WO1987005334A1

WO1987005334A1 - Nucleic acid detection using particle agglutination

Info

Publication number: WO1987005334A1
Application number: PCT/US1987/000414
Authority: WO
Inventors: Malcolm L. Gefter; Christie A. Holland
Original assignee: Angenics, Inc.
Priority date: 1986-03-04
Filing date: 1987-03-03
Publication date: 1987-09-11
Also published as: EP0259453A1; CA1313485C; JPS63502875A

Abstract

A method of detecting, identifying and/or quantitating nucleic acids in a sample through determination of agglutination or inhibition of agglutination of suspendable particles having nucleic acids bound thereto. The nucleic acids can be bound directly to the particle surfaces or attached through a spacer molecule which can, in turn, be either covalently bound or adsorbed to the particle surfaces. The suspendable particles are small enough to remain in suspension and will generally have a large particle size relative to the molecular weight of the DNA or RNA attached to the surfaces. The presence or absence of nucleic acid sequences in a sample is determined by detecting agglutination or inhibition of agglutination of particles having bound thereto nucleic acid sequences complementary to those of interest in the sample.

Description

NUCLEIC ACID DETECTION USING PARTICLE AGGLUTINATION

Description

Technical Field This invention is in the field of ligand assays and in particular relates to the detection and quantification of nucleic acid sequences through nucleic acid hybridization.

Background Information Nucleic acid hybridization is the basis for many methods used for the detection and identifica¬ tion of nucleic acids in a sample. Hybridization is the process by which a single stranded nucleic acid (i.e., DMA or RNA) recognizes its complementary strand and hydrogen bonds to it, forming a double stranded molecule. That is, when single stranded nucleic acids are combined under appropriate condi¬ tions, complementary base sequences pair and. double- stranded hybrid molecules are formed. In nucleic acid hybridization assays (e.g.,

DNA-DNA, DNA-RNA) , it is often the case that sample DNA or RNA is attached to a solid support (e.g., a cellulose nitrate filter) by simply allowing it to adhere to the support. A labelled probe DNA or RNA is then added under conditions appropriate for hybridization of complementary sequences to occur. The presence of sequences complementary to the probe sequence is determined by detecting binding of the labelled probe to bound (sample) DNA or RNA. Attachment of DNA to a solid support can be accomplished by non-specific physical adsorption of single stranded nucleic acid (e.g., to nitrocellu¬ lose papers) and by chemical bonding (e.g., to agarose/Sepharosef a inoethyl-Sepharose Sephadexes cellulose) .

Nucleic acid hybridization provides a very sensitive and specific approach to detecting and identifying nucleic acids in samples. However, methods presently available require enzyme - or radioactive tracer - labelled nucleic acid probes, time-consuming^' procedures and/or sophisticated equipment. Presently, nucleic acid hybrids are detected by observing a change in the absorbance of a DNA solution; by physically isolating hybridized DNA from nonhybridized DNA using chromatography or hydroxy patite and quantitating the hybridized DNA; or capturing the hybridized DNA on nitrocellulose. Generally, these methods require labeled nucleic acids because although a nucleic acid sequence will anneal only with its complementary sequence, the presence of hybrid double stranded molecules is undetectable unless the probe is labelled. For example, nucleic acid sequences are often radio- actively labelled using phosphorous 32 ( 32P) , which can be introduced into DNA molecules as phosphate groups while they are being synthesized by host bacteria or by an _irι vitro reaction. Radioactively labelled nucleic acid sequences are widely used, but radioactive material can pose a risk to the user.

Such materials typically have short half-lives and, therefore, limited shelf lives. In addition, expensive, sophisticated equipment is necessary for their detection.

In European Patent Office (EPO) Application 0,130,523, Dattagupta and Crothers describe a solid support for nucleic acids and an immobilized nucleic acid probe capable of hybridizing with complementary nucleic acids. The solid support, to which a nucleic acid can be bound by irradiation, is des¬ cribed as comprising a solid substrate which has reactive groups; a photochemically reactive inter- calator compound or other ligand capable of binding nucleic acids; and a divalent radical chemically linking the solid and the nucleicacid binding ligand. Upon photoactivation, the ligands chemi- cally link with nucleic acids. Specifically, the solid substrate is nitrocellulose paper having hydroxyl groups and linked by a bifunctional reagent to an amino-substituted compound, which in turn is photochemically linked to a nucleic acid. The resulting immobilized nucleic acid is described as being useful in hybridization assays in which the support with coupled DNA is mixed with an unknown (possibly containing sequences complementary to that on the support) and a detection (labelled) probe. Testing the solid support for presence of a label (e.g., radioactivity) shows whether hybridization has occurred or not (and thus whether complementary DNA is present) .

