatography.

In the following application examples, the method according to the invention is described in more detail. The method of the invention is not dependent on the nucleic acid-20 fragments used in the examples. It will be apparent to one skilled in the art how similar nucleic acid fragments can be prepared.

Example 1 Identification of adenovirus DNA from a cell lysate using homopolynucleotide 125

Purple-labeled recombinant phage mkTH 1206, a 30 Bgl II fragment of adenovirus (type 2) from position 42-45.3% of the adenovirus gene map, was used as a detector probe. This recombinant phage has been deposited in the strain collection Deutsche Sammlung von Mikroorganismen under number DSM 2827 and has a specific activity of 7 7 x 10 cpm / μg DNA. The probe is described in more detail in 35 Ranki et al., Gene 21., 77-85, 1983.

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The recombinant plasmid pkTH 1202, deposited in the strain collection Deutsche Sammlung von Microorganism under the number DSM 2824 and consisting of a BamHI D fragment of adenovirus (map position 29-42%) cloned into plasmid pBR322, 05 was used as a ligand probe. The recombinant plasmid pkTH 1202 (DSM 2824) was digested with the restriction enzyme Hae III and the poly A tail was ligated to the 3 'ends of the fragments using a terminal transferase enzyme. The tail length was measured by H-A incorporation. The average length was about 70 A residues. Prior to use, the fastener 10 was denatured by boiling. Adenovirus-infected A-549 cells were used as a sample. Infected cells were incubated for 2 hours. Cells were harvested and disrupted with 1% sodium dodecyl sulfate solution. The solution contained about 106 cells / ml. The viscosity was reduced by sonication.

Uninfected A-549 cells treated identically were used as controls. Prior to hybridization, the sample was boiled for 5 min. In 0.02 M NaOH, cooled to 0 ° C and neutralized with acetic acid.

For the assay, 500,000 cpm of detector probe, 50 ng of fixative DNA, and 10 sample were combined in a test tube. The volume was adjusted to 50 and 0.6 M sodium chloride, 0.06 M sodium citrate, 0.02 M sodium phosphate (pH 7.6) and 0.5% sodium dodecyl sulfate were used as buffer solution. Mixture 25 was incubated for 1 hour at 65 ° C.

After hybridization, the solution was cooled to + 20 ° C and slowly drained through a chromatography column containing 1 ml of oligo dT cellulose. The permeate was collected and drained again through the column. 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 attached DNA was finally cleaved with 1 ml of 0.02 M NaOH. This solution was recovered and its radioactivity determined.

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Result: I activity (cpm) 05 Sample: infected cells Control cells 5230 325 10

Example 2

Identification of Chlamydia trachomatis DNA using 15 bioti ini-streptavidin inia 125

I-labeled recombinant phage mkTH 1245 containing two BamHI-SalI DNA fragments from clone pkTH 1220, together linked to the M13mp8 vector, was used as a detector probe. Clone pkTH 1220 has been deposited in the strain collection Deutsche Sammlung von Microorganism under number DSM 2825 and is described in Palva et al., FEMS Microbiology Letters 23, 83-89, 1984.

The recombinant plasmid pkTH was used as a probe

1250. This plasmid consists of a 2.9 kb SalI-ClaI fragment from plasmid pkTH 1220 (DSM 2825) and vector pAT 153. Biotin molecules were covalently ligated to pkTH 1250 DNA using a known "nick translation" method 30 (Rigby et al. et al., J. Mol. Biol. 113, 237-251, 1977) and biotin-11-UTP as a substrate (Bethesda Research Laboratories, BRL). Fastener DNA was boiled in 10 mM Tris-Cl pH 7.6, 1 mM EDTA for 5 min before use.

The sample was the recombinant plasmid pkTH 1220 (DSM 2825).

7 72146 This plasmid contains approximately 10 kb of Chlamydia trachomatis-specific DNA ligated into the vector pBR322. The plasmid acts as model DNA, representing the genomic DNA of the bacterium. Prior to use, the plasmid was boiled for 5 min in 0.02 M NaOH, after which the solution was neutralized with acetic acid.

Streptavidin used as an affinity material was coupled to CNBr-activated Sepharoseert® (Axen et al., Nature 214, 1302-1304, 1967).

