JPH01501339A - Improved nucleic acid hybridization method and kit used therefor - Google Patents

Abstract

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

Description

[Detailed description of the invention] Improved nucleic acid hybridization method and kit used therefor 1-Once The present invention relates to nucleic acid hybridization (crossing) methods and uses in the methods. Regarding diagnostic kits.

All articles and references identified in this specification are hereby incorporated by reference. It will be done.

In traditional microbial diagnosis, the presence of a microorganism in a sample is determined by the isolation of the microorganism. It is determined by After enrichment culture of WL organisms, their biochemical properties or their immunological properties are determined. It is necessary that the quality be determined based on either. Such identification is done by hand. This is very time consuming and time consuming.

Advances in genetic research have enabled microorganisms to survive, even if they are not viable. , it has become clear that it can be identified by the specific nucleic acid it contains. Nucleic acid is It can be a deoxylipo-nucleus @ (DNA) which is usually double-stranded, or it can be usually double-stranded. may be ribonucleic acid (RNA), such as bacteria, fungi, viruses, yeast, etc. All living organisms are identified in this way. Generally, identification operations involve artificially induced lysis, which destroys the cell walls of the organism and is identified This includes releasing the desired nucleic acid.

The hydrogen-bonded structure of the double helix of a nucleic acid molecule (e.g. DNA) is It can be destroyed by reprocessing. There is a covalent bond connecting opposing amounts of lead. Because there are no hydrogen bonds in the two polynucleotide chains of a double nucleic acid molecule, all hydrogen bonds are Completely separates when destroyed. This strand separation step is called denaturation. Non-denatured nucleic acid An always useful property is that under appropriate conditions this reaction can be re-produced by two complementary strands from the same material. It is possible to form N-turns so that it can be formed into a double helix. This is again called raw. Regeneration involves two complementary nucleotide sequences separated by denaturation. Contains reactions. This technique also allows any complementary nucleotide sequences to be linked together. Knealing to create a double structure - between end-strand DNA and RNA or from two different sources Nucleotide sequences from different nucleic acid parts can also be generated, such as reactions between single-stranded DNA. This is indicated as hybridization (hybridization).

Nucleic acid hybridization is a well-known method for identifying specific nucleic acids.

The ability to hybridize two single-stranded nucleic acid products is the basis of current nucleic acid assays. Ru. The principle of these assays is that two single-stranded nucleic acid products are brought into contact with each other and then shaped. The objective is to measure the amount of double-stranded products formed.

Hybridization assays usually involve polynucleotide hybridization. Includes use of probes. Probes are generally single-stranded nucleic acid fragments, or consists of denatured double-stranded fragments. The probe is the target nucleic acid (to be identified). has a specific nucleotide sequence that is complementary to the nucleotide sequence on the nucleic acid (nucleic acid) It is characterized by: The probe and target nucleotide (-terminated) are - When brought together, complementary sequences pair to form a double molecule. The probe is one It is generally prepared in the form of a purified reagent and contains an easily detectable mark in the molecular structure of the probe. Knowledge is incorporated. Therefore, the presence of the target nucleotide indicates that the hybridization carrying the label This can be confirmed by the formation of double molecules.

Polynucleotide hybridization probes are “target” nucleotide sequences It is inexpensive to test, localize and isolate, and has an interesting background to diization. provides information on the subject, including a discussion of its preparation and use. Polinuku Known methods for preparing leotide hybridization probes and those probes The use of is well described in the literature. Southern, Journal of Mo. Recular Biology, 98:503-517 (1975), Falkow et al. , U.S. Pat. No. 4,385,535.

Seedinges of the National Academy of Sciences The Ninis A. 79:4381-4385 (1982) and Langer et al. Proceedings of the National Academy of Sciences See Of the Ninnis Oxgo, 6633-6637 (1981) Those known global technologies, as disclosed in these and other documents, The probe preparation method typically involves cloning the probe region into a double-stranded DNA plasmid. This includes Plasmids carrying the probe region are typically enzymatically It is made into a marker material using polymerization technology.

This technique includes, for example, the translocation method (Rigby et al., of Biol. G., 20: 290 (1976)) and the terminal addition method. These methods are performed in the presence of modified nucleotide triphosphates. plow Nucleic acids can also be labeled by chemical means using habten or biotin. (Cheno et al., P.N.A.S. 81:3466 (1984), Fo. Star et al., Nucleitsk Acids Research 1.3 Near 45 (1985) , Viscidi et al., Journal of Microbiology 23:3 11 (1986).

In principle, there are two methods to perform the hybridization test.

i.e., hybridization to a solution (or liquid) and hybridization to a solid carrier. There is dization. In solution hybridization, -end chains are The prepared nucleic acid preparations are mixed together. For large amounts of sample, the reaction is a change in optical density. It can be tracked by. In small quantities, the probe may be labeled with a radioactive label, etc. shall be marked with an easily detectable sign. Unreacted single strands are separated from the double strands, Double-stranded nucleic acids are identified by detecting the presence of a label within them. can do.