In EPO Applicaton 0,130,515, Dattagupta et a_. describe a method for detecting the presence in a sample of a particular nucleic acid sequence which involves dual nucleic acid hybridization. A sample containing unknown DNA is mixed with two nucleic acid probes which are complementary to two nonover- lapping portions of the nucleic acid sequence to be detected. One probe is labelled and soluble in the sample and the other probe is fixed to a solid support (e.g., nitrocellulose). The mixture is allowed to stand under hybridizing conditions; hybridization to both probes by DNA in the sample occurs only if it contains sequences complementary to both probes. Separating the dual hybridization product (by separating the solid support) and detecting the label attached to it is said to provide a method of determining the presence in a sample of the DNA sequence of interest. In U.S. Patent 4,486,539, Ranki and Soderlund describe a kit for use in detecting and identifying viral or bacterial nucleic acids. The one-step sandwich hybridization procedure on which the kit is based requires two nucleic acid fragments from the genome of the microbe to be identified, which have no sequences in common. One fragment is fixed to a solid carrier (e.g., a nitrocellulose filter) and the other is labelled. Contact between nucleic acids to be identified and nucleic acids on the solid carrier results in annealing of complementary base pairs to form a hybrid. Annealing the second (labelled) fragment to the fragment to be identified results in labelling of those fragments formed on the solid support and thus allows their detection and quantification.

In Patent Cooperation Treaty (PCT) WO84/02721, Kohne describes a method for detecting and quantifying bacteria and viruses containing RNA. After the nucleic acids in a sample and a marked probe (radioactively labelled nucleic acid sequences complementary to the RNA of the organism to be detected) have been incubated under hybridization conditions, the degree of hybridization with the marked probe is measured. The method is described as being useful for in solution hybridization or hybridization with an immobilized nucleic acid probe.

At the present time, there is a need for a method of detecting the presence of nucleic acid sequences in biological samples which has the specificity of nucleic acid hybridization tech- niques, but does not require the use of radioactive materials, time-consuming preparation and sophisti¬ cated equipment.

Best Mode of Carrying Out the Invention

The method of this invention has very broad application, both in terms of the types of samples for which it is useful and the types of organisms which can be detected in such samples. The nucleic acid content of any type of biological sample (e.g., blood and other tissues; urine; and foodstuffs such as milk) can be determined using the present inven¬ tion. The presence in biological samples of bac¬ teria and viruses can be detected using particle agglutination. In addition, because bacteria have common nucleic acid sequences, as well as sequences specific to a strain or class within the species, it is possible to detect all bacteria in a sample by using a shared nucleic acid sequence or to detect specific bacteria by using a nucleic acid sequence unique to that strain or class.

Disclosure of the Invention The present invention is based on the discovery that nucleic acid segments attached to a suspendable solid support, such as latex particles, and comple¬ mentary nucleic acid segments in solution will hybridize and cause particle agglutination. The nucleic acid segments, which can be either DNA or RNA, thus initiate particle agglutination (i.e., cause particles to agglutinate) .

The invention described herein is a method of detecting, identifying and/of quantitating nucleic acids in a biological sample, as well as particles having nucleic acids bound thereto. The nucleic acids can be either bound directly to the particle surfaces or are attached through a spacer molecule which can, in turn, be either covalently bound or adsorbed to the particle surfaces. In the method of this invention, nucleic acid sequences are used either to produce agglutination of inert particles having bound thereto nucleic acid sequences comple¬ mentary to nucleic acid sequences to be detected in the sample or to interfere with agglutination of such particles.

That is, if a sample contains nucleic acid sequences complementary to those attached to the solid support, hybridization will occur and cause particle agglutination. Alternatively, inhibition of agglutination can be used to detect the presence of nucleic acid sequences of interest in a sample. In this case, two different nucleic acid sequences (e.g., + and -) are attached to the solid support; that is, some of the solid support particles have (+) strands attached to them and others (-) strands. If the sample contains nucleic acids complementary to either of the attached sequences, agglutination of the solid support is inhibited. In either case, detection of the degree of agglutination can be carried out visually or by another method known in the art. The degree of agglutination is indicative of the extent of hybridization of complementary nucleic acid sequences, which is, in turn, indica¬ tive of the presence of nucleic acid sequences in the sample.

