JPH05507613A - Methods and compositions for performing DNA assays - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Perform DNA assay and set Ryogodan Mutual Viewing The present invention generally relates to nucleic acid hybridization assays. For more information, A complete method for performing nucleic acid hybridization assays using magnetically responsive particles. relating to automated methods.

Many current diagnostic and epidemiological methods rely on culturing clinical specimens; This can be time consuming and difficult. Immunization such as RIA and ELISA The biological assay is not very time consuming and allows for virus typing and culture confirmation. It is often used for authentication. However, many viruses have sufficient antigen produced and therefore cannot be detected by immunoassay. Such pathogens Another method of identifying nucleotides uses nucleic acid hybridization analysis.

Nucleic acids are carriers of genetic information between generations - nucleotides arranged in a straight line. It is composed of individual units called chidos. Each nucleotide is a sugar phosphate salt, including pyrimidine or one of the purine bases, adenine (A), thymidine (T), uracil (U), guanine (G), or cytosine (C) ing. - Zero-value nucleic acids are double-coated by highly specific binding between the bases on the two mirrors. Form a spiral. A binds only to T or U, and G binds only to C. Therefore, two A double-stranded or hybridized nucleic acid is one in which the base sequences on the two strands are if the hybridization is sufficiently complementary that such hybridization is possible, and It is formed only in such cases. Depending on the form of sugar phosphate present, nuclear The acid is called deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

Nucleic acid hybridization assays are based on this principle of complementarity. representatively hybridizes a single-null nucleotide sequence that is complementary to the sequence in question. Combine with the sample under conditions that allow for The latter sequence above is called a “probe”. , the former sequence is called the "target". If a double-stranded nucleic acid containing a probe is present, the target The sequence is shown to be present in the sample. Such assays can be used to detect bacterial, Testing of biological samples for the presence of pathogens such as viruses or parasites, genetic Diagnosis of diseases related to abnormalities, indication of genetically mediated susceptibility to certain conditions, e.g. Parentage or other blood relationships, such as in forensic analysis, and dietary It has a wide range of applications, including biological contamination of goods or other products.

When the amount of target nucleic acid in a sample is initially small, methods can be used to amplify the target. Ru. Such amplification methods include polymerase chain reaction (PGI2) and There are amplification methods based on ligation. For example, U.S. Patent No. 4.683.195 No. 4.683.202 and $4,800.159, and PCT See patent publication no. WO 39/12696.

Various methods of determining the presence and extent of hybridization are familiar to those skilled in the art. It is knowledge. In such assays, the hybridized probe It is necessary to be able to distinguish it from the undiscerned probe. nucleic acid hive The majority of redization assays rely on isotopes, primarily 32p. Use detection and want to separate (manual process involving a few steps) . For example, traditional membrane-based assays involve sample pretreatment, denaturation, and nucleic acid Immobilization on solid supports such as nitrocellulose or nylon filters and is included. Such methods are imprecise, labor-intensive, time-consuming, and automation is difficult.

In addition, hybridization probes are typically used with high It is radiolabeled to have a high specific activity. Such probes are unstable and difficult to handle. It is frowned upon in clinical laboratories because it poses significant problems in preparation and disposal.

As a result, practical use in clinical laboratories is limited due to the small number of such reagents commercially available. Applications are limited.

In the nucleic acid hybridization assay, ar m is also used. For example, U.S. Pat. No. 4.711.9 by Ward et al. There is a method disclosed by No. 55. In this method, alkaline phosphatase Coloring using a biotin-labeled probe using a Z-Avidin polymer complex A specific precipitable dye is produced enzymatically and immobilized on the filter. NA sequences can be detected. However, these non-isotope indirect detection systems The membrane is contaminated by intermediate, background-sensitive binding and washing steps, and Limited to hybridization.

Several membrane-based non-isotopic direct assays that utilize covalent enzyme-DNA system is disclosed. Renz and Kurz (Nucl, Ac1 ds Res, 12:3435 (1984)) on the long probe method. However, these methods have long hybridization times and cloning problems. This method is limited due to the difficulty of mapping methods. Alternatively, well-established synthetic oligonucleotides Otide probes may be used. For example, directly labeled enzyme-synthetic oligonucleotides. The otide complex is Nucl, Ac1dsRes, by Jablonski et al. 14:6115 (1986); Am, Soc by Kertchner et al. , Microb, Abst, 56:309 (1987>, McLaug Lancet 714° March 28 (1987) by hlin et al. It is shown. These documents are incorporated herein by reference. These multiples The conjugate is easily synthesized for specific analytes and does not cause non-specific binding. Hybridization properties remain unchanged and have high sensitivity and stability.

Due to the high probe concentration, oligomer hybridization is fast on the membrane support. complete. However, full utilization of these enzyme complexes is difficult to achieve in conventional membrane formats. has not been realized.

Other attempts at membrane-based assays include, for example, U.S. Patent No. 4.486.53. There are sandwich assays such as those disclosed in No. 9. on solid matrix by hybridization to immobilized complementary sequences (direct capture) or by reactivation. After hybridization with Gund-labeled probes, the parent (indirect capture), by removing unbound probe. , remove the target DNA from the original sample. The bound target is unique to neighboring sequences. Detected by a second, directly or indirectly labeled probe.

By adding this second hybridization step, the selection for the target selectivity is increased, and the chances of non-specific interactions are reduced. long cloning pro This is the most appropriate for routine diagnostic methods, although the use of It is one of the most popular formats. This is because sample preparation, purification, and This is because target immobilization on a specific filter can be omitted.

Direct capture hybridization supports contain nucleic acids, usually cloned DNA. nitrocellulose, cellulose, etc. by chemical modification, adsorption, or enzymatic methods. base, nylon, polystyrene, Teflon polyacrylamide, polypropylene , adhered to various solid phases including agarose, sephacryl, and latex It is synthesized by Hybridization efficiency of immobilized DNA and Details of their capabilities are disclosed. For example, J, Cl1n by Miller et al. , Microb, July 198g.

p, 1271-1276 and Molecular an by Yehle et al. d Ce1lular Probes 1:177-193 (1987). Light. These documents are incorporated herein by reference. However, as mentioned so far Supports are difficult to prepare and are difficult to load with the exception of oligonucleotide resins. capacity is relatively small. Mixed phase direct capture hybridization is also stoichiometric can be inefficient due to limited availability and inability to access immobilized DNA. Ru.

In contrast, hybridization in solution is very efficient and also in solid state. It takes many times less time than hybridization on a support. 125■ Solution hybridization is performed with a probe labeled with Methods for non-specific adsorption to droxyapatite (HAP) are well known in the art. It is. However, nonspecific background binding to RAP is a function of probe concentration; Also, capture is non-specific. Therefore, probe input is limited and hybridization zation rate is suboptimal.

All of the DNA assay methods briefly described above are performed manually and are generally effective. It has not been proven that. Current methods also provide lasting, accurate, and repeatable results. The disadvantage is that it does not. For example, the relative amounts of various reagents, the binding tags of these Due to variations in timing and required number of agitation of bound reagents, some Accurate and non-repetitive results will result.

Furthermore, the potential for contamination as well as the numerous lengthy steps of manual assay manual This is yet another disadvantage unavoidable in DNA assays.

Therefore, it is highly recommended to perform DNA assays while minimizing the possibility of contamination. provides accurate and repeatable results, and highly skilled intervention and monitoring is the most There is a need for fully automated methods and reagents that require only a limited amount of . The present invention satisfies this need and also provides related advantages as well.

2nd day! ” L giant The present invention is highly accurate, efficient, reproducible, and fully automated. A nucleic acid hybridization tool that requires minimal human intervention. This invention relates to an improved method of performing an assay. The method of the invention provides a programmable XY 2 pipettors and multiple connected wells, each accommodating a test tube. an interconnected array of thermally controlled reaction tube holders, centered around the longitudinal axis of each reaction tube. and a means for transposing or rotating the magnetic field. this Using an xyz pipettor, separate tubes housed in multiple reaction tube holders can be Transfer the nucleic acid sample to a test tube and add /% ibuidization solution to multiple test tubes. and the suspension of magnetically responsive particles a predetermined number of times in succession, and then the reaction tube is placed over them. It is positioned a predetermined number of times with respect to the coupled magnetic field. Target nucleic acid sample, no\ipridase of probes and oligonucleotides and magnetically-responsive particles in a chemical solution. A binding reaction occurs between nucleic acids that can be visualized with high precision and reproducibility. cation assays are now available.

In particular, using XYZ Bipefter, first add the hybridizing solution Place the suspension of magnetically responsive particles in two wells and the wash buffer solution in the third well. Add the substrate buffer to the fourth well and the quench buffer to the fifth well. Ru.

Separate nucleic acid target samples are placed in multiple reaction tubes, each contained in a separate reaction tube holder. Put it in the tube. First, remove the hybridization solution using an xYz pipettor. The solution is sequentially transferred from the first well to multiple test tubes where the hybridization process is performed. cause a reaction. Then, using an XYZ pipettor, remove the magnetically reactive particles. The suspension is sequentially transferred from the second well to multiple reaction tubes, then magnetically responsive particles Place the reaction tube in such a way that the samples remain in liquid suspension and bind to separate nucleic acid samples. Selectively rotate or remove the magnetic field. Selectively stop the rotation process. By doing so, the magnetically responsive particles are moved to selected locations within the reaction tube by the magnetic field. Transfer the unbound nucleic acid sample and the hive in the liquid phase using an xYz pipettor. redization solution is removed from the plurality of reaction tubes. Alternatively, the electromagnetic field can be fixed. The particles can be removed from the suspension by directing them around a defined tube.

Next, use an xYz pipette to transfer the wash buffer solution from the third well to the reaction tube. vinegar. After resuspending and separating the magnetically responsive particles, then using a pipettor, Remove the wash buffer solution and remove the bound DNA sample and hybridization molecules. , leaving magnetically responsive particles behind. Then, using a pipettor, add the substrate buffer to the fourth The wells are then transferred to multiple reaction tubes where the enzyme probe label generates a detectable reaction. catalyze. Alternatively, if the label is luminescent or fluorescent, the probe dehybridize and detect in solution. Next, use a pipettor to Transfer the quench buffer solution from the fifth well to multiple reaction tubes, then add the sample to each reaction tube. Transfer to separate wells of a microtiter plate. In this way, hybridization The degree of binding reactivity between an oxidation solution and a separate nucleic acid sample can be conveniently measured. .