Hydroxyapatite (HA) removes hybridized probes in solution from unhybridized probes. It has been used as a standard method for separation from lobes. Under normal conditions, HA It binds selectively to hybridized DNA probes, but does not bind to unhybridized lid probes. It doesn't match. Other methods are used to separate hybridized probes from unhybridized probes. The method described above can also be used. One method involves the use of specific enzymes, viz. The use of S1 nuclease to selectively degrade unhybridized probes into small fragments. Includes usage. Double-stranded molecules are unaffected by this enzyme. The tube is a well-known size separation method. Page 363, edited by Grossman and Mordashi, Academic Press, New York. 1974 Solution hybridization has advantages in terms of speed and reaction efficiency. Ru. However, it also has one major drawback. Pros that did not cross the crossed probes The separation stage is generally labor-intensive and time-consuming and cannot be automated. For example, the size separation techniques described above are relatively non-specific and ineffective. It is efficient and has poor reproducibility. Although it is more specific than the HA component force method, This method is generally used when the target polynucleotide is present in large amounts relative to the probe. useful only when the target is RNA or both. be. Additionally, unpurified samples such as plant and animal tissue homogenates, blood , feces, Js and urethral mucus, etc., it is very difficult to perform effective separation. .

In the case of hybridization to a solid carrier, one of the terminal-stranded nucleic acid preparations is transferred to a solid carrier. It is fixed by fixing it on the rear. at either end of the polynucleotide There are numerous ways to connect the ends to a given support, e.g. , A., and Boonian, M., Methods in Enzymology, Volume XXX. See also Volume IV, Part B, 463-475.1974; Derivatives of 1 such as those carrying a terminal aminohexyl nucleotide are easily It can be deposited on a variety of supports. Mossback, K. et al., Methods In Enzymology, Chapter X1. See Volume IV, 859-886, 1976. Oligoribonucleotides can be immobilized on borate derivatives of various supports. can. Shot-Etch, et al., Bio’ryox Tree, 12,932.19 See 73.

As an example, nitrocellulose adsorbs single-stranded DNA, but not RNA. Furthermore, further DNA adsorption on nitrocellulose can be achieved by well-known processes. can be inhibited. Then, if a second denatured DNA or RNA preparation is If added, it can establish a pair with the initially adsorbed DNA. The rubber is fixed to a solid carrier. generally nuclei that are not initially associated with a solid carrier Acid products are labeled. solid carrier after the hybridization reaction is complete. The carrier is separated from the reaction mixture and the degree of hybridization is determined by the solid carrier. Determined by measuring the label affixed to.

A refinement of solid carrier hybridization was published in Lanki's U.S. Pat. , 486, 539, by sandwich hybridization. be. The sample is prepared under conditions that make the target nucleotide (nucleic acid to be identified) into a -terminal chain. It is processed. The sample is then mixed with two purified nucleic acid reagents. The first nucleic acid reagent is Has a nucleotide sequence consisting of at least 10 bases and is fixed to a solid carrier Contains a single-stranded fragment of a nucleic acid that has been isolated. The second nucleic acid reagent contains at least 10 A piece of nucleic acid that has a nucleotide sequence consisting of bases and is labeled with a radioactive isotope Contains chain fragments. Nucleic acid reagents interact with target polynucleotides through complementary base pairing. hybrid molecules. Two types of nucleic acid reagents cross each other The solid carrier is then washed and incorporated into the hybridizing molecules. The outside markings are substantially removed. The presence of the target nucleic acid is then detected on the washed solids. Identification is made by measuring the label on the body carrier. Also, sandwich high Hybridization is a method that uses two specific hybrids compared to methods that use a single nucleic acid reagent. It has improved characteristics because it includes a diization step.

The advantage of solid carrier hybridization is that the target nucleic acid and probe are It is easy to separate hybrid molecules from the reaction mixture. However, The conventional solid carrier hybridization method is a sandwich according to the above ranking. Both hybridization methods have significant drawbacks. solid carrier The reaction rate of hybridization is that all hybridization components Solution hybridization is much slower than solution hybridization because there is no diffusion; In solid carrier methods, the reaction can be completed within hours or even days. moreover , some nucleic acids cannot be used for base pairing, so hybridization efficiency may be low. Moreover, the degree of preparation required is also important. In general, the hive Sample preparation for redization requires several hours, followed by 1-2 hours of washing. is necessary. Also, a relatively large amount of probe is required.

Therefore, solution hybridization and solid carrier hybridization A nucleic acid assay method that combines the advantages of both methods is desired.

Diagnosis of genetic diseases is usually time-consuming and labor-intensive with the current state of technology. technology is used. Diagnostic methods that have recently gained acceptance for identifying genetic diseases is a method known as restriction fragment length polymorphism (RFLP). RFLP method In general, genes generated by treating sample DNA with restriction enzymes are A somewhat tedious separation step based on fragment size is used. Systemic cell anemia and This special example is the most easily understood modification of the RF LP method, and the page shows recent fetuses. An editorial is provided that describes how the early calcaneus anemia test is performed.

Generally, a DNA sample contains two groups of different sizes for the normal and heel alleles. It is treated with a restriction enzyme (MstII) that generates a Robulin gene fragment. Cut off The pieces are then separated according to their size, e.g. on an agarose electrophoresis gel. These fragments were made into single strands and transferred onto filter paper. The probe identifies the exact fragments in similarly sized piece molasses. Gel for this test Both the electrophoresis and transition steps require highly skilled individuals and are time consuming. This is a time-consuming stage.