As a result of this discovery, it is possible to detect, identify, quantify and/or isolate nucleic acids of interest in biological samples (e.g., body fluids, tissues, foodstuffs) and other samples using techniques and equipment which do not require highly skilled personnel for successful operation. An important characteristic of the use of in solution or in suspension hybridization as described herein is that the reactants are not immobilized as, for example on large particles or filter membranes, and, as a result, hybridization occurs more rapidly because the reactive (hybridizable) sites can diffuse together more readily. The resulting speed and the specificity with which nucleic acid se- quences of interest can be detected (e.g., in diagnosing infectious diseases, detecting bacterial contaminants in foodstuffs) are important advantages of the present invention. In addition, no separa¬ tion of bound and unbound phases is required before the reaction mixture can be evaluated for hybridiza¬ tion. Separation of the bound and unbound phases is required in other methods of performing hybridiza¬ tion. In addition, because nucleic acid hybridiza¬ tion is highly specific - that is, a nucleic acid sequence will hybridize only with a complementary sequence - the particle agglutination method of the present invention is a very reliable means of

"selecting" nucleic acid sequences of interest from among the numerous sequences found in biological samples. The nucleic acid sequences to be detected can be characteristic of or shared by all members of a bacterial or viral species; as a result, all bacteria (or viruses) in a sample can be detected. This is particularly useful, for example, in detect¬ ing all bacteria in a foodstuff (e.g., a complete plate count for bacteria present in a milk sample) . Alternatively, the nucleic acid sequences to be detected in the foodstuff can be specific to members of a given strain or class.

Brief Description of the Drawings

Figure 1 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which nucleic acid sequences are bound to particles.

Figure 2 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which probe nucleic acid sequences are bound to particulate support material and complementary nucleic sequences are bound to other particles.

Figure 3 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which nucleic acid sequences comple¬ mentary to those to be detected in the sample are bound to particles.

Figure 4 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which probe nucleic acid sequences are bound to inert support material and complementary nucleic acid sequences are bound to inert support material of relatively smaller size.

Figure 5 is a block diagram of one embodiment of the method of detecting nucleic acid sequences in a sample in which sample nucleic acid sequences are bound to particles. Detailed Description of the Invention

According to the method of the present inven- tion, detection of nucleic acids in a sample using agglutination of particles to which nucleic acid sequences are attached can be carried out directly or indirectly.

In the direct method, an example of which is represented in Figure 1, nucleic acid sequences complementary to those of interest are attached to latex particles and contacted with an appropriately treated sample (i.e., one which has been treated by methods known to those skilled in the art to make the nucleic acid sequences available for hybridiza¬ tion) . Hybridization of the bound nucleic acids with those in the sample causes particle agglutina¬ tion; agglutination does not occur if the sequences of interest are not present in the sample. For example, if a solution containing one set of latex particles having nucleic acid sequences ABC and a second set of latex particles having nucleic acid sequences DEF is contacted with a sample containing the nucleic acid sequence complementary

I I I t f I to ABC and DEF (designated A B C D E F ) , hybridiza¬ tion of the complementary sequences will occur. Particle agglutination will result. If the sample does not contain the complementary sequence, hybridization and particle agglutination will not occur.

In the indirect method, an example of which is represented in Figure 2, the presence in a sample of the nucleic acid egments of interest inhibits agglutination of particles bearing complementary nucleic acid sequences. Two sets of nucleic-acid- bearing latex particles are used. For example, latex particles bearing strands of DNA (designated +) and other latex particles bearing DNA strands complementary to (+) strands (designated -) are contacted with a sample to be analyzed for the presence of a particular DNA sequence. The DNA strands attached to some of the latex particles are complementary to the DNA to be detected in the sample. If the sample tested (e.g., blood) contains the DNA sequence of interest, that sequence will hybridize to its complement (which is bound to latex particles) and prevent hybridization of the particlebound (+) and (-) DNA segments. Agglutination of the particles will thus be inhibited or prevented and its absence will indicate that the DNA sequence sought is present in the sample.

Nucleic Acid Segments

Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) to be used as the probe can be attached to a solid support or included in a solution which is contacted with the sample DNA-particle complex. It can be any gene or nucleic acid sequence (DNA or RNA) of interest. For example, it can^"be sequences complementary to a ribosomal RNA sequence that is present in either all bacteria (inclusive) or sequences complementary to a ribosomal RNA sequence that is characteristic of a single type of bacteria (exclusive) . If an inclusive ribosomal nucleic acid is used as the probe, contact with a sample contain¬ ing RNA from any bacteria will result in hybridi¬ zation and agglutination of the particles. An exclusive ribosomal probe will only agglutinate when RNA from a specific group of bacteria is present. Because RNA is rapidly degraded when an organism dies, only viable cells will be detected. In the first case, all viable bacteria in a sample will be detected; in the second, only the one type of viable bacteria having RNA complementary to the probe selected will be detected. This could be used, for example, in detecting viable bacteria in a milk sample pretreated to release bacterial RNA; it provides a simple, rapid and specific alternative to the standard plate count presently used in the dairy industry. As described in greater detail in Example 1, any single strand of DNA and its complement can be attached to the solid support. In this case, one clone provides one DNA strand (designated +) and a second clone provides the second (or complementary) DNA strand (designated -) . For example, the src gene and the beta globin gene can be attached to the latex particles.