In other more detailed features of the invention, the xYz pipettor further includes an optical sensor. The process of selectively replacing and fixing the magnetic field is performed using the pipettor's support. by moving the sampling tip to a location where it can be detected by an optical sensor. It can be accomplished. With proper programming, the magnetic field can be applied selectively. Precise timing of changing and fixing steps and hive using pipettor Time difference between transferring the redization solution and transferring the magnetically responsive particle solution is precisely controlled.

In other more detailed features of the invention, the hybridization solution contains a a combined nucleic acid probe and a nucleic acid probe bound to a biotin or habten molecule; and magnetic reactions attached to avidin, streptavidin, or antibody molecules. Contains both sexual particles. In this way, this method Sei is performed. The label is a fluorescent or luminescent substance, or a labeled enzyme. obtain. The labeled enzyme advantageously exhibits fluorescent or luminescent properties, and the method further The sample contained in the microtiter plate is measured using a spectrometer, fluorometer, or The method may further include assaying using a light needle.

In other aspects, the invention provides a method for hybridization assays in which: Magnetically responsive particles immobilized on ligands are used to separate targets from foreign nucleic acids. use Magnetically-responsive particles are stably attached via a covalent spacer. toavidin. The length and composition of deposits on particles are critical to their effectiveness. It is essential.

Other features and advantages of the invention will be apparent to those skilled in the art, embodying the principles of the invention while referring to the accompanying drawings. This will become clearer from the following description of the method of preferred embodiments, illustrated by way of example.

[iyui supervision gori] FIG. 1 shows a nucleic acid hybridization assay according to a preferred method of the invention. xyz pipettor 1 and the reagent well and reaction tube connected to it for performing FIG. 3 is a perspective view of the array with reaction tube holders.

FIG. 2 is a top view of the array of reagent wells and reaction tube holders shown in FIG. 1. FIG.

FIG. 3 shows the X of FIG. 1 when performing the nucleic acid hybridization assay of the present invention. Comprehensive flowchart showing the working steps performed by the YZ pipettor It is.

Figure 4 shows a process for mixing hybridizer 9 buffer with multiple individual nucleic acid samples. In the process, a more detailed description of the working steps carried out by the XYZ pipettor is provided. It is a flowchart.

FIG. 5 shows that x A more detailed flowchart showing the working steps performed by the Yz pipettor It is the default.

Figure 6 shows the process of washing the reacted nucleic acid sample with an xYz pipettor. 3 is a more detailed flowchart showing the operational steps performed;

Figure 7 shows how to transfer the reacted nucleic acid sample into a microtiter plate for the next reading. The action steps carried out by the XYZ pipettor during the transfer process are shown below. This is a more detailed flowchart.

Light 1st eye 2jlL Figure 11 Tsui Reagents suitable for use in the method of the invention and methods for their preparation are described herein. I will post it. It is understood that other reagents and methods well known to those skilled in the art may also be utilized. I want to be.

Synthesize oligonucleotide probes using a variety of standard methods and reagents. obtain. Preferably, the probe is chemically prepared by a person skilled in the art (using methods known to those skilled in the art). Synthesize into See, for example, Ruth, PCT Open Publication, which is incorporated herein by reference. Please refer to report number WO34703285. Described in WO34103285 internal base, such as A spacer and a C-67 amino modifier (C-6 amino mod Prepare a capture probe containing a biotin moiety attached by Ta. Applied Biosystems, Inc. (Foster C Using an automatic DNA synthesizer such as the Madel 380 manufactured by 1ty, CA), An amino-terminated oligomer was synthesized. Prior to the start of synthesis, the spacer phosphor amidite (O-dimethoxytrityldiethylene glycol-0°-(77 noe Chi-N,N-diisopropyl-phosphoramidite)) and 5°-aminomodification Decorative factor C6[3-(4-monomethoxytrityl-amino/)hexyl-(2-cya noethyl)-(N, N-diisopropyl)-phosphoramidite], (Gl en　Re5earch　Corp, ■erndon, VA) from anhydrous acetic acid. Resuspend in tonitrile.

More specifically, spacer phosphoramidites were synthesized as follows. under vacuum diethylene glycol (10 g, 94;2+u+ole) was made anhydrous. 4. in anhydrous pyridine (20ml). 4-dimethoxytrityl chloride (3.19 g, 9.4 mmole), Added with stirring over 1 hour. Stir vigorously in ice water (too ml) After that, the reaction mixture was extracted three times with 75 ml of methylene chloride. 75ml Back-extracted with water three times, dried with sulfuric acid 1-IJ chromium, filtered, and rotary evaporated. The capacity was reduced by Baboleshicon. The resulting mixture was applied to a 5i02 column (2, 5X 30 cm) l, -, ethyl acetate:cyclohexane (1:2 v/v > and eluted in the same solvent. TLC of 5i02 (acetic acid ethyl Chill: Cyclohed beef, 2+1 v/v), then UV absorption and HCL evaporation. The appropriate fraction was identified by the color reaction after exposure to air. The above The filtration was combined and dried by rotary evaporation.

The residue was dissolved in 20 ml of benzene, extracted three times with water and lyophilized. (3.16 g, 7.75 tole O-dimethoxytrityldiethylene Recall, X5182%).

The anhydrous residue was redissolved in tetrahydrofuran (20ml) and evaporated under vacuum. I let it happen. The residue was dissolved in tetrahydrofuran (25 liters) and diisopropylethyl. redissolved in 3 ml of amine (3 ml) and placed in a dry ice-ethanol bath under argon. It was cooled down. 2-cyanoethyl-N,N-diinpropylchloro-phosphoramide Add dyte (2 ml, 9 mmole) with stirring (20 minutes) and After that, the mixture was further stirred at room temperature for 20 minutes. Insoluble salts are vacuum filtered under dry argon. and washed with anhydrous tetrahydrofuran. After vacuum concentration, ethyl acetate (50 ml ) was added to the syrup and extracted three times (1M sodium carbonate, 150 μl each).

After drying with sodium sulfate, filtering and concentrating, ethyl acetate:cyclohexane (1:2゜5ml>) was eluted from the silica column and subjected to TLC (described above). After that, 1 liter of Varakushi was combined and dried by 1 liter of rotary evaporation. . The residue was lyophilized from benzene to give an oily residue (2.9 g, 62% yield). I got it. The residue was redissolved in benzene and filtered via a septum under N2. and lyophilized and stored at -20°C.

The oligomer was purified by reverse phase HPLC on a C-8 silica column and Analyzed by acrylamide gel electrophoresis. Antioxidants containing oligonucleotides The reactive amine spacer arm was prepared in 0.2M sodium bicarbonate, pna, S. , 100- to 1000-fold molar excess of NHS-X-biotin (N-hydroxysulfate) Cinimidyl-aminocaproic-biotin; CalBiochem, L a Jolla, CA) for 1 hour. acetonitrile gradient The free probe and The biotinylated probe is purified from excess biotin and then desalted and purified with ethanol. was allowed to precipitate. Product purity was determined by analytical gel electrophoresis.

Jablonski et al., Nucl, Ac1ds, incorporated herein by reference. Res, -14:6115 (1986) according to the process, Directly labeled alkaline phosphate-DNA complexes can be prepared. Simply put, The linker-arm nucleoside terminates the C-5 methyl within the primary amine. Analogs of thymidine modified by substituting an atom with a linker arm be. Such probes are purified using conventional methods to form unmodified probes. By comparison, they exhibit essentially unchanged behavior in terms of physical properties. For more details, see The method of Ruth et al., DNA 4:93 (1985) is incorporated herein for consideration. The linker arm nucleotide 3-phosphoramidite was prepared by App Lied Biosystems, Inc. (Foster C1ty, CA) Oligonucleotides are synthesized using an automatic DNA synthesizer such as Model 1380 manufactured by It was taken into account. This oligomer was analyzed by reverse phase HPLC on a C-8 silica column. It was purified and analyzed by 20% polyacrylamide gel electrophoresis. gel for analysis Product purity was determined by electrophoresis.

The linker arm oligomer is first treated with disuccinimidyl suberate (DSS). guided. The volume of oligomer in 0.2M sodium bicarbonate is expressed as the volume of DMSO2. and a 30-fold excess of DSS. After holding at room temperature for 3 min, the reaction was transferred to FP Applied to LCG-25 gel filtration column and eluted with 1mM sodium acetate, pH 5.0. did. Fractions were monitored and collected by flow absorption measurements at 260 nm. Microconcentrator (Centricon IOK; Am1c on, Danvers, MA>.

The final concentration was determined by adding sodium chloride crystals to the activated probe. It was set as 3M. 3M NaCl in 1,0,1M bicarbonate, pH 8,25, 2x excess calf intestinal alkaline phosphatase (Boehringer Mannh eim, Indjanapolis, IN) and maintained at room temperature for 16 hours. did. The complex was purified by FPLC anion exchange and NaCl gradient It was eluted with Peak Valaxin 1 was determined by the absorption rate of 2607280 ntx. collected. Product purity is determined by analytical non-denaturing polyacrylamide gel electrophoresis. Check the degree.

The present invention utilizes ligand-induced magnetically responsive particles, including magnetic or paramagnetic particles. do. Various substances, including oxides of iron, chromium, titanium, or other metals can be used. The particles are small enough to remain dispersed and the average settling time is approximately It must be longer than 3 minutes. The particles preferably have a diameter of about 0.1-1 oμ, more preferably about 1μ. nucleotide or oligonucleotide A variety of Gantts other than Gantts may be used, including, for example, Avidin or Strain. putavidin, biotin, hapten (e.g. with carrier f=’) nitro containing phenol (DNP) or digoxigenin), lectin, or antibody ( (e.g., antibodies against biotin, fluorescein or gigo-bovine sigenin) .