What is needed is a simple method to diagnose the presence of genetic mutations due to genetic diseases. be.

summary The present invention satisfies the following requirements. Specifically, the present invention provides Includes methods for quantitatively or qualitatively detecting single-stranded target polynucleotides The method consists of the following steps.

(a) The sample is exposed to at least two different probes, a first probe and a second probe. lobes to form a reaction mixture, each probe contains a target polynucleotide. consisting of a single-stranded polynucleotide having a nucleotide sequence complementary to a portion of the Both probes form complementary base pairs with the target polynucleotide. Forming a hybrid molecule, none of the probes are immobilized on a solid carrier. , the probes do not hybridize with each other, and the second probe is tagged with a detectable label. ing.

(b) The reaction mixture is then combined with the first probe, but not with the second probe and the target polymer. contact with a solid carrier that does not bind the polynucleotide.

(C) The presence or absence of bound label on the solid carrier is then determined.

Single-stranded target polynucleotides are formed by denaturation of double-stranded polynucleotides be able to. Preferably, the two probes do not compete with each other and are Attach to different parts of the polynucleotide. Preferably the two different types The parts are adjacent or nearby.

The method of the invention is suitable for microbial diagnosis. Also, genes like sickle cell anemia Suitable for disease diagnosis, cancer diagnosis, and other uses. One invention related to the latter Methods include cleavage steps such as through the use of restriction enzymes and sandwich hybridization. For example, this method combines standard polynucleotide Can be used to assay target polynucleotides in samples that may also contain However, if the nucleotide sequences of the two polynucleotides are substantially the same and the standard Polynucleotide has A part and B part, target polynucleotide has A' part and B' part The difference between the standard and target polynucleotides is the standard polynucleotide. At least one nucleotide located between the A and B parts on the nucleotide different from the nucleotide located between the A' and B' parts of the target nucleotide . This method consists of the following steps.

(a) Both the standard and target polynucleotides present are In this case, there is no segment containing both A and B parts, and the target polynucleotide At least one segment includes both an A' portion and a B' portion, respectively. cut into single-stranded segments; (b) The treated sample in step (a) is at least a first probe and a second probe. is also mixed with two different probes to form a reaction mixture, but each probe The first probe consists of single-stranded polynucleotides and cannot hybridize with each other. is complementary base-paired with the A portion and the A′ portion, and the second probe is complementary base-paired with the B portion and the B′ portion. Both probes have complementary base pairs with the A' and B' parts. By complementary base pairing with a single-stranded segment of a target polynucleotide that has both form a hybrid molecule, (c) The presence of hybrid molecules with both probes is then determined.

Preferably neither probe is immobilized on a solid carrier and the second probe has a detectable label and measures the presence of a hybrid molecule containing both pronibs. Each stage is one stage after another.

(a) the reaction mixture is bound to the first probe, but only to the second probe, part B; The segment 1 containing only the B' portion and the segment containing only the B' portion are bonded to each other. (b) the label bound on the solid carrier with bc Measure whether it exists or not.

drawing These and other features, forms, and advantages of the invention will be apparent from the description and accompanying claims below. It will be better understood by the scope of the request and the drawings: FIG. 1 is a schematic explanatory diagram of each stage of the assay method of the present invention.

FIG. 2 is a schematic illustration of the sickle cell assay using the method of the present invention.

Figures 3 and 4 are plots of label assay relative signal values and target DNA concentrations. be.

Figure 5 shows the binding of avidin-cellulose solid carrier and biotin-labeled probe. The characteristics are plotted.

ILΩ”LJ Methods and kits having features of the present invention can be applied to viruses, bacteria, fungi, yeast, etc. Polynucleotides (DNA or R) containing organisms such as microorganisms and infectious agents Examples of methods and kits that can be used to detect the presence of For example, used in food hygiene research, drug diagnosis, and some microbial diagnosis. be able to. Suitable samples include animal and plant tissue homogenates, blood , serum, feces, nasal and urethral mucus, water, dust, and soil. A solid sample is first transformed into a liquid. The method of slurrying and homogenizing test samples in a body medium also allows normal This method is also suitable for diagnosing genetic diseases where genes have been mutated. Suitable for diagnosis.

In the method of the present invention), the polynucleotide to be detected (target polynucleotide) Samples containing cells suspected of containing Either the target polynucleotide is released from the cell into solution or the cell wall is exposed to the target polynucleotide. Allows the reagents used to detect cleotide to pass through. target porine Creotides are generally DNA or RNA, or derivatives or fragments thereof.

Hybridization occurs between single-stranded polynucleotides. However, at 9 If the target polynucleotide is double-stranded, the test sample is One condition such that the cleotide can be denatured, e.g. 100°C for 5 minutes, or O,S Heat or heat and high pH such as 20-40 °C for 5 min using molar NaOH. Processed with conditions. Denaturing conditions for polynucleotides are well known.