The nucleic acid sequences attached to the solid support can be of almost any length. It has been demonstrated that a stable bond or hybrid is formed when the nucleic acid has five or more base pairs. Generally, therefore, the nucleic acid sequences bound to the solid support will be five or more bases in length.

Nucleic acid, sequences to be attached to the solid supports for use as probes can be obtained by cloning of isolated DNA or RNA segments according to methods well known in the art. See, for example, Maniatis, T. et al. , Molecular Cloning - A Labora¬ tory Manual, Cold Spring Harbor Laboratory (1982) . For example, any restriction enzyme, such as EcoRI, can be used to produce a desired DNA fragment for use as a probe. Bacterial DNA can be cleaved at selected sites on either side of the DNA fragment of interest; the resulting fragments of interest can be isolated from other fragments (and thus purified) electrophoretically. The isolated DNA fragments can then be amplified by inserting them into a plasmid or a bacterial virus (bacteriophage) , which is in turn inserted into an appropriate bacterial host cell. As the cells containing the plasmid prolifer¬ ate, the plasmid also replicates, producing many copies of the DNA fragment to be used as a probe. After the cells have been allowed to proliferate, the hybrid plasmids are isolated and purified, resulting in the isolation of many copies of the DNA fragment. Nucleic acid sequences to be attached to the solid supports could also be obtained synthetically or, if they occur in nature in sufficient quanti¬ ties, simply by isolation and purification.

It is also possible to attach nucleic acid sequences from an appropriately treated biological sample to the solid supports. In this case, the nucleic acid sequences in solution would be those complementary to the sequences of interest in the sample; that is, the probe sequences would be in solution.

Solid Support Materials

The solid support to which the DNA or RNA is attached can be essentially any insoluble material to which the DNA or RNA can be covalently attached or irreversibly adsorbed; that is, the material must be reactive with the DNA or RNA or must adsorb a substance which can be covalently linked to the DNA or RNA. The solid support can be, for example, latex, charcoal, colloidal gold, bentonite or glass. in addition, silica gel, controlled pore glass, red blood cells and liposomes can be used. In fact, any such particles to which nucleic acids can be attached can be employed in the application of the present invention. Nucleic acids have reactive amino functional groups, as well as terminal phos¬ phate groups and reactive hydroxyl groups in the sugar "backbone". In addition, ribose in RNA can be partially cleaved oxidatively (e.g., with periodate, as in Fischer, U.S. Patent 4,264,766) to produce a reactive aldehyde function. These re¬ active groups on the nucleic acid can be reacted with reactive groups on latex or other particles.

Glass or other particles can be derivatized to form reactive functional groups (see, for example, Weetall, U.S. Patent 3,652,761; Koster, et al. , Tetrahedron Letters 2_\ 747 (1983)) capable of reacting with nucleic acids and proteins. Nucleic acids can also be linked directly to polystyrene (e.g., Ito, et al. ; Nucleic Acid Research 10; 755 (1982)). The solid support need not have a particu¬ lar shape (configuration) but will often be spheri- cal. It must be small enough to remain in suspen¬ sion and will generally have a large particle size relative to the molecular weight of the DNA or RNA probe (e.g. , less than 500 microns) .

Binding of Probe to Solid Support Nucleic acid sequences, either DNA or RNA, are attached to a solid support. For example, they can be covalently bound; the binding can occur randomly t along the length of the DNA or RNA, at the 5 end or at the 3 end. The sequences can also be bound to the solid support through a protein or other spacer material. Previously, a carbodiimide coupling reaction has been used by those in the field for the purpose of covalently linking DNA to solid supports, such as agarose (Sepharose ®) , ammoethyl-Sepharose Sephadexes and cellulose. Allfrey, V.G. and A.

Inoue, In: Methods in Cell Biology 1_8: 253-270

(1978) . This reaction produces a phosphodiester bond between the phosphoryl group at the 5' end of the DNA and hydroxy1 groups on the solid matrix.

In one embodiment of this invention, nucleic acid sequences bound to solid supports are linked to latex particles along the length of the fragments.