The length and composition of the covalent bond between the magnetically responsive particle and the ligand determine the effectiveness of this method. important for efficient execution. According to traditional methods, proteins are using glutaraldehyde, carbodiimide, or cyanogen bromide cross-linking. to crosslink the solid matrix. Both methods detect proteins bound to particles. generate. However, the activity decreased significantly and subsequent particle hybridization The efficiency and strength of binding in binding assays are generally unacceptable. for example, In PCT Publication No. 88702785, Beebe et al. cellulose using N,N'-carponyldiimidazole to obtain the binding site. Avidin-derived beads bound to base beads, using 33B beads per assay. It is necessary to do so. PC publication publication No. 10867013B1. S In NiSnlt et al., an anti-fluorescein antibody was prepared by adsorption at 174-in one bead (approximately 120 ng) per assay. ) is used. In the method described below, magnetically reactive particles are For every 1++ g of particles, by attaching non- or streptavidin, Has a binding capacity of about IX tG-” moles (1 nanomole) of biotin binding sites Generate particles. This means that only 15 μg of beads per assay, i.e. The amount of particles used is less than 1/1000 of the amount of particles in the prior art method. means. Therefore, prior art methods produce particles suitable for use in the present invention. cannot be provided. Because, from a cost and efficiency perspective, how many milligrams per assay? It is impractical to use beads with such large amounts (milligrams). This is because magnetism cannot be removed by using a lens.

Covalent bonds formed in the manner described below can be formed by multiple, alternating hydrocarbon and Contains mido (including urea) residues.

The linker has at least 3 residues in total, preferably more than 8 residues in total. Has fewer residues. The method described below has mixed hydrophilic/hydrophobic properties. , tannins bound to magnetically responsive particles via relatively long bonds (20-60 atoms). Generates protein. After testing the bond length and composition of has also proven to be favorable.

Additionally, traditional glutaraldehyde, carbodiimide, or cyanogen bromide crosslinking Attachment of proteins to particles using conjugates is possible under storage conditions in aqueous solution. Hydrolyzable, may not be stable under storage conditions in mild acids and bases It also provides a strong bond. The present invention is suitable for use in aqueous solutions, e.g. between pH 4 and 8. chemically stable to mild acids or bases; Stable against many amine-containing buffers, such as Tris, which is often used in It also provides binding. The resulting particles can be stored and used much more conveniently. Ru.

Strept-avidin-derived magnetically responsive particles were prepared. To put it simply: , amine-induced magnetic particles, approximately 1 μm in diameter (Advanced MagneHcs, Inc., Cambridge, MA) (50 mg/+il) in an aqueous suspension. 10 ml of the solution was centrifuged at 12.00 rpm for 30 minutes. Discard the supernatant; The precipitate was completely resuspended in 20 rsl of H2O by portex and centrifuged again. The heart was separated. The particles were further washed with 20 ml of water and twice with methanol. Wash with 10% methanol/triethylamine, wash with methanol 2@, and finally Washed twice with ether. The particles were completely resuspended after each wash.

The particles were thoroughly dried with P2O5-NaOH in vacuo.

The precipitated particles were suspended in anhydrous dioxane (10 ml) and then collected. centrifuged in a box tube. After removing the supernatant, the particles were soaked in fresh dioxane (1 0 ml) and then resuspended in 1,6-diisocyanate (2 ml) were quickly mixed and the reaction tube was placed on a rotator and kept overnight. The next day, magnetic particles Centrifuge, remove the supernatant, and portex the particles in anhydrous dioxane. Carefully washed three times using centrifugation.

Finally, the precipitate was resuspended in dioxane 101 and 1 g of 1. A solution of 6-diamituhexane in dioxane (5 ml) was added. reaction tube, It was kept on a rotator overnight. The next day, the particles were centrifuged and then treated with dioxane (20 ml) three times. Particles moistened with dioxane were treated with glutaric anhydride (1g> , p-dimethylaminopyridine (1 g), anhydrous acetonitrile (15 ml), and anhydrous pyridine (5 ml). Transfer the reaction mixture again to the rotator. It was kept on top overnight. After centrifugation and removal of the supernatant, the solution-wet particles are dried Resuspend in a mixture of pyridine (15 ml) and acetic anhydride (3 ml) for 3 hours. It was kept on a rotator for a while. Particles were centrifuged and mixed with pyridine-water (3:1) 20% each. Washed twice in ml and then four times in acetonitrile. lastly , the particles were resuspended in anhydrous acetonitrile (>10 ml) and the carboxy-induced magnetic particles A mixture of 50 g/ml of filtrate was obtained. The resulting mixture was stored at -20°C for up to 1 year. or longer.

3 ml of the above suspension was centrifuged. After removing the supernatant, the particles were soaked in dimethylforma Suspended in Mid (1.5 ml). N-hydroxysuccinimide (1115m g), trifluoroacetic anhydride (140 μm), dichloromethane (2.5 ml) Prepare a solution and add 1-methylimidazole (320 μl) to it under cooling. Added without moisture. The resulting mixture was dimethylformamide (2,5 ml) t' diluted and then added to the magnetic particles prepared above. the resulting mixture The solution was stored in a Teflon-coated screw cap vial for 6 to 12 hours. Hold on rotator for an hour. The particles were centrifuged and washed six times with dimethylformamide ( 10 μl each) were washed in a container. The particles are then eppendorff (eppendorf). The mixture was transferred to a do → tube and further washed three times with anhydrous acetonitrile. Finally, the particles Resuspend in anhydrous acetonitrile (1.5 ml).

1 volume of the suspension was obtained by drying 100 μl of the suspension in a desiccator in vacuo. The weight of the particles was determined for each amount.

Activated particles were collected to the test tube wall by a magnet and the supernatant was transferred to another container.

Streptavidin, o, 1+olJ ester, O, Q5 azide, solution 20 g/11 becomes 0.24 g of streptavidin per 1 g of particles. was added to the wet particles in the following proportions: The particles were rotated overnight. Particles, I Washed 4 times in X SSC, 0.1% SDS, then washed in storage buffer (l SC, l mg/ml BSA, 0.05%! J door X100 and 0.0 5% azide) and set the goal to 501. Protein and L4C-bi By performing an Othin binding assay, each particle IB Jin loading and biotin binding capacity were determined respectively.

appropriate drugs, including antibodies against biotin, fluorescein, and digoxigenin. Other ligands, such as antibodies, can be attached to magnetic particles in a similar manner.

Below, general methods for nucleic acid hybridization are described. Like Alternatively, to detect the presence of specific nucleotide sequences in a sample, Use a touch format. Suitable samples include, for example, cell or tissue extracts. , body fluids such as blood or blood products, urine, saliva, containing nucleic acids or specific nucleic acid sequences. Contains food or other substances that may be present. In such samples, the nucleic acids are dissolved. be present in the liquid and must be handled in such a way that hybridization can take place. do not have. Typically, sample preparation involves performing cell lysis if cells are present. , followed by some separation of the cellular nucleic acids from other cellular material. typical Cells can be disrupted by osmotic pressure, membrane disruption by detergents or heat, mechanical shearing, ultrasound, Alternatively, it is dissolved by a combination of these methods. Nucleic acids were dissolved in JI [Methods, P For example, phenol-chloroform, ion exchange, or dialysis or gel filtration. using size exclusion, centrifugation, or a combination of these methods, such as Can be purified from other cellular materials. Some samples do not require nucleic acid purification.

- In the example, incubation in a buffer containing sucrose and a non-ionic detergent lysis to dissolve the outer cell membrane. Then, the nucleus was pelleted by centrifugation. Cytoplasmic debris is removed by oxidation. Addition of detergent and protein The nuclear membrane was lysed and the chromosomal nucleic acids were released by incubation using enzyme K. let These steps are performed automatically, as described below.

Amplification techniques can be used to increase targeting, especially when nucleic acids or target sequences are present in low abundance. can be adopted. Amplification typically involves polymerase chain reaction (PCR). Alternatively, this can be accomplished using standard methods, such as ligation amplification or other amplification methods. child Such methods are well known to those skilled in the art. In general, P incorporated herein for reference CRTECHNOLOGY (Edited by H, A, Erlich, 5tockton) Press, 1989) and U.S. Patent No. 4.683.195, No. 4.683. ．． See No. 202, and No. 4.800.159.

In PCR, each target sequence that is adjacent to the target sequence is A primer nucleotide sequence is prepared that is complementary to the strand. Appropriate NA polymer in the presence of a enzyme and a nucleotide precursor and under appropriate reaction conditions. Multiple copies of a specific target sequence defined by adjacent primers is generated exponentially. If appropriate, such as biotin or enzymes A label can be covalently attached to one of the primers, so that the amplified labeled ol Ligonucleotides are obtained. Alternatively, the primers interfere with the PCR process It may contain long or short linker arms without any linker.

More specifically, the target DNA free of interfering substances is heated to approximately 95°C. method and hybridize with primer oligonucleotides. taq 1 (Cetus Corp, Emeryville, CA). Merase binds to the hybridized primer sequence and removes the excess nucleotides. Catalyzes the synthesis of new complementary strands in the presence of triphosphate. newly generated The resulting strand is separated from the template strand by thermal denaturation. When the temperature drops, A new primer binds to the template and the process is repeated. These anti The redaction is carried out automatically in a thermal cycler, as described below.

The invention can be successfully utilized in a variety of nucleic acid hybridization formats. includes some specific novel features. Magnetic reaction that can bind target DNA The use of synthetic particles effectively facilitates solution hybridization of targets and probes. On the other hand, subsequently hybridized nucleic acids and unhybridized nucleic acids Provides an effective vehicle for separating In addition, magnetically responsive particles The use facilitates automation of the assay system. Direct labeling of probes allows Use of ligands/ligands such as biotin/avidin or hubten/antibodies Immobilize the probe/target complex on magnetically responsive particles with the desired affinity It can be used for.

Preferably, the sandwich format is used to analyze specific nucleotide sequences in a sample. Used to detect. For example, a sequence complementary to or close to the target sequence A biotinylated capture probe having a sequence of Combine with the sample under conditions that allow hybridization. target sequence a detection probe containing a labeled sequence complementary to, or a sequence close to, a labeled sequence; can also be hybridized with the sample. detect the presence of particles or If quantifiable, target/capture probe/detection probe complexes are Addition of biotin to avidin or antibody to antibody to bind to the particles. Utilizes the affinity of butene.