The method of the present invention includes a solution sandwich hybridization stage as shown in FIG. It consists of a floor and a harvest stage. Solution Sandwich Hybridization Smell the test sample contains at least two different specific nucleotide piperidases. -combined with shicon probe to form a reaction mixture. For example, two types of probes are Using a first blow-boo binding probe and a second probe/discrimination probe. can be done. Each probe is complementary to a portion of a single-stranded target polynucleotide It consists of a single-stranded polynucleotide containing a nucleotide sequence. therefore , each probe is complementary to the corresponding nucleotide sequence on the target polynucleotide. Can be group-paired.

Preferably, the two probes are placed on the target polynucleotide so that they do not compete. and preferably the portions are closely spaced (approximately 300 nuclei). and more preferably directly adjacent to each other (about 10 (no more than nucleotides apart), the probes cannot hybridize with each other. In the reaction mixture, the two probes and the end-stranded target polynucleotide Tiids hybridize with each other to form complex two-stranded hybrid molecules. Fast and effective hive Due to redization, neither probe is immobilized on a solid carrier , the second probe, i.e. the identification probe, has a label that can be easily detected. What you do can be further characterized. This method is sandwich hybridization. tion, which is the difference between the target polynucleotide and the resulting hybrid molecule. This is because they are sandwiched between species probes.

Probes can be contained in separate reagents. Probes should not hybridize with each other. Therefore, all probes can be kept in a single reagent. probe The order in which the test sample is combined with the test reagent is generally not important; It is also possible to add all probes at the same time or in some other combination. Can be added in combination.

The reaction mixture is maintained under conditions that promote hybridization. child The conditions for It is being Hybridization is allowed to proceed until substantially complete. most For example, for a polynucleotide of about 1 hour, the time is no longer than about 1 hour. The concentration of plasmid probe was approximately 0.15 molar and the plasmid probe concentration was 10 micrograms at a temperature of 65°C. For ram/ml, the time to reach 1/2 reaction completion is within 20 minutes.

During the harvest stage, the 5 reaction mixture is bound to the first probe (binding probe). However, the second probe (identification probe) and the target polynucleotide are not bound together. is brought into contact with a solid carrier that cannot be combined. Here "combine" and The term "bond" refers to a strong bond due to a special functional group that is symmetrical to a non-special bond. Taste.

The hybrid molecule formed from the binding of the target polynucleotide with two probes is As such, the probe is bound onto a solid carrier by a binding probe.

The second probe itself or the second probe itself on a solid carrier is almost non-specific. Selected not to combine. In this way, the target polynucleotide is present in the test sample. The presence of is a measure of whether any of the labels are bound on the solid carrier This is confirmed by Quantitative measurement of the amount of target polynucleotide in test reagents It is also possible by measuring the actual amount of label bound on a solid carrier. .

To determine whether any label is bound on the solid carrier, at least There are two basic methods for and measuring the distribution of the second probe in the liquid phase. In the first method, incorporation into the hybrid molecule Wash, aspirate, and decant excess identification probe that was not removed. The amount of label bound on the solid carrier is then removed by conventional methods such as Measure. The presence of the label on the solid carrier indicates the presence of the target nucleotide in the sample. It shows. In the second method, the solid carrier is removed from the liquid phase of the reaction mixture. It can be separated or left in the liquid phase. solution in liquid phase of the second probe added to the reaction mixture. Compare with the total amount. A second probe with some difference between the two quantities is placed on the solid carrier. binding, in other words, the presence of the target polynucleotide in the sample. show.

Preferably, the first probe (binding probe) comprises a first binding group and is attached to a solid probe. The carrier comprises a second linking group, and the linking groups are capable of forming a strong bond with each other. more connected to each other. Both binding groups constitute a binding pair. One bonding group of the bonding pair is rigid. The other binding group is part of the ff1l probe. Ru. The binding pair may be, for example, one of the following pairs: avidin-biotinylated; Hapten-antibody, antibody-antigen, carbohydrate-lectin, riboflavin-riboflavin Rabin binding protein, metal ion-metal ion binding substance, enzyme-substrate, Bolognese Tosis diol, Staph A protein-antibody, enzyme-inhibitor, binding pair are also listed above. including pairs of derivatives of the above moieties.

The selection criteria for each bonding pair are (1) the speed at which the bonding groups in that pair bond with each other; (2) the bonding strength between the pair of binding groups, (3) the solution concentration of the binding group on the first probe. (4) the bonding group is a solid carrier substrate; These include being able to easily bind to substances and being easy to use to form the first probe. Ru.

The substrate material of the solid carrier is, for example, agarose or cellulose.

Glass, latex, polyacrylamide, polycarbonate, polyamide Iζ ( For example, nylon TM), polyethylene, polypropylene, polystyrene, silica It can be chosen among gels, silicas and their derivatives. What is the above list A solid to which one bonding group of a bonding pair can be immobilized is not completely Are suitable.

Solid carriers can be in a variety of forms, e.g. Fine particles of 100 microns or less (e.g. micronodular cellulose), particle size 0. 1-2-Omrn macro particles, sheets, tubes, bed tips, plates, filter media, beads It may be ``Z'', etc.

Methods of fixing the second binding group to a solid carrier are well known. 1st pro Methods of directing the polynucleotide portion of the probe to the first binding group-containing body are also known. There is. Cheno et al., P.N.A.S.