One method which can be used for covalent attachment is described in detail in Example 1. Nucleic acid sequences bound to latex particles using this method appear to be covalently attached randomly along their length. The method in Example l'is based on the method for forming an amide bond between latex and protein described by Dor an in U.S. Patent 4,045,384. There are three steps to the procedure. First, carboxylated latex particles are activated; that is, an active ester is formed at the latex surface through reaction with a water-soluble N-hydroxy compound (e.g., N-hydroxybenzotriazole or N-hydroxysuccinimide) and a water-soluble carbodi- i ide (e.g., l-cyclohexyl-3-[2-morpholinoethyl]- carbodiimide methyl-p-toluene sulfonate (CMC) ) . These materials are combined, stirred and cooled (e.g., to about 2°-50C) . As a result, an anhydride link is formed between the carboxyl group of the latex particle and the hydroxy nitrogen (NOH_. group of the hydroxybenzotriazole. Next the reaction mixture containing the hydroxybenzotriazole deriva¬ tive attached is cleaned by dialysis. In this way, unchanged reactants (e.g., carbodiimide and by¬ products) are removed. Single stranded DNA (or RNA) can be covalently bonded to the activated latex particle by combining the latex-hydroxybenzotriazole complex with single stranded DNA (or RNA) and agitating the combination (e.g., by rocking) at room temperature. The product of this procedure has been shown to be single stranded DNA covalently attached to the latex particle at random along the length of the DNA. Similarly, this can also be done using RNA.

As mentioned, nucleic acids can also be at¬ tached to particles by being attached to other "bridging" molecules which can be adsorbed or covalently attached to latex. Nucleic^'acids can be covalently linked to polysaccharides through one of many known reactions (Allfrey, V.G. and A. Inoue, In: Methods in Cell Biology 18: 253-270 (1978)). Polysaccharides can be partially oxidized to yield reactive aldehyde groups which can, in turn, be reacted with particles having suitably reactive groups such as amino groups (e.g., amino functional latex) .

DNA can be covalently attached to proteins by numerous methods (Nucleic Acid Research, 12: 3435

(1984)) and the proteins attached to latex or other particles using well known techniques. Synthetic polymers can also be used (Nucleic Acid Research 12: 3435 (1984) ; Litchfield, et al. : Clinical Chemistry 3_0: 1489 (1984)) .

Hybridization of DNA Bound to Solid Support

With Complementary DNA

There are several methods which can be used to detect nucleic acids in a sample using DNA (or RNA) probes bound to a solid support according to the present invention. These are best described with reference to the figures. As shown in Figure 2, the test or probe DNA or RNA (designated ABC(+)) can be bound to support material, such as latex particles, and DNA (or RNA) complementary to the probe DNA (designated

I t I A B C (-) ) can be bound to other latex particles. When the sample containing unknown nucleic acid (e.g., ribosomal RNA) is contacted with a mixture of the two nucleic acid-bearing particles under condi¬ tions appropriate for hybridization to occur, if the

I t t sample nucleic acid is complementary (e.g., A B C ) to that of the probe DNA, it will interfere with agglutination of the particles bearing the comple¬ mentary nucleic acid sequences. Particle agglutin¬ ation will be inhibited. The extent of interference with particle agglutination is directly related to the amount of complementary nucleic acids in the sample. If there are only a few complementary sequences in the sample, particle agglutination will be interefered with to a lesser extent than if the sample contains a large number of sequences comple¬ mentary to^* sequences on the latex particles.

In another embodiment of the method of this invention, represented in Figure 1, probe nucleic acid is covalently bound to all latex particles. The presence of complementary nucleic acid sequences in a sample will be detected when the latex parti¬ cles agglutinate. For example, one group of latex particles can have the nucleic acid sequences represented by ABC bound to them and another group sequences represented by DEF bound to them. If the sample nucleic acid contains the complementary

I I t I I I sequences to ABCDEF (e.g., A B C D E F ) hybridization of the sample nucleic acids A B C with the ABC on one particle and of the sample t I I nucleic acids D E F with the DEF on another particle will occur. As a result, agglutination of the particles will also occur. As shown in Figure

3, this would also occur if all latex particles have the entire sequence (ABCDEF) bound to them and the sample nucleic acids have the complementary

I I I I I I sequences ( B C D E F ). In a further embodiment, represented in Figure

4, particles of two different sizes can be used for the attachment of probe DNA. For example, probe DNA can be attached to the larger particles and its complement to the smaller particles. In the pre- sence of complementary DNA or RNA in the sample, small particles would not be agglutinated to large particles and would pass through a filter of selected pore size. If the complementary sequences are not present in the sample, the probe DNA (bound to the larger particles) and its complement which is bound to the smaller particles will hybridize, preventing the small particles from passing through the filter.

It is also possible using the method of this invention to attach the unknown or sample nucleic acid DNA or RNA to an activated latex particle. This is represented in Figure 5. In this case, a specific probe in solution is contacted with the sample DNA-particle complex; the probe used can be tagged for purposes of detecting its presence. If the sample DNA or RNA is complementary to the probe DNA or RNA, hybridization of the nucleic acids and agglutination of the particles will occur.