As one skilled in the art will appreciate, other sandwich assay formats may also be used. obtain. For example, magnetic or paramagnetic microspheres include biotin, habten (e.g. , dinitrophenol (DNP), or BSA. avidin lectin, or an antibody (e.g., biotin Other substances can be derivatized, such as capture pro Abs also include non-containing molecules on microspheres, such as avidin, antibodies, saccharides, or habten. It may be derivatized with a non-oligonucleotide moiety that is complementary to the oligonucleotide moiety. difference Additionally, radioactive components of enzymatic reactions; chemiluminescent, bioluminescent, and fluorescent parts; A variety of labels can be used in detection probes that include molecules.

Preferably the target and probe contain DNA. However, the target and/or Or the probe can be used by DNA-RNA or RNA-RNA hybridization method. It is understood that RNA may be included, so as to utilize the

With reference to the drawings, particularly Figures 1 and 2, Santoy, Chia.

magnetically responsive particles and biotin/avidin as described above. A complete guide for performing nucleic acid hybridization assays using hybridization complexes. A fully automatic device is shown. The above equipment includes xYz vipelter 11, test tube rack 13, reaction tube array 15, reaction tube A plurality of optical sensors 17 coupled to an array, a plurality of reagent wells 19, a thermal cycle 21, and a microtiter plate 23. The xYz pipettor is Conventional services for transferring various NA samples and reagents from one location to another A sampling chip 25 is provided to perform the assay. The device is fully automatic, with virtually no intervention by trained personnel. The assay has a high degree of precision and a high degree of repeatability. It is done sexually.

Nucleic acid hybridization performed by the apparatus of Figure 1 can be performed using the sandwich technique. Use. According to it, D While the NA probe is bound to biotin, its binding partner, avidin, is bound to biotin. Bind to magnetically responsive particles. The DNA sample to be tested is first bound with biotin. the combined DNA probe and a second adjacent DNA probe labeled with a reporter group. Mix with lobe and let react. The resulting mixture was then mixed with magnetically labeled avidin. Combine and react.

The magnetic label is then isolated from the unreacted DNA sample and the DNA probe. and then detect the reporter-labeled 1tDNA probe bound to the DNA sample. and complete the assay.

The xYz pipettor-11 is a conventional device available from several commercial sources. . It is operated under the control of an interlocked personal combicoater, thereby , which provides greater flexibility in selecting process details. This pipettor is Click on the pulling tip 25 and the liquid level sensor (not shown) on the tip. Direct current (pos it we-f 1ov) washing used for A cleaning section 27 is provided.

As mentioned above, this apparatus includes a reaction tube array containing reaction tubes 29 in which binding reactions occur. Including A15. More specifically, this array consists of 48 temperature-controlled reaction tubes. The wells are arranged in a 6x8 arrangement, and a permanent magnet is provided adjacent to each well. Ru. Each well further includes a 400 to 100 Orpm connected thereto; With degrees of rotation from 45 to 1080, the hour It has a variable speed motor that can alternately rotate clockwise and counterclockwise. reaction The tube well is located slightly off-center on the vertical motor shaft. 6 units Control of a group of motors including the motors is performed using the adjacent charging switch 17. The photoelectric switch is then controlled using a pipetter tip 25. This method The motor can be simply connected to the pin without the need for a separate convenience interface. It is controlled solely by moving the Petter tip.

Furthermore, as described below, a plurality of further reagent wells 19 may be provided. A variety of buffers and reagents are accommodated. To supply portex operation A particle mixer 19a connected to a motor (not shown) collects magnetically reactive particle samples. Provided to accommodate medicine. This particle mixer has a motor on and off. a photoelectric switch 31 connected to the mixer for selectively switching the However, other types of switches may also be used.

The magnetically responsive array 15 holds the various reaction tubes 29 at selected temperatures and is magnetically reactive. functions to selectively disperse and collect reactive particles. The tube containing the suspension is The particles remain in suspension as long as they are rotated by a motor connected to it. Ru.

However, when the vibration ends, the particles move toward the reaction tube side under the magnetic force of the adjacent permanent magnet. is quickly collected so that excess liquid is removed by pipettor tip 25. removed efficiently. Subsequently, more liquid reagent is added and the vibration is resumed. , instantly the magnetic particles are homogeneously resuspended.

Thermal cycler 21 is provided to perform a thermal cycle amplification reaction, and is configured to adjust the temperature. A programmable microcentrifuge test tube holder. Temperature control is preferred Specifically, it is carried out between 25°C and 110°C, and the rate of temperature change is 1'C per It takes about 3 seconds.

The amplified sample can be obtained from a thermal cycler, using a pipettor tip 25. , directly transferred to the hybridization tube 21 and reaction tube 29.

Perform nucleic acid hybridization sequence analysis with reference to the flowchart in Figure 3. A preferred method will be described below. In a first step 41, various nucleic acid samples are manually into individual test tubes in test tube holder 21. hive containing oligomers Add the hybridization solution containing the redization buffer to the reagent wells. 19b and the solution of magnetically responsive particles into the particle mixer well 19a. It will be done. After this has been done, the xYz pipettor device is used for nucleic acid hybridization. ready to start the fully automated part of the assay.

Thus, in the next step 43, the aspiration/dispensing tip of the xYz pipettor 11 is pump 25 aspirates the hybridization solution from well 19b and subsequently Continuously aspirate the nucleic acid samples from the test tubes in the tube rack 21 and combine them into a solution. into individual test reaction tubes in reaction tube array 15. hybridization solution After a predetermined time period in which xY A z-pipettor aspirates a solution of magnetically responsive particles and transfers it to the reaction tube array. Continuously transfer to a reaction tube.

Avidin molecules attached to magnetically responsive particles are then present in each solution sample. After a predetermined period of time has elapsed, in step 47, each sample is subjected to a binding reaction with biotin. The liquid portion of the liquid is removed and the remaining magnetic particles (bound DNA probe and (with the DNA sample to be obtained) using the washing buffer solution contained in well 19c. and then washed. In the next step 48, using the pipettor tip 25, The wash solution is removed from the reaction tube and the substrate buffer solution is transferred to the tube. fermentation After a predetermined period of time has elapsed, in step 49, the step 2 is performed. Turch 1 pump 25 injects the quench buffer solution from well 19d and the sample into each well. from each reaction tube to individual wells of microtiter plate 23. after that , the labeled DNA probes in these samples are read using, for example, a fluorometer. taken.

FIG. 4 shows the hybridization solution and various reaction tubes in the reaction tube array 13. Flowchart illustrating in more detail step 43 of FIG. It is the default. More specifically, in the first step 51, the pipettor suction/ The dispensing chip 25 is controlled to move to the well 19b, and the dispensing chip 25 is controlled to move to the well 19b. The chip 25 then aspirates a predetermined amount of hybridization solution. after that , in step 53, immediately transfer the chip to the first test tube in test tube 13. where the chip dispenses a predetermined amount of the nucleic acid sample contained in the test tube. ,Suction. Thereafter, in step 55, the chip is placed in the first column of the reaction tube array 15. into a reaction tube, where the chip is injected with hybridizer into the tube. Combine the solution and the nucleic acid sample and dispense. After that, on Ste.1bu57 Then, the chip is washed in the washing section 27.

Then, in step 59, the last nucleic acid sample is aspirated from the test tube bottom 13. Determine whether or not the The last tube in the row that is not being aspirated and is in operation is If not, in step 61 the sample number (n> is incremented by 1 and the sample number is The program shows the first step of aspirating hybridization solution from well 19b. Return to step 51. Finally, in step 60, hybridization Ensure that the solution and sample are dispensed into the last tube in the row of tubes being operated. So Once this has been done, the program proceeds to step 63 where the pipettor The chip 25 is attached to the optical sensor 17 for the row of reaction tubes that has just been completed. The movement activates a motor connected to the column. Step 6 5, the chip is cleaned in the cleaning section 27. Then increment the sample number. The program then returns to step 5I. There, the chip receives a predetermined amount of Aspirate the hybridization solution. Finally, in step 59, the last n Nucleic acid samples are aspirated from 13 test tubes and placed in the reaction tube array 15. Make sure it is transferred to one of the reaction tubes. The program then proceeds to step 63 , where the bipenotarch knob 25 is adjusted to the operated column (single and multiple). Move the optical sensor(s) for the reaction tube(s) 17 and correspond Start stirring the solution by switching on the motor. last Next, in step 65, the chip is cleaned in the cleaning section 27.

FIG. 5 illustrates the process of FIG. 3 in which a magnetically responsive particle solution is added to the reaction tubes of reaction tube array 15. It is a flowchart explaining step 45 in more detail. In more detail, In a first step 67, the Hihenoter aspiration/dispensing tip 25 is connected to an optical sensor. -31 to the particle mixing well containing the magnetically responsive particle solution. Start stirring the particle mixer for 19a. After a predetermined, relatively short period of time , in step 69 move the chip to the optical sensor(s) 17 The motors of the corresponding row(s) of the reaction tube array 15 are - to stop. Then, in step 71, the chip is attached to the optical sensor 31. By returning, the agitation of the particle mixer is terminated. Then, in step 73 The tip then aspirates a predetermined amount of the magnetically responsive particle solution. In step 75 , the chip stirs the particle mixer again by switching the optical sensor 31. Stir. In the next step 77, the chip transfers the magnetically responsive particle solution to the first reaction. After that, in step 79, the chips are washed in the washing section 27. do.

The program then proceeds to step 8I, where the magnetically responsive particle solution is is distributed to the last tube of the reaction tube containing the nucleic acid sample. distribution If not, the program proceeds to step 83 where the solution is It is determined whether distribution has been made to the last reaction tube among the reaction tubes in the row currently being operated. distribution If not, in step 83, increment the reaction tube number by one. The program then repeats the subroutine starting at step 71 for the next reaction tube. Revenge.

In the above subroutine, stirring of the particle mixer is ended.

Finally, in step 83, the magnetically responsive particle solution is applied to the tube cavity of the row being operated. Then, confirm that it has been distributed to the last reaction tube. Once that is done, the program proceeds to step 87 where the pipettor tip 25 finishes the operation. By switching the optical sensor 17 for the row of freshly prepared reaction tubes, Activate the motor of the column connected to. Thereafter, in step 87, the column The number is incremented and the program returns to step 69. At step 69 uses a viptor to stop the motor of the next row of reaction tube array 15.

Finally, in step 81, the magnetically responsive particle solution contains the nucleic acid sample. Make sure it is distributed to the last tube of the reaction tube. Once this is done, the program proceeds to step 91 where the pipettor tip 25 is inserted into the last reaction tube. of the reaction tube array 15 by switching the optical sensor 17 for the column of Then, the motor of the column connected to it is activated.