517 (1975); Falkov et al., U.S. Pat. No. 4,385,5320: 290 (1976).

゛The second probe (identification probe) contains 0 different types of probes containing easily detectable labels. Labeling methods for specific polynucleotide probes are known. easily detected labeling that can be used and does not unduly interfere with the solution piperidization step. Suitable labels include radioisotopes, photolabels, and enzymes, which can be used with any enzyme cofactors, habten, antibodies, avidin, biotin, carbohydrates, These include cutin, metal chelators, and their derivatives. Detection method is radiation either directly as in the case of sexual isotopes or with subsequent use of enzyme-labeled antibodies. There are indirect ones, such as in the case of

32 Radioactive isotopes such as 125N can be used to label probes.

Radioactive labels can be can be detected using well-known methods. However, radioactive isotopes The use of signs is expensive and dangerous. Special training for attendants when handling radioactive materials and safety measures are required. Furthermore, radioactive isotopes have a certain half-life. It has a period of time (generally a period of several weeks).

For the reasons mentioned above, other labels such as photolabels and enzymatic labels, etc. system was developed. European Patent Application No. 82303701.5 of Heller et al. chemiluminescent agents, bioluminescent agents, fluorescent agents or Phosphorescent agents can be mentioned, which under appropriate conditions can be contrasted with radioisotopes. The sensitivity is as high as possible. Photolabeling of the single-stranded polynucleotide segment of the globe Although it can be attached anywhere, the terminal position is known to be more preferable. ing.

Examples of optical labels include the following = (1) Chemiluminescent agent: peroxidizer (2) bioluminescent agent: bacterial luciferase, luciferase, flavin mononucleotide (FMN), adenosine tri Phosphate (ATP), reduced nicotinamide adenine dinucleotide (N ADH), nicotinamide adenine dinucleotide phosphate reduced product (NA DP) () and various long-chain aldehydes (such as decylaldohyde); (3) Fluorescence Agents: Fluorescent agents such as adenosine nucleotides, etheno-cytidine nucleotides, etc. Nucleotides, or functionalized nucleotides (amino-hexane adenosine nucleotides) nucleotides, mercury nucleotides, etc.), these were initially labeled with fluorescent agents. , which is then covalently attached to the single-stranded polynucleotide segment of the probe. (4) Phosphorescent agent = 2.3-butadione, ■-carboxyphenyl ]-1,2-pentanedione, and 1-phenyl-1,2-propanedione. Eel 2-lactone. Methods for attaching optical labels to probes are well documented in the art. It is listed. The label can be activated by activating the label and transmitting its light sensitivity to a light detection device. It is measured by measuring. Fluorescent and phosphorescent agents are irradiated with light of the appropriate wavelength. It can be activated by irradiation. Chemiluminescent or bioluminescent labels are It can be chemically activated by known methods.

Identification probes can also be labeled using enzymes. hybridization The reaction product is detected by the action of the enzyme on the substrate for the enzyme, e.g. Enzymes that can act on chromogenic substrates can be selected. of the substrate The conversion rate can be monitored by optical analysis equipment. This conversion rate is then the target related to the presence or absence of polynucleotides. Avidin and biotin identified It is well known that suitable methods can be used to attach enzymes to nucleic acid probes. Examples of suitable enzymes include beta-galactosidase, alkaline phosphatase, etc. luciferase, horseradish peroxidase and luciferase.

The major advantage of optical labels and enzymatic labels is that their qualitative detection does not require expensive detection equipment. It is something that can be done in a visible manner without the need for physical placement. Of course these signs Quantitative measurements of can also be carried out by the use of photometric equipment.

The first and second probes each have substantially mutually exclusive positions on the target nucleotide. In other words, the first and second probes are preferably complementary to the sandwich probe. Competing with the same nucleotide sequence to the extent that hybridization is hindered. It should not be.

The probe is a polynuclease treated with an appropriate restriction endonuclease from the organism of interest. from leotide or enzymatically such as Exom digestion or RNA polymerase transcription. can be prepared from double-stranded polynucleotides by methods. In these cases, Probes are fragments of RNA or DNA. The base sequence of the unique part is known In some cases, probes can be synthesized by organic synthesis techniques (Stabinski -, J. et al. Nucleitsk Acids Research 4, 353.1977, Go. U, G, R, et al. Nucleitsk Acids Research 6,1557,1 979; Go, G, R.

et al., Nukleitsk Assidzu Research 7, 1955, 1979; Narang, Niss, A., Methods in Enzymology, Volume 65, Part 610- 620.1980), and under appropriate conditions polynucleotide phosphorylated oligodeoxyribonucleotides with a specific sequence using E. coli 1980), and also cloning, e.g. plasmid or M13 Recombining a specific sequence into a bacteriophage vector and then Probes can be produced in large quantities by cloning samples using standard methods. Both are possible. Cloning methods are preferred.