Measurement of Agglutination or Aggregation of

Particles Having Bound DNA or RNA as an Indica- tor of the Presence of Complementary Sequences in a Sample

Determination of agglutination or aggregation of particles having covalently-bound DNA or RNA can be carried out by any method capable of detecting the degree of agglutination present after sample and probe have been contacted under conditions appropri¬ ate for hybridization to occur. For example, it can be carried out visually using the unaided eye (e.g., visualization against a black or other dark back- ground) ; microscopically; or by turbidimetric measurements. In addition, a particle counter having a size threshhold can be used to detect aggregated/unaggregated particles. Selective counting techniques, which are well known in the art, make it possible to count the number of part¬ icles in a given size range and thus allow quanti¬ tative assays to be carried out. See, for example, U.S. Patent 4,184,849 to CL. Cambiaso et al. , in which such techniques are described. It is also possible to use a filter having a defined pore size; the pore size is selected so as to allow nonaggre- gated particles to pass through but to prevent aggregated particles from doing so. See, for example, U.S. Patent 4,459,361 to M.L. Gefter. Although it is possible, using filtration, to separate agglutinated particles from unagglutinated particles (and thus detect hybridization of complementary sequences) , this approach is limited in that the number of particles present must be large enough to allow visual detection. It is possible, however, using known techniques, to enhance the visibility of the particles through selection of such properties as color, optical density and fluorescence. The particles can be enzyme labeled in such a way that the enzymes attached to the particle surfaces catalyze a color-producing change (thus aiding particle detection) . Such amplification techniques are particularly useful when the nucleic acid sequences of interest are present in a sample in low concentrations. This invention will now be more specifically described by the following examples.

Example 1

A. Preparation of single stranded complementary DNA To prepare single stranded complementary DNA, the following method was used. A DNA sequence (i.e., a portion of the src gene) was cloned into an M13 bacteriophage using protocols described previ¬ ously. Hu, N. and J. Messing, The making of strand- -specific Ml3 probes. Gene, 1/7:271-277 (1982);

Messing, J., New Ml3 vectors for cloning, Methods in Enzymology-Recombinant DNA Techniques 54:20-77 (1981) . Two clones were isolated which contain the sequence cloned in opposite directions. The bac- teriophage produced by one of these clones contains single-stranded DNA molecules which is partially co plementary to the DNA molecule of the other clone. The methods for using, cloning and growing commercially available Ml3 bacteriophage vectors exist (A ersham) . For simplicity, (+) is used to describe one M13 bacteriophage cloned DNA and (-) to describe the cloned DNA which contains its comple¬ ment. Therefore, (+) and (-) strands of DNA can hybridize by hydrogen bonding. Another method is to isolate the complementary DNA strands by preparative electrophoresis as described by Maniatis and co- workers. Maniatis, T. et al. , Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory (1982) .

B. Activation of Carboxylated Latex Carboxylated latex used as the solid support in covalent binding with DNA was prepared according to the following technique, which is based on the method described by Dorman in U.S. Patent 4,045,384.

Activation of the carboxyl groups results from the reaction of the carboxylate latex v/ith N-1-hydroxy- benzotriazole in the presence of CMC, according to the following reaction:

Specifically, 0.2 ml. of an N hydroxybenzo¬ triazole solution was added to 1 ml. of carboxylate latex (1 micron in diameter, 2.5% solids). (The N hydroxybenzotriazole solution was made by dissolving 93 mg. of Aldrich N-hydroxybenzotriazole in 1.6 ml. dimethylformamide; this was diluted to 4 ml. with water.) The combination was placed in an ice water bath and 0.1 ml. of CMC (100 mg. CMC solution in 2 ml. water at 0°C) was added dropwise. The mixture was stirred in the cold for four hours ^'or overnight.

Cleaning of the latex (removal of the unreacted products of the reaction, e.g., urea, as shown above) was accomplished by dialyzing the mixture overnight against 0.1 molar (M) sodium chloride (NaCl) at 4°C. An additional 2 ml. of 0.1M NaCl was added to the dialysis bag.

The mixture was placed in Eppendorf tubes (1 ml. each) and sonicated for 10 seconds. They were spun and resuspended three times in water and then resuspended in 50 mM phosphate buffer at pH 8.0.

Covalent Bonding of DNA to the Modified Latex

DNA to be bonded to the latex particles was added to the dialyzed particles described above. The DNA prepared as in part A was added while the particles were being stirred in an ice water bath or at 5°. Also present at that time was 1 ml. of pH 8.0 phosphate buffer and 0.1 M NaCl solution. About 0.06 ml. of the N-hydroxybenzotriazole solution (prepared as described above) was also added to aid the forward reaction. The pH of the mixture (6.8) was raised to 7.2 by the addition of 0.5M dibasic sodium phosphate. The mixture was stirred for 5 days at about 4°C and washed with distilled water containing azide.