In step 93, the chip is cleaned in the washer 27.

FIG. 6 is a flowchart explaining the cleaning step 47 of FIG. 3 in more detail. Ru. More specifically, in a first step 95, the pipettor's aspiration/dispensing tip is turned on. By moving the tube 25 to the first optical sensor 17, the reaction tube array 15 Stop the first row motor. This column is designated by the reference number X.

When the motor stops vibrating the reaction tube, the magnetically reactive particles will It is attracted to the walls of the reaction tube by the stones.

While this is happening, in step 97 the chip 25 is washed in the washing station 27. Purify. Then, in step 99, the chip is moved to the next optical sensor 17. The motor of the second or X+1 row of the reaction tube array is to stop.

This allows a wash cycle to be carried out in the first or X row of reaction tubes. While the magnetically responsive particles are in the buffer solution for this second row of reaction tubes, can be separated from Next, in step 101, the chip 25 The buffer solution is aspirated from the center of the reaction tube C1 of No. 1, and in step 103, Then, the suctioned buffer solution is discarded and washed in the washing section 27. Then the program In step 105 of Determine whether or not suction has been made from the latter tube. If not, step 10 7, the reaction tube number (n>) is incremented by one and the program The process returns to step 101 where the liquid is sucked.

Finally, in step 105, the buffer solution is added to the six reaction tubes of the column X in question. Make sure that the last tube is aspirated, then the program will go to step 1 Proceed to 09. There, the chip 25 removes the washing buffer solution from the reagent well 19c. The liquid is aspirated, and in step 111, the washing buffer solution is applied to all six reaction tubes. distribute. After that, in step 113, the chip is cleaned in the cleaning section 27 and cleaned. In step 115, by moving the chip to the optical sensor 17 of the The motor for column X of the reaction tube array 15 is activated. This will result in 6 in this column. Stirring of the reaction tube is started, which homogenizes the solution it contains. , improves cleaning during execution.

Thereafter, in step 117, the column for the six reaction tubes that have just been operated on is It is determined whether the number X indicating the number of is the maximum value. If it is not the maximum value, the At step 119, the number X is incremented by one, and the program Return to page 99. In step 99, the optical sensor 17 for column X+t of reaction tubes is , thereby stopping the motors of the rows connected to it. again By the time you return to step 99, the motor for the reaction tube in the previous row stopping for a long enough time to allow separation of the reactive particles and the buffer solution. There is. The program then runs in a similar manner as above for the next row of reaction tubes. Repeat the subroutine.

Finally, in step 117, it is determined whether the operation was performed on the reaction tube in the last row. Check. Once this is done, the program proceeds to step 121 where it Then, it is determined whether the reference number k has become 4 or not. If it is not 4, At step 123, reset number X to 1 and increment number k by 1. do. The program returns to the first step 99, which switches the optical sensor 17. , thereby stopping the first row motor. Then the program Repeat the program subroutine in a similar manner as above, so that number k Perform 3 wash cycles until 4. Once that is done, the chip Rather than aspirating the wash buffer solution from well 19c, the substrate is drawn from well 19d. Modify step 109 to aspirate the buffer solution. Finally, step 1 At 21, it is confirmed that the four iterations of the program subroutine have been completed. , then this program subroutine ends.

Figure 7 Quench the reaction with substrate buffer and transfer the reaction sample to a microtiter plate. 23 is a flowchart illustrating step 49 in more detail. More details Specifically, in a first step 125, the aspiration/dispensing channel of the XYZ pipettor 11 is By moving the dropper 25 to the first optical sensor 17, the reaction tube array 15 is , stopping the connected first row motors. This column is designated by the reference number X . After the motor stops vibrating the reaction tube, it transfers the magnetically reactive particles to the adjacent permanent magnetic It is attracted to the reaction tube wall by the stone. While this is being done, step 125b In the step, the chip 25 is cleaned in the cleaning section 27. In step 141, the number Increment X by one. Then, in step 125, the chip is Stop the next row of reaction tube array by moving to optical sensor 17 . This ensures that the samples in column 1 are transferred to the microtiter plate. During this time, particles are allowed to separate from the buffer for this second column.

Thereafter, in step 127, the chip is cleaned in the cleaning section 27. After that, In step 129, the chip is moved to reagent well 19e, where the chip is The pump aspirates a predetermined amount of quench buffer solution. Then the chip will step 13 1, the chip is moved to the first reaction tube in the row of tubes, and then the chip is moved to the first reaction tube in the row of tubes; aspirating a sample from the sample, dispensing the sample in step 133, and dispensing the sample in step 11 At 7, place the microtie 9 into the first well in the plate 23. after that , the program proceeds to step 135 where the sample is found in the six columns of the column. It is determined whether suction has been made from the last reaction tube among the reaction tubes. Not being sucked in If so, in step 137, the number n is incremented by one and the program Then, the process returns to step 127 where the chip 11 is cleaned in the cleaning section 27.

Finally, in step 135, the sample is extracted from all n reaction tubes in the column of tubes. Confirm that it has been aspirated. Once this is done, the program goes to step 139. Then, it is determined whether the last row of reaction tubes has been operated. manipulated If not, in step 141, the column number X @ is incremented by one. , the program returns to the first step 125. There, the aspiration/dispensing tip 25 is an optical sensor for the next column of reaction tube assay 15, namely column X. Switch 17. The program then runs the same subroutine as above. Repeat, but this time for the next row of reaction tubes.

Finally, in step 139, the sample is added to all rows of reaction tubes in assay 15. Confirm that the suction has been taken from the Once this is done, the nucleic acid hybridization The fully automatic portion of the assay method is completed. Then, in step 143, Transfer the microtiter plate 23 containing n reaction samples to a suitable fluorometer. This provides an accurate and precise way to measure the degree of reaction in each DNA sample. provided.

As mentioned above, with reference to the flowchart in Figure 3-7, for interfacing continuous operations to the specific xYz pipettor being used. This can be accomplished using a suitably designed computer program. Attachment The record is [BM PS-2 Model 30-285 Personal Convenitor, or a software for some suitable program written to operate on damaged objects. This is a printout of the code. The above program is the conventional Pack Written in C for use with the Ard XYZ Pipettor.

With appropriate programming of the XYZ pipettor, other operations can also be performed. Ru. As mentioned above, an amplification step can be performed before placing the sample in the test tube. Similarly, Samples that require preparation prior to hybridization, e.g. cell lysis It is also possible to add the appropriate solution directly to the reaction tube and remove it from there. Ru.

The following examples are illustrative of the invention and are not the invention itself.

(Margin below) Example! Cloned UtV A model model in which the human immunodeficiency virus (Hm fragment) was inserted into a cloning vector. built the stem. Briefly, 5P6-1 (IV vector (pBllo -R3) to E, 1. Du Pont & De Nemours.

Obtained from Wilmington, DE. PSP-64 5st-1 restriction section A vector was constructed by cloning a 9 Kb HIV genomic insert at position.

pal (1G-R3 restricted with Baa-[1, then induced deletion of the 3-LTR region) Plasmid pMB1107 was constructed by subsequent religation. Transform cells and selected those resistant to ampicillin. The plasmid was passed through a CsC1 gradient. Cardiac isolation method (Maniatis et al., Molecule, incorporated herein by reference) ar Cloning: A Laboratory Manual, 2nd edition, C o1d Spring Harbor% New York (1989)) Purified.

9MB1107 was linearized by restriction with BamHl. BaaHl- Serial dilutions of 9MB1107 to 10mM Tris% pH 7, 5, 1mM E Manufactured using DTA (TE). 10 μl of each target dilution was added to 10 ttl in TE. ．． 01 mg/a of human placenta ([lP) DNA. Control is equal amount of T It contained E and HP DNA. Denature the sample at 95°C for 10 minutes and place on ice. Cooled in a water bath for 1 minute and briefly centrifuged to collect the concentrate. Sample on workbench into the tubes in a magnetic sample rack on top.

biotinylated and phosphatase label The oligonucleotides were prepared as described above using the sequences in Table 1. xY Initialize the processing program for the z-pimeter and set the 7 parameters such as the number of samples and number of replicates. operated on the Say variable. Using the XYz pipettor, carefully pipet 102 μl of high Bridization buffer (6XSSC, pH 8, 0, 10% formamide, 0 ．． 1% SDS, 0.1 B/+I BSA, 3 nM Biotin-N231 , -N226, -N224, -N211, -N253 and -N229, and to 3 nM AP-N218, -N220°-N227, -N233, and - N234) and 20tt1 samples were placed together in a reaction tube on a magnetic rack. each Clean the sampling tip itself between samples to reduce the risk of carryover. Lost. Hybridization was carried out for 30 minutes at 37°C with rotation.

The XYZ pipettor will automatically transfer the 6XSSC, pH 8,0° 10% solution as described above. prepared with Lumamide, 0.1% SDS, 0.1 mg/ml BSA, 1 Add 5 μl of 1 mg/ml streptavidin particles (SA particles) to each reaction. Added to the reaction tube. 15 μg of SA particles has a biotin binding capacity of approximately 60 pmole. do. Therefore, the biotin probe has a 35-fold molar excess of biotin binding sites. . Entrapment of the ternary hybridized complex onto the particles was performed for 30 min with rotation at 37°C. went.

(Margin below) Table■ probe array N178 5’-dATA ATCCACCTA TCCCAG TAG G AG AAA TN279 5l-dTTT GGT CCT 'I'GT C TT ATG TCCAGA ATG CN280 5th grade-dCAT TCT 'I'AC'l'AT TTT ATT TAA 'I'CCCAN231 5 嘗-dCTA GGT GAT ATG GCCTGA TGTN226 5 '-accc TAT CAT TrT TGG TTT CCA TN22 4 5f-dTGT TGA CAG GTG TAG GTCCTA [215 '-dCTG GCr TTA ATT TTA CrG GTA CAN2 53 5 Lightning-dTGCCAT TTG TACTGCTGT CTTN229 5+-dTGCCACACAATCATCACC'rGC[2185l-dTA GAGGGTTGCTACTGTATTATN220 5l-dTAG TTC CTG CTA TGT CACT’rCCN227 5l-dCTGTCTB TACTTTGATAAAAACCTCN233 5l-dTATTCTTTCC CCTGCACTGTAN234 5-dcrG TAA TAA ACCC GA AAA TTT TGAA105' 51-dCCCGAG CCG ATG ACT 'I'AC'I'GG CA214 5l-dGATATCT CACCCTGGTCGAGGCGGTA209 5l-dTGT GTG G TG TAG ATG TTCGCG ATT CN174 5’-dCAG GAG CAG ATG ATA CAG TAT TAGC1035l-d AGG CGT TTCCACATCTAT ATA GTC2045'-d AGT ATCATCACCCACGAT GTG CTXYz pipettor is The rotation of the reaction tube was stopped by automatically activating the light switch on the sampling chip.