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For the assay, 500,000 cpm of probe, 50 ng of fixative DNA, and 10 ng of sample DNA were combined in a test tube. The volume was set at 20 and the buffer solution was the same as in Example 1. The control DNA was calf thymus DNA.

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The mixture was incubated for 60 min at 65 ° C. Then, 500 μl of a buffer solution consisting of: 0.1 M Tris-Cl pH 7.5, 0.1 M NaCl, 2 mM MgCl 2, 0.05% Triton x-100 was added. The solution was finally fractionated on a Streptavidin-Sepharos column so that the biotinylated DNA adhered to the Streptavidin but other DNA passed through the column. The radioactive probe adhered only as a result of hybrid formation. Adherent radioactivity was determined by transferring the entire column to a gamma counter counting tube.

25 125

Result: I activity (cpm) 30 Sample: 10 ng pkTH 1220 10 ng control DNA 1350 115 8 721 46

Example 3

Identification of plasmid pBR322 DNA using antigen-antibody pair, 05

A derivative of plasmid pBR322 from which the PstI-SalI fragment (3613-3651) has been removed was used as a detector probe. The plasmid was labeled with photobiotin using a known method (Forster et al., Nucleic Acids Res. 13, 745-761, 1985) and 10 commercial reagents.

Recombinant phage M13mp111 DNA to which the PstI-SalI fragment of plasmid pBR322 was ligated was used as a probe probe. DNA was sulfonated using a known method (Orgenics Ltd. Yavne, Israel).

The sample was E. Coli HB101 to which plasmid pBR322 had been ligated. The amount of plasmid pBR322 was increased using chloramphenicol amplification (Maniatis et al. Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory 1982). Bacterial cells were disrupted with lysozyme and boiled in NaOH as described in Palva, J. Clin. Microbiol. 1J3, 92-100, 1983.

Monoclonal antibodies were attached to the wells of polystyrene microtiter plates by standard methods (McKearn, Hybridomas: A New Dimension in Biological Analyzes, ed. Kennett et al., Plenum Press, 1980).

For the assay, 5x10 6 disrupted E. coli cells (both airless and ligated into pBR322), 100 ng of fixer DNA, and 100 ng of detector DNA were pooled into a hybridization solution adjusted in volume to 50. The hybridization conditions were the same as in Example 1 except that 5% polyethylene glycol (PEG 6000) was added and the sodium dodecyl sulfate content was 0.1%.

After hybridization, the solution was diluted to 250 with the addition of 0.02 M sodium phosphate (pH 7.6), after which the solution was transferred to a well of a microtiter plate to which the antibody was attached. It was then incubated for 2 hours at 37 ° C. The well of the microtiter plate was then washed with 0.15 M sodium chloride, 0.02 M sodium phosphate, 0.05% Triton-10 X-100 solution.

The detector probe was detected by the addition of streptavidin (BRL), washing, addition of biotinylated alkaline phosphatase (BRL) and washing as described in Leary et al., Proc.

15 Natl. Acad. Sci. USA, 80, 4045-4049, 1983. Finally, 250 μl of paranitrophenyl phosphate solution (35 mg / ml, Sigma) in diethanolamine buffer (pH 10) was added. After 60 minutes, the reaction was stopped and the absorbance was measured at 410 nm.

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Result: Δ 410 nm 25 Sample: cells to which Control cells were attached pBR322 (without pBR322)> 2 0.1

Claims (7)

A method for identifying nucleic acids by a hybridization method using two 05 probes in the same solution phase, of which e.g. the detector probe is labeled with a suitable, measurable tracer characterized in that the so-called the fastener is attached to the probe component of the affinity pair can act as a party, wherein the hybridization reaction, the resulting fastener probe: Sample: 10 detektorikoe-tin-hydride can be isolated from other components of the hybridization mixture for the second party attached to the solid support by means of an affinity pair. Process according to Claim 1, characterized in that the affinity pair is biotin-avidin or biotin-streptavidin. The method of claim 1, characterized in that the affinity pair is a homopolynucleotide pair. 20 Method according to Claims 1 and 3, characterized in that the affinity pair is poly dC -poly dG. Method according to Claims 1 and 3, characterized in that the affinity pair is poly dA -poly dT. Method according to Claims 1 and 3, characterized in that the affinity pair is poly dA -poly U. The method of claim 1, wherein the affinity pair is an antigen-antibody.