Evaluation of the latex particles prepared according to the method described above was carried out. The latex particles were spun down in Eppen- dorf tubes and washed three times with 0.1% SDS; each time they were resuspended in 1 ml. of 0.1% SDS. Microscopic assessment showed that most of the particles were monomers or dimers.

D. Agglutination of Latex Particles by Hybridiza¬ tion of (+) Stand Covalently Bound to Latex to (-) Strand DNA in Solution

Hybridization of (-) strand DNA to (+) strand DNA covalently bound to latex particles was carried out. About 0.1 ml of latex (2% solids) to which (+) strand DNA was covalently bound was placed in each of three tubes. The solid latex particles were pelleted by centrifugation and resuspended in 50 microliters of 0.4M sodium phosphate buffer (pH 7.8) and 0.1% SDS containing one of the following:

(1) No DNA

(2) (+) strand DNA (0.5 micrograms)

(3) (-) strand DNA (0.5 micrograms)

The three tubes were incubated at 60°C for 10 to 12 hours. After the incubation the latex was pelleted again and resuspended in 0.1% SDS. Approximately 10 microliters were placed on a glass microscope slide and examined microscopically under oil immersion at 100 X magnification under a cover slip. The examination showed the following: (1)

Mostly monomeric; a few dimers (2) Mostly monomers; a few dimers (3) Few monomers; many particles in which 4 to 10 latex particles have agglutinated. These results demonstrate that (-) strand DNA in solution can cause latex which has (+) strand DNA covalently attached to it to agglutinate. Example 2

The procedure described in Example 1 was used except that the latex preparation was resuspended in 50 microliters of 0.4 M sodium phosphate buffer, 0.1% SDS containing

(1) Denatured calf thymus DNA (1.5 ug)

(2) (-) strand DNA (5 ug)

(3) (-) strand DNA (0.5 ug) + calf thymus DNA (1.5 ug) Microscopic observations showed the following:

(1) Mostly monomers; a few dimers

(2) Very large aggregates; many greater than 50-100 particles, visible using the unaided eye (3) Few monomers; many aggregates (having) 4 to 6 latex particles. These results demonstrate that: (1) non-complemen¬ tary DNA will not cause DNA which has (+) strand DNA covalently bound to it to agglutinate; (2) a large amount of (-) strand DNA (5 ug) in solution will cause a lot of visible agglutination while a little (-) strand DNA in solution (0.5 ug) will cause only a little agglutination; (3) heterologous , noncomple- mentary thymus DNA will not interfere with agglutination of latex with (+) strand DNA covalently attached to it by (-) strand DNA in solution. The main conclusion to be drawn from both of these examples is that under the conditions des¬ cribed DNA in solution can hybridize to comple¬ mentary DNA attached to a solid support and cause the latex to agglutinate.

Example 3 Detection of Nucleic Acid Sequences In A Biological Sample Using Covalently Bound DNA Sequences

RNA can be extracted from a sample of milk and used to cause the agglutination of a latex that has a probe specific for ribsomal RNA (vRNA) attached to it. Such a probe is DNA or RNA complementary to the sequences of vRNA of organisms which occur in milk. This can be carried out as follows: (1) To 10 ml of skim milk add SDS to 0.1%, Proteinase K to 200 ug/ l

(2) Incubate at 60°C overnight

(3) Add 10 ml of liquified equilibrated phenol

(4) Extract and centrifuge (5) Remove aqueous phase and add DNase to 100 ug/ml

(6) Repeat steps 3 and 4

(7) Take aqueous phase; add 10 ml of isoamyl alcohol and chloroform mixed in a 1 to 24 volume/volume mixture

(8) Repeat step 4

(9) Repeat steps 7 and 8

(10) Remove aqueous phase and bring to 0.1 M sodium chloride. Add 20 mis ethyl alcohol (li) Bring to -20°C for 10-12 hours

(12) Centrifuge at 15,000 g.

(13) Resuspend pellet in 50 microliters containing 0.4 M phosphate buffer, 0.1% SDS and latex with a rRNA probe covalently attached as described in example IB.

(14) Incubate at 60°C for 10 to 12 hours

(15) Observe the latex microscopically or visually

Visual (microscopic) evaluation shows that there are few monomers; most are 5 to 100 agglutinated latex particles.

Industrial Applicability Nucleic acid probes and hybridization assays according to this invention have a variety of possible applications in which the ability to detect, quantify and/or identify complementary nucleic acid sequences of interest in biological samples of all kinds is of great value. For ex¬ ample, they are useful in a research context as tools for studying gene structure and inheritance. In addition, they are useful in clinical settings for the detection and identification of infectious agents and for prenatal diagnosis of genetic dis¬ orders. Finally, DNA probes have utility in the diagnosis of cancer (by providing information on the structure of oncogenes) ; in tissue typing; in veterinary and plant diagnostics; and in food testing (by providing a quicker, more convenient means of testing for the presence of pathogens) .