When the rotation is stopped, the particles are quickly removed from the solution by permanent magnets attached to all sides. It was removed. The XYZ pipettor automatically aspirates buffer from each reaction tube and places it in place. Two samples were washed between each sample in a given row.

The xYz pipettor automatically aspirates and dispenses 200μl of wash buffer (4XSSC, O,Lχ5DS) was dispensed into each reaction tube to wash the chip between each sample.

The xYz pipettor then activates a light switch to begin rotating the tubes in a given row. I let it happen. The xYz pipettor cleans the next row of tubes as described above until all samples have been processed. I continued until I was done. Repeat this process two more times for a total of three washes each. The test was carried out at 37°C for 5 minutes.

The above washing step was carried out using 0.1 M jetanolamine, ( DEA), pH 9,0 (JBL), 150 μm substrate relaxation in S mM MgCh. Solution (30aM 4-methylumbelliferyl phosphate (4-MUBP) , JBL 5 scientific1 San Louts 0 bispos The procedure was repeated one more time by dispensing CA) into each reaction tube. using a substrate The incubation was performed at 37°C for 60 minutes with rotation.

Again, the sampling tip pipenoter automatically activates the light switch to place the sampling tip in the predetermined column. The rotation of the reaction tube was stopped and the particles were removed from the solution. xYz pipettor is a reagent Add 40 μl of 10°0 mM EDTA from the rack and 110 μl from the reaction tube. Aspirate the substrate buffer and dispense the liquid into the wells of the microtiter plate. did.

The xYZ pipettor cleaned the tip between each sample. The xYz pipettor is Quenching/transferring rows of tubes continued until all samples were processed.

An alarm sounds at the end of the program to notify the user that the assay is complete. I set it. Fluorescent plate that automatically scans each well of a microtiter plate Toreader (Pandax FCA% Baxtar Healthara, Pandex Division% Mundelein.

IL). Alternatively, a manual single-well fluorometer may be used.

Plates can be read immediately or within 16 hours after completion. So The results are shown in Table II.

The results show that the minimum extrapolated detection limit is 9MB1 based on a noise-to-signal ratio of 2.0. It is shown that 107 plasmid targets 4XIO' copies. This result is the target It is linear over three orders of magnitude with respect to level. Large number of acquisition/revolution lobe pairs This increases the overall sensitivity by increasing the signal without proportionally increasing the noise. let it go up

Table■ pMB I 107 serial dilution: Composite Santo Ibuchi Pair 500, Goo 10 2.5 5,000.000 66 16.5 50,000.000 522 130.5500,000.000 451? 1129.3 Example ■ of viral DNA - and This embodiment of the invention provides a semi-rotating target amplification assembly in an HSV model system. It was shown that the method can be combined with automated sandwich assays. open up Amplification in tubes can be performed using XYz pipettes without serious contamination or carryover. It has been shown that this can be done by a computer.

The high sensitivity of target amplification makes the assay unduly susceptible to internal and external contamination. Easy to access. The amplification reaction is clean (i.e. not exposed to the amplification product) and biological The test was carried out in a safe indoor hood, isolated from the XYZ pipettor. disposable piston Clean positive displacement pipette (FDP) with pipe and capillary (Rainin , Woburn, MA), sterile tissue grade water (Sigma Che 1ca1, St., Louts, MO), sterile light mineral oil (Sigma), and a sterile 0.6 ml snap cap tube (Rob binsScientific, 5unnyvale, CA) to minimize contamination. It was used to limit the

New Taq l thermostable recombinant DNA polymerase (3,1 units Ampli Taq Cetus, Emeryville, CA) with 80 μm amplification buffer. solution (62.5mM ICI, 12.5mM Tris-HCI, pH8, 3,1,88mM MgC1a, 0.13% (W/V) gelatin, 250aM dNTP, 0.31aM biotin-A105 and 1,25aM A214) added to. 20 μl water, and 20 μl template (IX104:ff Bee pH 5V106 (Bethesda Re5earch Laboratory s, Gaithersburg MD)) for negative and positive samples, respectively. added to. The final reaction mixture consisted of 50 mM KCI, 10 mM Tris -CI, pH 8, 3, 1, 5mM MgCl2.0.1% (v/v) gelatin, 200μ each dNTP (Pharvacia, Piseatavay, N J), 0.25aM biotin-A105 forward primer, and 1.0aM Contains A214 reverse primer and 2.5 units of AspliTaq. Ta. The sample was overlaid with 100 μl of light mineral oil. Put a lid on the control test tube to be amplified. Place the test tube to be tested uncovered on the workbench of the xYz pipettor. A thermocycler modified as follows (Erico 6. San Diego, CA ). Amplification and sandwich protocols were run simultaneously. sa Ntoifuchi was delayed by 210 minutes. Completion of the amplification protocol takes approximately 2 Denaturation at 94°C for 1 min; 55°C for 1 min and 94°C for 1 min. 30 amplification cycles; extension at 72°C for 5 minutes; and denaturation at 95°C for 10 minutes. The process was carried out. As a result, the amplification product is 109 times larger than the target IX with 100% efficiency. was produced.

The amplified products were subjected to agarose gel electrophoresis, dot plot method, sine plot method, and analyzed by the Sand-Deutsch method. The Sanderutsch method was as described in Example I. AP-A209 was used as a detection probe. This obtained quantitative results It was the only way.

The closed tubes were opened manually at the end of the amplification program. xY2 pipettor is 1 0 mM Tris-CI, pH 7,5, equilibrated with 1*M EDTA, Automatically invert aqueous and oil phases by dispensing 200 μI of chloroform I let it happen. The oil/chloroform phase sank to the bottom and the aqueous phase rose to the top. Next, XYZ Bi Pefter prepared 102 μl of hybridization containing 3 nMAP-A209. Gon buffer (6XSSC% pH8.0, 1O%FAM, 0.1$ SOS, 0.1 mg/ml BSA) and 20 u1 of amplification product from the thermal cycler. Combined. An automated sandwich assay was performed as described in Example I. sun Biotin in the German assay (free biotin primer and biotinylated It is necessary to ensure that the total amount of amplified products does not exceed the binding capacity of the SA-particles. 2 0 μl of 0.25 μM biotin primer solution has a particle binding capacity of 60 pm. 1e This is equivalent to a total of 5 pHoles of biotin, which is 12 times less.

The results are subjected to target amplification on the bench of an xYz pipettor, followed by automated sampling. It is shown that a German assay can be performed. By phase inversion process, sample Using a sampling tip, the sample can be aspirated without coming into contact with the oil phase. The frequency of carryover is significantly reduced. The frequency of contamination (cf) is twice the relative fluorescence unit (rfu) of a closed negative sample In the number of open negative samples giving rfu greater than stipulated.

C = # rfu of open tube negative sample > 2x rfu of closed tube negative sample # open tube negative sample sum of CF=O/47 (average result of 9 different experiments) Typical open tube amplification assay results is shown below.

+ or - mean relative fluorescence units idan thick base once bean 51 - Yui 1 vol 8 [shi - closed 20 (22; 1g> +22,742 (22,900; 22.5δ4) -43 (44:43) (Average of negative samples in closed tubes = 32 rfu) Open 42 (26; 5 g) + 23178 (22,610: 23,745) - 50 (56; 44) +22991 (22,952; 23.030) -54 (54; 54) + 22,694 (22,894: 22.694>-3632・40 Example I11 of HIV ■IV-infected CEM-C with approximately 1 to 20 copies of old V per cell M3 cells (ATCC accession number Tl8195) were collected and used for PCR as described below. Prepared. Alternatively, reverse transcriptase can be used to form cDNA from RNA. , this cDNA may be used in PCR.

CEM-0M3 cells were grown in RMPI 1640 medium (M, A) in a 75 cc flask. , BioproductsSWalkersville%MD), 10% of fetuses Calf serum, 1% penicillin-streptomycin, 1% Fungizon e (M, A.

Bioproducts), and 1% Shiichi Glutamine in 5% CO2, Cultured at 37°C. Cells were subcultured every 5 to 6 days at a ratio of 1=6. contrast Cells were subcultured in a ratio of 5 ml C5M-CN3:30 ml RMPI medium. fed. HIM-infected CEM-0M3 cells, 5 ml HIV-infected CEM-0M3 Cells: Passaged in the ratio of 10ml CEM-CN3 and 20ml RMPI medium Cultured.

Add uninfected CEM-0M3 cells to infected cells to increase virus titer. I set it. Old V CEM-CM3 continuous passage cell line without increasing virus titer In order to maintain the added to the ground. Cells were harvested after 4 to 5 days, when titer was maximal. cells The pellet was pelleted in a clinical centrifuge at 11,000 rpm for 15 minutes. Discard the supernatant. The pellet was then resuspended in 5 ml of IXPBS. Quantify cell concentration with blood cell needle The usual result was 2-5X106 cells/ml. Adjust concentration to suit did.