The size of the probe ranges from 10 to too or oo nucleotides.

can be made to a length of If the number of nucleotides is less than 10, hybridization will not be stable; It begins to denature at temperatures above ℃. The number 12 of complementary polynucleotide combinations indicates proper bonding. The minimum length required to obtain a force of 0 is usually the lowest practical value of about 15. Nu When the number of cleotides exceeds 100,000, the speed of hybridization (regeneration) increases. See pages 213-219, 197], all probes are scaled per base. It is not necessary for the globe to be complementary to the target polynucleotide in the target polynucleotide; from about 15 to about so, ooo, more preferably from about 15 to about 10,000 nuclei. It is the length of the punch line.

The number of complementary (base-paired) nucleotides in the probe preferably ranges from about 200 to about s , oooO. Complementary nucleotides are particularly useful when the number of complementary nucleotides is small. If the number is small (less than 200), it is preferable that they be adjacent to each other, but the It is not necessary that all base-paired nucleotides of the target and probe be complementary in an 1:1 ratio. stomach. The small globe Hs-1oe) itself is manufactured using automated organic synthesis technology. . Probes ranging in size from 100 to 10,000 nucleotides are suitable for enzymatic methods. Alternatively, it can be obtained by the recombination DNA method.

A relatively bulky label moiety (e.g. chemical compound) on a relatively small polynucleotide segment In some cases, labeling agents such as chemiluminescent agents may interfere with the hybridization process. harm Therefore, it is important to choose the label correctly. An example of label selection criteria is , easily incorporate labels into polynucleotides without hybridization inhibition easy and sensitive detection of the label, and the specificity of the labeled probe. The proper hybridization conditions are the first binding of the first probe (binding probe). The nature of each label attached to the group and the second probe (identification probe), the two types The size of the probe, the content of [G] + [C] (guanine plus cytosine) in the probe complementary nucleotide sequences on the target polynucleotide, and before the test sample. Determined by processing method. The label is attached to the temperature at which the hybridization reaction is performed. For example, chemiluminescent agent catalysts can absorb/emit It can be sensitive to the temperature and salt concentration tolerated by the part. of the probe Size affects the temperature and time of hybridization. Salt concentration and test If the drug concentration is the same, the nucleotides aio, ooo~ioo, ooo hybridization using a range of reagent polynucleotide sequences at 67°C. Hybridization with 14 to 100 nucleotides takes 0 to 80 minutes. ization is 5-30 minutes at 25°C. Similarly, [G] + [C] content For sequences with high [G] + [C] content, the temperature is higher than that for polynucleotides with low [C] content sequences. It is interbred with degrees. Ultimately, the conditions used to prepare the test samples and the target polynuclear Conditions that maintain the cleotide in a single-stranded form are used in the hybridization reaction. This will affect the temperature for 1 hour and the salt concentration. The preparation conditions for the test sample are those for polynucleotides. Affected by length and [G] + [C] content. Generally, if the array is long The longer the length or the higher the [G] + [C1 content, the higher the temperature and/or or the concentration of probe or target in the reaction mixture where low salt concentrations are required. Determine the time required for redization to occur. glove or target The higher the concentration, the shorter the incubation time required for pipelidized cidin. The basic rate of diization also depends on the type of salt present in the wet layer mixture, its concentration , and wet layer temperature. Sodium chloride, sodium phosphate and que Sodium chloride is the salt most often used for hybridization. , the salt concentration can be as high as 1.5-2M. Same as salt above Equivalent polynucleotide hives are equivalent for each species of C. redization rate is obtained. Priten et al. (1974) (Methods in Enzymology% Volume XXIX.

Part E, edited by Grossman and Moldip, Academic Press, Ku 2, Nyuko 1 Minato , p. 349) is a standard hybridase obtained with commonly used salts. provides data indicating the basic speed of the application. Hybridization of DNA with RNA What is the rate of hybridization of RNA by DNA? It's a little rough. The degree of difference is 10 times or more, which is a product, for example, DNA or It varies depending on which RNA is used in excess. Gaff Volume 4, 16. No. See page 2306).

Although not the optimal condition, almost all probes with nuku1/octide number of 100 or more Preferred general methods for hybridization with target and target combinations There are conditions. These conditions are approximately 0.75 mol of sodium chloride, sodium citrate, Mu 0.075, sodium phosphate 0.025 (pH 6, s), as℃ and poly The nucleotide concentration is o-ooi ~ 1.0 micrograms/ml. Also, low temperature methods to obtain similar conditions, e.g. using a denaturing agent like formamide during the reaction. (Casey et al., Nuclear Acids Research) Chi 4:1539 (1977)), other reagents during the hybridization reaction. The reaction rate can be increased by adding sulfuric acid, an example of which reagent is sulfuric acid Kistrin (Warn Glycol (Amazino, Analytical Biology 1) 52=304 (1986)) and phenol (Kohn et al. 16: 5329 (1977)).

Assay of single-stranded standard target polynucleotide in a test sample by the assay method of the present invention There are at least 2,1 kits to use! different probes, the first Brogoux binding one or more probes that are a probe and a second blowboo identification probe; liquid polynucleotide reagent. Each probe represents one target polynucleotide. A single-stranded polynucleotide containing a nucleotide sequence complementary to a moiety Ru. None of the probes are fixed to a solid carrier. Probes hybridize with each other The second probe has a label that can be detected. tree The cut is also. It binds to the first globe, but the second probe and target polynucleotide It consists of a solid carrier that does not bind to tide. Other probes and solid carriers The properties of are generally described in the description of the method of the invention above. The kit is even more It can also consist of means for qualitative and/or quantitative measurement of the label.