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experi- mentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of determining the presence or absence of nucleic acid sequences in a sample, compris¬ ing detecting the agglutination or inhibition of agglutination of particles having bound thereto nucleic acid sequences complementary to those whose presence or absence in the sample is to be determined.

2. The method of Claim 1 in which the comple- mentary nucleic acid sequences are covalently bound to the particles or to a material which is adsorbed to or covalently bound to the surface of the particles.

3. A method of determining the presence or absence of nucleic acid sequences in a biological sample, comprising the steps of: a. contacting the biological sample with particles having covalently bound thereto nucleic acid sequences complementary to those whose presence or absence is to be determined in the biological sample; and b. determining the degree of agglutination of the particles.

4. A method of determining the presence of nucleic acid sequences in a sample, comprising detect¬ ing the hybridization of nucleic acid sequences bound to particles and nucleic acid sequences in the sample which are complementary to the bound sequences by detecting the degree of agglutination of the particles.

5. The method of Claim 4 in which the nucleic acid sequences bound to particles are covalently bound to latex particles or are covalently bound to a material which is adsorbed to or covalently bound to the surface of latex particles.

6. A method of detecting nucleic acid sequences in a sample, comprising the steps of: a. covalently bonding to particles nucleic acid sequences complementary to the nucleic acid sequences to be detected in the sample, the diameter of said particles being less than about 500 microns; b. treating the sample to make the nucleic acids present in the sample available for hybridization with complementary nucleic acid sequences; c. contacting the particles having covalently bound nucleic acid sequences and the treated sample under conditions appropri¬ ate for hybridization of complementary nucleic acid sequences; and d. detecting hybridization of nucleic acid sequences bound to the particles with complementary nucleic acid sequences in the sample by determining the degree of agglutination of the particles.

7. A method of Claim 6 in which the particles are latex particles.

8. A method of detecting nucleic acid sequences in a sample, comprising the steps of: a) contacting the sample with 1) particles having bound thereto nucleic acid se¬ quences complementary to the sequences to be detected and 2) particles having bound thereto nucleic acid sequences comple- mentary to the sequences bound to the particles in (1) , under conditions ap¬ propriate for nucleic acid hybridization and b) determining the degree of agglutination of the particles.

9. The method of Claim 8 in which nucleic acid sequences are covalently bound to the particles or are covalently bound to a spacer molecule which is adsorbed to or covalently bound to the surface of the particles.

10. A method of Claim 9 in which the particles are latex particles.

11. A method of detecting nucleic acid sequences in a sample, comprising the steps of: a. bonding to particles nucleic acid se¬ quences complementary to nucleic acid sequences to be detected in the sample; b. bonding nucleic acid sequences which are complementary to those bound to the particles of (a) to inert particles of smaller size than the particles of (a) ; c. contacting the particles of (a) and the particles of (b) with the sample under conditions appropriate for hybridization of complementary nucleic acid sequences to occur; and d. determining the degree of agglutination of the particles of (a) with the particles of (b) .

12. In a method of detecting nucleic acid sequences in solution using nucleic acid probes, the improvement comprising the use of particles having bound thereto nucleic acid sequences complementary to nucleic acid sequences to be detected in the solution.

13. In a method of detecting nucleic acid sequences in solution using nucleic acid probes, the improvement comprising detecting the degree of agglutination of particles having bound thereto nucleic acid sequences complementary to the nucleic acid sequences to be detected.

14. A method of detecting nucleic acid sequences in a sample, comprising the steps of: a. contacting particles having bound thereto nucleic acid sequences from a sample with nucleic acid sequences complementary to those to be detected and b. determining the degree of agglutina¬ tion of the particles.

15. A kit for detection of nucleic acid sequences in a sample by dtermination of agglutination, comprising a container and latex particles having covalently bound thereto nucleic acid sequences complementary to the nucleic acid sequences to be detected.

16. Particles having bound thereto nucleic acid sequences.

17. The particles of Claim 16 to which the nucleic acid sequences are covalently bound to the surface of the particles or covalently bound to a spacer material which is adsorbed to or covalently bound to the surface of the part¬ icles.

18. The particles of Claim 17 in which the part- icles are latex particles having a diameter of less than about 500 microns.

19. Latex particles having nucleic acid sequences bound thereto, said sequences being either covalently bound to the surface of the latex particles or covalently bound to a spacer material which is adsorbed to or covalently bound to the surface of the latex particles.

20. In a solid support having attached thereto nucleic acid sequences, the improvement com¬ prising latex particles having a diameter of less than about 500 microns and having the nucleic acid sequences either covalently bound to the surface thereof or covalently bound to a meterial which is adsorbed to the surface thereof.