Serial dilution of HIV-infected CEM-0M3 cells from 1X10' to 1X10' The solution was spiked into 1X10' uninfected CEM-CM3 cells. cells (Vf=65 μl) was dissolved as follows. 500μl outer membrane lysis buffer (10mM T ris-CI, pH 8, 0, 1, 5mM MgCl2.140mM NaCl and 0.5% NP-40) and portex the mixture into a microcentrifuge tube. and centrifuged. The pellet was mixed with 50 μl of nuclear membrane lysis buffer (SOmM [C L 10 rxM Tris-C 1% pH 8, 3, 2, 5mM MgC 1g, 0 ．． 1% (v/v) gelatin, 0.05 mg/ml proteinase K, 20 d Resuspend in DDT and 1.7 gM 5DS) and incubate for 1 hour at 55°C. I started. Samples were boiled for 20 minutes to inactivate proteinase K and centrifuged. The concentrate was collected by separation machine. In a laminar flow hood, transfer 25 μl of the sample mixture to 75 μl. μl of amplification buffer (671M KCI, 13raM Tris-CI, pH 8 ,3,2,0g+M MgCl2.0.13% gelatin, 267gM each dNTP , 0.33 μM biotin-N174, 1.3 μM 8224, and 3.3 mono Newly added AmpliTaq; Cetus, Emmeryvilie , CA) to make a final concentration of 50mM Il:C1, 10mM Tri s-C1, pH 8, 3, 1, 5mM MgCl2, Q, L% (w/v) Zera Chin, 200 gM each dNTP, 0.25 μM Hio f:/-NL74 forward protein. primer and 1.0 gM N224 reverse primer and 2.5 units of new A thermostable recombinant DNA polymerase (AmpliTaq; Cetus Company, Emeryvilles CA). Place the sample tube on the thermal cycler , denaturation at 94°C for 1 min; amplification cycle of 1 min at 55°C and 1 min at 94°C. 30 times; and extension at 72° C. for 5 minutes. of the amplified product Using AP-N227 as a detection probe, 20 μl of the sample was used for Example ① and The samples were assayed by the Sand-Deutsch method as described in [11] and [11].

Experimental results are inconsistent, but after target amplification, automated sandwich assays Indicates that 0 old V-infected cells could be detected. The total amount of signal produced is is not proportional to the amount of target present. This may depend on the restriction reaction conditions and/or This may be due to inhibition by cell components. In the latter case, IXIG' uninfected cells This is supported because it was completely inhibited. Sandwich assays are not limiting It was. This is because there is a 10-fold molar excess of biotin-binding sites on the particles, and M The turnover of UBP is approximately 70.0OOrfu. It is et al.

Table II+ 10. Old V-CEM unit of relative fluorescence, average fluorescence, and standard error per OOOCEM (rfu) Unit 1.000 22060 10.000 23470 100, Goo 8 Example! V The old V of the trial This embodiment of the invention allows an automated system to detect HIV infections in blood samples of seropositive patients. It has been shown that the virus can be detected.

Blood was collected from the donor and was heparinized or treated with EDTA. The mixture was placed in a 0.10 liter vacuum container. Maximum at room temperature after sample collection Stored for up to 24 hours.

The blood container was inverted several times to mix thoroughly. Approximately 7.0 liters of blood in total Removed as a pet. Transfer the blood to a cell separation tube (LeukoPrep Cat, No. , 2750-2752, Becton-Dickinson, Ruther Ford, NJ) and capped with the rubber stopper that came with the product.

Further 2. hl's 1x PBS (120mM NaCl, 2, 7mM KCI.

10 mM phosphate buffer) was added to bring the total volume to 9.0 ml. Leukopre Infectious material was inhibited by incubating LeukoPrep tubes at 1600Xg for 20 minutes at room temperature. centrifuged in rotators and centrifuges designed to After centrifugation, blood The upper layer containing serum was removed and discarded. Remove the "white blood cell layer" containing mononuclear cells, 1 ．． Transferred to a 5 ml Eppendorf tube. From here, collect 0.5 ml of mononuclear cells. The mixture was transferred to a 1.5-all container Enovenodorf tube with a screw cap. Discard remaining cells. It was. Other methods for isolating mononuclear cells include, for example, 5eplacel I density gradient. and Ficoll-Hypague.

Half') Nottle's lysis buffer (50mM MC1, 10mM Tris-CI, pH 11.3 (25℃), 2.5M MgC1z, o, xmg 1 layer 1 gelatin 0.45% MP40, 0.45% Tween-20, and 60 μg/sl Proteinase K) added immediately before use was added to the nuclear pellet. Pouring the cell nucleus Disintegrated by lutex and/or by mixing with a pipette resuspended. Incubate for 1 hour at 55°C for optimal proteinase activity. This was promoted by Proteinase K was inactivated and the sample was The genomic DNA was completely denatured by boiling for minutes. Centrifuge the sample briefly It was collected.

Amplification of target DNA was performed as follows. 75 μl amplification as described in Example III Transfer the buffer solution into a 0.6 ml Eppendorf tube (Robbins 5cienti). fic). 25 μm cell lysate (approximately 100 cells or lag (l) og) genomic DNA) was added and mixed with a pipette. The final reaction mixture was 10 mM Tris-CI, pH 8.3 (25°C), 50 mM KCI, 1. 5mM MgCh, 0.1% (v/v) gelatin, 200ttM each dNTP (Pharmacia), 0.25 μM biotin-8178 forward same primer , IμMN279 reverse primer, and 2.5 units of A11)l1Ta q (Cetus). Add 100ttl of oil to the sample (Sigma light) was overlaid. Samples were prepared as Er1coa+p as described in Example III. Programmable Cyclic Reactor (San Di ego, CA).

Amplification for assays on the automated Santoi Fuchi hybridization system The product was prepared as follows. Pour the 25 μm HIV amplification product into a 2.0 ml volume microtube. Added to 25 μm of 10 mM TrlSsp 8.0 in a small centrifuge tube. this rare The dilution solution provided a 1/10 analysis of the PCR reaction. Example I This was performed using AP-N280 as the detection probe as described in . The result Shown in Table IV.

Omoteka V 11 Jodan Kansa” Shishi 4th place Negative 1 2986 Negative 2 2620 The above data are from one experiment. A positive cutoff result is this The assay was twice as high as the highest negative control. Although the negative control is significantly higher, 4 All results were serologically correlated.

Example V Camlobacter In accordance with this embodiment of the invention, automated sandwich assays It has been shown that 5s RNA purified from ER-infected stool samples can be detected.

Several Ca. pylobacter-infected stool samples were analyzed using the conventional dot plot method. It was analyzed using Briefly, 75 μm denaturing reagent (0.2 N HCI) An ion exchange column (Extractor-10”, Molecular Bi 2++1 eluate from San Diego, CA) and denatured for 5 minutes at room temperature. The denatured sample was transferred to a trans′′ membrane ( Equilibrated centrifuge tube with transverse me + *brane) (Gene 5clean Plus; DuPont/NEN, Bosto ns MA). The apparatus was rotated at 750×g for 20 minutes. take out the membrane and placed in fixation reagent (1M sodium carbonate) for 30 seconds. Add this in distilled water for 3 Stir for 0 seconds and blot dry onto Whatman 3MM paper. Hybrid membrane Transfer to a hybridization bag and add 2 ml of hybridization buffer (0, 75M sodium citrate, 1% sarcosine, 0.5% BSA, 0.6% Ka thon (Rohm and Haass Ph1ladelphia, P A)) Brehybridization was carried out at 50° C. for 15 minutes. Discard the buffer, 2. 2 ml hybridization buffer containing 5 nM AP-C103b has been newly added. Hybridization was carried out for 15 minutes at 50°C. produce Buffer 1 (2x SSC, pH 7,0,0,5% Sarcosine, 0.12% Kathon) for 20 minutes. , then at room temperature working membrane wash buffer 2 (lx SSC% pH 7, 0, 0, 5% tr It was washed for 10 minutes with iton (X100.0, 12% Kathon). plastic membrane I put it in a stick bag and added the newly added nitro blue tetrazolium (NBT; Ameresco, 5olon, OH) and 5-bromo-4-chloro- Solution (3.7uM NBT, 4.6uM BCIP, 0.1M tris -CI, pH7,5,0,1M NaCl1,0,05M MgCl2.0.02 % NaN5) for 4 hours at 37°C. Weak positive and A tonic as well as a negative control was identified.

Traditional manual column and semi-automated bulk affinity chromatography purification methods The automatic magnetic sander was compared with Germany Chia, Sei. In manual column format, 2 00 μl sample, 800 μl dilution reagent (10% formalin, 0.1M sodium phosphate) thorium, pH 7,0) and 2000 μm lysis reagent (8M urea, 0.25% SDS, 0.25% sarcosine, 0.05M EDTA) for 30 minutes. Incubation was carried out at 50°C. Samples were prepared in advance containing 1 ml of anion exchange resin. It was applied to an equilibrated extraction column. Wash the column with 15 ml of Wash Reagent 1 (40% ether). Nor, 0.2M NaCl, 0.02M Tris-CI, pH 7.5) , 5ml of washing reagent 2 (0,25M NaCl, 0,02M tris-CI .

pH 7, 5, 0,05% NaN5) and 21 elution reagent (0,5M NaN5). CL　O,02M　Tris-C11pH7,5,0,05%　NaN5) I let it out.

The eluate was concentrated to 330 μl by lyophilization. The final concentration of the sample is In a volume of 400 μl, 10 volumes of formamide, 0.1% SDS.

It was adjusted to 3.75 nM biotin-C204b. sample for 10 minutes9 Denature at 5 °C, cool in ice water, briefly rotate to collect concentrate, xyz Pipettor - placed in test tube rack.

Semi-automatic bulk format includes 200 μl sample, 100 μl diluent reagent, and 600 μm lysis reagent together and equilibrated for 30 minutes at 50°C. Offered to Rix. The sample tube was placed on a magnetic rack and stirred for 10 minutes at 37°C. tube was stopped intentionally and the matrix was allowed to stand for 5 minutes. Sampling chip is the best 300 μl of liquid remaining in the lix bed was aspirated. sampling chip Then, 300 μl of Wash Reagent 1 was added and the tube was agitated for 5 minutes before being stopped. Matri The mixture was allowed to stand and 300 μl was aspirated. Repeat this step using washing reagent l. Repeated once and then twice using Wash Reagent 2. 400 μl of target It was eluted from the matrix with 8°33X SSC. Aspirate 330 μl of sample Ta. Samples were prepared at a final concentration of 10% formamide, 0.1% in a final volume of 400 μl. SOS was adjusted to 3.75 nM biotin-C204b. trial Denature the sample at 95°C for 10 minutes, cool in ice water, and collect the concentrate by gentle rotation. and placed it on the sample rack.

xYz Pipettor - Runs the program to allow the operator to change the indicated variables. I input it. The sampling chip automatically contains 15 nM AP-C103b , Lot) μl of hybridization buffer to 400 μl of reaction tube! put in I put it together. The remaining assays were carried out in wash buffer at 1X5 as described in Example ■. This was done using 2XSSC instead of SC.