One or more probes can be present in each polynucleotide reagent For example, probes do not hybridize with each other, so they can be conveniently combined in one reagent. be able to.

It is preferable that the kit contains one or more of the following components.

(a) each such as sodium dodecyl sulfate or sodium lauryl sarcosinate; (b) Proteinase K and Prona (C) a salt that promotes denaturation of the target polynucleotide; (d) reagents used to detect the label on the second probe; medicine.

(a), (b), (c) and probe reagents may be combined as a single reagent if necessary. It is also possible to use multiple reagents.

The methods and kits described above can be applied for testing a range of target nucleotides. can. A separate set is prepared for each target polynucleotide, and each set has its own specific for that target polynucleotide. Each set of probes is less Both probes contain two different types of probes, i.e., the combined discriminating probes as characterized above. The solid carrier used consists of a large number of probes (first and second probes). The binding groups of each binding probe identified on each of the target polynucleotides are can bind to any discriminating probe or any target nucleotide. It has a bonding group that cannot be removed. Therefore, one type of general-purpose solid carrier is All of the different binding probes are used. In the kit, each probe is The components are preferably contained in separate reagents.

The methods and kits described above are for simultaneous testing of several target polynucleotides in a test sample. It can be applied to Separate probe sets for each target nucleotide and each set is specific for the corresponding polynucleotide. all Probes cannot hybridize with each other, each probe set has at least two species different probes, i.e. the first probe characterized above (binding probe ) and a second probe (identification probe), all the first probes are carrying the same linking group so that they are all harvested on the same solid carrier There is. The solid carrier is compatible with any identification probe and with any target polynucleotide. The second probe contains a variety of labels, each of which binds to a specific target nucleus. It corresponds to cleotide. For detection of labels on solid carriers after the harvest stage information on the presence and/or amount of the corresponding target polynucleotide in the sample. It will be done. In the kit, all probes are contained in a single liquid reagent That's preferable.

The advantages of the assay method and kit of the present invention are numerous. It provides the reaction rate and reaction efficiency of the solid carrier hybridization method. The method also provides ease of isolation of hybridization-generated breasts from test samples. Furthermore, this assay method uses at least two types of probes that target polynucleotides. It is highly specialized, requiring it to be complementary to Tido. solution sandwich Hybridization can generally be completed within an hour and can be completed before the harvest stage. does not require more than 5 minutes (optimally about 0.5 minutes or less), thus providing substantial Save time.

Additionally, the Kira 1- of the present invention is easy to use. The user can attack all target Polinukus. There is no need to stock a single solid carrier reagent that can be used for the reotide assay. It just is. Only one liquid agent is required for each target polynucleotide. Ru. A third test may be necessary to detect the label.

Another important advantage of the method of the invention is that it eliminates the need for expensive test sample preparation. That is. Conventional solution hybridization methods remove other cellular components and The prepared target nucleic acid is assayed. Nucleic acids in cells and viruses are usually associated with other cellular components, such as Generally, it is a complex tightly bound to a protein, and in this form, hybridization is difficult. It cannot be used to destroy cells or viruses, only to destroy them and release their contents. Opening requires that the polynucleotide be used for hybridization. Polynucleotides are not intended to be able to be removed from cells or viruses. Even if released, it remains in a complex form with other cell or viral components and is not actually the same. can be severely degraded by nucleases released by On top of that , when the probe is added to this mixture, the probe will stick to the sticky cell or virus formation. may become complexed with molecules and become unusable for hybridization, or It is decomposed by the action of enzyme. Various conventional techniques are used for the purification of polynucleotides. There is a method. All these methods are time consuming and take up to 11 hours. Such a method is said to be very quick and requires a large number of operations. be. Surprisingly, the assay method of the present invention can be performed using unpurified samples. It has therefore been found that labor- and time-consuming purification steps can be eliminated. The 0-line test method itself is easy to automate, further simplifying the user's work. It is. Assay results can be either characteristic or quantitative.

Another feature of the method and test kit of the present invention is that it is used for genetic disease diagnosis and cancer diagnosis. It is to be discontinued.

Genetic diseases are the result of mutations in the nucleotide sequence of polynucleotides . Such mutations are also a factor in cancer progression. Therefore, that Normal polynucleotides taken from a patient as a result of a genetic disease or cancer Samples containing polynucleotides also contain normal polynucleotides. It may also contain an abnormal polynucleotide (target polynucleotide) that is a mutant. It becomes. The sample may also contain only abnormal polynucleotides. normal condition and abnormal polynucleotides typically have a mutation point sequence on the polynucleotide. have substantially the same nucleotide sequence except for Therefore, normal and abnormal Polynucleotides react differently to certain reagents.

The methods of the invention explore such differences between normal and abnormal polynucleotides. This method can be used to replace the cleavage step with solution sandwich hybridization. It is combined with the tion method.

The cleavage step consists of a segmentation step (7) and if the target polynucleotide is double-stranded. If the condition is severe, it may include a degenerative stage. The order of these two steps is not important .