As a result, semi-automated sample preparation and sandwich hybridization The method was found to be feasible using a magnetic rack and an XYZ pipettor. Ta. Bulk chromatography formats are preferred compared to column formats. Yes. Sample #1270c is more viscous than the others, which indicates that the bulk format contributes to the reduction of the signal in

Table V Campylobacter Sandwich Column vs. Bulk Sample Purification Relative Fluorescence position (actual value) ・・LL column bulk column method negative 1780 52 (36: 68) 35 (34: 36) 67% slightly positive 1791 176 (184; 168) 293 (252; 334) 167% strongly positive 1270c 5 319 (5424; 5214) 3140 (3072; 3208>59% Example Vl ■Due to the affinity of v″・: In this embodiment of the present invention, sample preparation Preparation and enrichment were performed using automated systems and affinity capture methods. this Advantages of the method include target enrichment without the need for amplification and compatibility with automation. can be mentioned. Old V DNA in blood was detected in the examples listed below.

Blood samples were collected and transferred to Leucoprep tubes as described in Example IV. , centrifuged. Remove the cap and place the tube on the workbench of the xYz pipettor. I placed it on the sample rack above and started a program that quickly performed the following steps. sa Using a sampling tip, transfer 1 Ill of mononuclear cells from the "white blood cell layer" to a reaction tube on a magnetic rack. Moved to. Lyse the cells and add 1111 of 4.0 M guanidine thiocyanate, 1 Nucleic acids were denatured by adding 0 mM EDTA, pH 8.0, and Incubate for minutes at 37°C with stirring.

Hypuridase buffer (15Xssc, 25% formamide, 0.1 25% SOS, 0.125% sulfucin, 0125 mg/ml BSA, and 7, adding biotin capture probe in SnM biotin-N178). The tube was stirred for 30 minutes at 37°C. Stress of 30 μg of paramagnetic material derived from Fe5Oa Add putavidin particles (Advanced Magnetics) for 30 min. Stirred at 37° C. for minutes. Stop the agitation and remove the bound biomolecules that have hybridized to the target nucleic acid. Particles with Chin capture probe were collected on the side of the tube. Heterologous nucleic acids and cellular debris The supernatant containing li was aspirated. Particles were injected twice over 5 minutes each in 200 μl of 20 Wash with stirring in raM TrisSpH 8, 3, 250mM KCl. Ta. Add target nucleic acid to 20 μl of capture probe:target hybrid at 0.25 M It is released from the particles by denaturation with stirring in KOH and then without stirring. 20μm of 0.25M HCL, 150mM Tris, pH 8.3 Neutralize for 5 minutes. If RNA is the intended target, the addition of base and acid It was reversed. The supernatant (40 μm) was transferred to a new tube and amplified as described in Example IV. Sandwich assays were performed as follows.

This step concentrated the target approximately 200 times in an automated format. The method is compatible with various types of samples and sample preparation processes.

Although the present invention has been described in terms of preferred embodiments, the present invention does not depart from the spirit of the invention. It should be understood that various modifications may be made within the scope. Therefore, the present invention is covered by the following patent claims. limited only by the scope of the request.

FIG.3 FIG.4 FIG.7 abstract The present invention performs a fully automated nucleic acid 7% hybridization assay. Provide an improved method. In this method, magnetically responsive particles with immobilized ligands are Use children.

Copy and translation of amendment) Submission (Article 184-7, Paragraph 1 of the Patent Act) October 1992 6th (to)

Claims (21)

[Claims] 1. 1. A method of performing a nucleic acid hybridization assay, the method comprising: A programmable XYZ pipettor with an aspiration/dispensing tip and also a linked providing a plurality of linked wells and a linked array of test tube holders in a magnetic field; wherein each test tube holder accommodates a test tube and holds the test tube along its longitudinal axis. a process adapted to selectively rotate the DNA sample; A binding reaction occurs between the hybridization solution and the magnetically responsive particles, increasing the precision to provide a well-reproducible nucleic acid hybridization assay. Nucleate separate test tubes housed in multiple test tube holders using an XYZ pipettor Transfer the DNA sample to the test tube and add the hybridization solution to the multiple test tubes a predetermined number of times. transfer the solution of magnetically responsive particles to the plurality of test tubes a predetermined number of times; selectively rotating the test tube a predetermined number of times. 2. 1. A method of performing a nucleic acid hybridization assay, the method comprising: (a) a programmable XYZ pipettor with an aspiration/dispensing tip; and , with multiple connected wells and each test tube holder to accommodate a test tube. a connected array of test tube holders; a connected array of test tube holders; means for causing relative rotation about the longitudinal axis of the test tube; and an associated microphone. (b) providing a rotator plate with a hybridization solution; one well, the solution of magnetically responsive particles in the second well, and the wash buffer solution in the third well. placing the substrate buffer solution in the fourth well and the quench buffer solution in the fifth well; (c) transferring separate DNA samples to multiple samples contained in the array of the test tube holder; (d) using the XYZ pipettor to place a predetermined amount of the hive into a test tube; Transfer the redization solution from the first well to each of the plurality of test tubes sequentially. (e) using the XYZ pipettor to cause a binding reaction there; A quantitative solution of the magnetically responsive particles is connected from the second well to each of the plurality of test tubes. The process of subsequently transferring (f) said magnetically responsive particles remain in liquid suspension and bind to said separate DNA sample; A relative rotation is caused between the test tube and its coupled magnetic field to cause a reaction. (g) the magnetic field causes the magnetically responsive particles to stopping the process of causing relative rotation so as to move to a new position; (h) Hybridize the unbound DNA sample using the XYZ pipettor. wash buffer solution from each of the plurality of test tubes, and then remove the wash buffer solution from each of the plurality of test tubes. , from the third well to each of the plurality of test tubes, and then the wash buffer solution: removing from each of the plurality of test tubes, (i) using the XYZ pipettor, (j) transferring the substrate buffer solution from the fourth well to each of the plurality of test tubes; The magnetically responsive particles remain in liquid suspension where the enzymatic reaction takes place. (k) causing relative rotation between the test tube and its connected magnetic field; A magnetic field causes the magnetically responsive particles to move to a selected location within the test tube. a step of stopping the step of causing relative rotation of the sea urchin; (l) Use the XYZ pipettor to remove the quench buffer from the fifth well. into each of a plurality of test tubes and then into separate wells of the microtiter plate. transfer the sample from each of the plurality of test tubes to the hybridization tube. The degree of binding reaction between the solution and the separate DNA samples can be conveniently assayed. A process that can be done, How to include. 3. A method for performing the nucleic acid hybridization assay according to claim 2, , The XYZ pipettor is further coupled to an optical sensor to cause relative rotation. the above steps (f) and (j), and the steps (g) and and (k) the sampling tip of the XYZ pipette by the optical sensor. This is accomplished by moving the device to a selected location where it can be detected. Method. 4. A method for performing the nucleic acid hybridization assay according to claim 2, , The step (e) using the XYZ pipettor uses the XYZ pipettor. carried out in a predetermined first period after the step (d), and the first period includes the hybridization any binding reaction between the reaction buffer and the DNA sample is substantially complete. It is defined as the time to ensure that The step (h) using the XYZ pipettor is performed before using the XYZ pipettor. It is carried out in a predetermined second period after the step (e), and the second period is performed in which the fine particles and the DNA as a time to ensure that any binding reactions with the sample are substantially complete. stipulated, and The step (1) using the the third phase is defined as allowing the enzymatic step to take place; Method. 5. Step (h) using the XYZ pipettor and transferring the wash buffer solution. and the sub-step of removing the wash buffer solution is repeated multiple times in succession. A method for performing the nucleic acid hybridization assay according to item 2. 6. A method for performing the nucleic acid hybridization assay according to claim 2, , The XYZ pipettor further cleans the sampling tip. from one of the plurality of wells to one of the plurality of test tubes. the sampling to transfer the solution and remove the solution from one of the plurality of test tubes; Immediately after each use of the tip, apply a treatment for cleaning the sampling tip. further comprising the step of using a cleaning section; Method. 7. A method for performing the nucleic acid hybridization assay according to claim 2, , The hybridization solution contains an enzyme-conjugated DNA probe and a biotinylation solution. a DNA probe bound to a DNA molecule, and A method, wherein an avidin molecule is bound to the magnetically responsive particles. 8. the enzyme exhibits fluorescence, and Using a fluorometer, measure the sample contained in the microtiter plate. The nucleic acid hybridization method according to claim 7, further comprising the step of assaying. How to perform a cation assay. 9. Before the step c, performing a step of amplifying the sample in a thermal cycler. Performing a nucleic acid hybridization assay according to claim 2, further comprising: Method. 10. 10. The method of claim 9, wherein the amplification step is repeated at least once. 11. Before step c, performing a sample preparation step using affinity capture. Performing the nucleic acid hybridization assay according to claim 2, further comprising: How to do it. 12. Using magnetically responsive particles to separate single-stranded nucleic acids from double-stranded nucleic acids An improved method of performing a nucleic acid hybridization assay, comprising: 13. The improvement has avidin stably attached via a covalent spacer. 13. The magnetically responsive particles of claim 12, further comprising using the magnetically responsive particles to Method. 14. Attached via a covalent linker containing multiple alternating hydrocarbon and amide residues 1. A composition of matter comprising magnetically responsive particles having a ligand thereon. 15. 15. The linker of claim 14, wherein the linker comprises a total of at least about 3 residues. Composition of substances listed above. 16. 14. The ligand is avidin or streptavidin. Composition of the substances described in . 17. 15. A composition of matter according to claim 14, wherein the ligand is an antibody. 18. Approximately more than 100 pmole of ligand binding sites per mg of dry particles A composition of matter comprising magnetically responsive particles immobilized in a composition of matter. 19. 18. The ligand is avidin or streptavidin. Composition of the substances described in . 20. 19. A composition of matter according to claim 18, wherein the ligand is an antibody. 21. Producing magnetically responsive particles useful for nucleic acid hybridization assays A method, coupling an amine or carboxyl end group to the magnetically responsive particles; and terminal amines or carbons to form multiple alternating hydrocarbon and amide residues. successively adding moieties containing carboxyl groups.