Restriction reagents, such as restriction enzymes, are known. Restriction enzymes can cleave polynucleotides under the right conditions. At the segmentation stage where Otido is divided at a very specific point on Nuku 1/Otide spine. used. The standard and target polynucleotides are woody identical. standard poly A nucleotide has an A part and a B part. Target polynucleotide is A' portion and B' portion. The two types of polynucleotides are each of the polynucleotides at least one null located between the A part and the B part and between the A' part and the B' part of the It differs depending on the cleotide. Restriction reagents are used to limit standard polynucleotides to any segment. The target polypeptide is also segmented so that it does not contain both A and B parts. At least one segment of nucleotides contains both A' and B' moieties Selected to segment as follows. If the target polynucleotide is a standard polynucleotide In a typical case of a leotide genetic variant, A part, A' part, and B part and B' part are the same.

Existing target and standard polynucleotides or segments thereof (segment stage or ) may be subjected to denaturation treatment, if necessary, after or before the segmentation step, i.e. - It is chained at the end. The processed sample is thus exposed to target and/or standard polynucleotides. Next, this treatment contains a single-stranded product for hybridization. The treated sample is combined with at least two probes to form a reaction mixture. . The two types of probes are a first probe and a second probe. Each probe is a single-stranded Consists of polynucleotides. The probes do not hybridize with each other, the first probe A salt complementary to the A portion of the standard polynucleotide and the A′ portion of the target polynucleotide. Base pair. The second probe consists of the B portion of the standard polynucleotide and the target nucleotide. It forms a complementary base pair with the B' portion of the base. Both types of gloves have A' part and B' part. base pairing with a single-stranded segment of the target polynucleotide containing both to form a hybrid molecule. For standard polynucleotides, segment I- After the conversion process, the A part and the B part exist in separate segments, so the first probe and No hybrid molecules are formed that bind both of the second probes.Both probes are in separate cells. adheres to the product. A hybrid molecule that has both a first probe and a second probe is Formed only when an abnormal (target) polynucleotide is present in the sample Become.

The remaining hybrid molecules bound to both probes in the reaction mixture are the targets in the sample. This indicates the presence of polynucleotides. Iburi to this sandwich Identification of hybrid molecules formed by the diization method is performed by conventionally known methods. For example, as disclosed in Lanki U.S. Pat. No. 4,486,539, A method equivalent to fixing one probe on a solid carrier can be used. Wear. Other methods, i.e. not all probes need to be in solution is also known. Alternatively, a solution solution with a harvesting step as described above may be used. Hybridizable simine can also be used. In the latter case, the top Another limitation to the method of the invention described above is that none of the probes are mounted on a solid carrier. is not immobilized and the second probe has a detectable label. 0 reaction mixture binds the first probe, but binds the second probe and the B moiety or B' Contacted with a solid carrier without bonding with any segment containing only the moieties Ru. This subsequent measurement is carried out with the label attached to the solid carrier as described above. This is done to see if.

For example, the 5-order method is suitable for a paired red blood cell assay. Mutant gene DNA strands (e.g. For example, a test element suspected of containing a heel allele) is a structural residue in the DNA strand. It has been used in gene mapping to detect gene deletions. Sudden changes in DNA This type of direct identification of hematopoietic diseases due to a single point mutation in the DNA This was possible due to the special advantages of restriction enzymes. A single nuc at the enzyme cleavage site Changes in leotide can be easily detected using appropriate enzymes. limit The enzyme Mstn cleaves DNA on average and separates it from the β8 and β5 genes, respectively. This is known to generate fragments of 1.1 and 1.3 kb. MstII is sequence (CC) that is a subset of the Ddel site (CTNAG) (7) cleave TNAGG). DNA from the sickle cell gene It has nucleotide variations at the beginning. Therefore, the mutated DNA strand Restriction enzyme treated test sample that is not cleaved by the Mstn enzyme at the site The material is then subjected to conditions that render the polynucleotide single-stranded. single target poly Nucleotide-single-sample hybridization Mst of the site where the mutation occurred There is an additional restriction that it binds to the DNA site on the opposite side of II. target poly The only case in which a complex hybrid molecule is formed consisting of a nucleotide and both probes is This is the case when the gene is mutated, such as sickle cell. normal gene is cleaved between the two probe binding sites. This model of the assembled red blood cell assay The formula diagram is shown in Figure 2, and this method is similar to the gel electrophoresis stage in the conventional assay method. This eliminates the step of transfer to a solid support (filter paper). Simple like this ization is very significant. This assay is useful for other genes that contain genetic mutations. It can also be used for disease diagnosis as well.

In the case of cancer diagnosis, the same test as in the calcaneal erythrocyte example is used, but this test This may take the form of a quantitative test of the level of metsenger RNA from the tumor gene.

This example shows the relationship between the harvested signal value and the target DNA concentration. The target polynucleotide is mpB1017, probe 1 is biotinylated as the first binding group. P)IBC6 with 32P-labeled M2S 10W, probe 2 The solid carrier that binds the biotin of the 1 probe is avidin-cellulose.

Yuiya SSC is 0.15 moles of sodium chloride and ois moles of sodium citrate. It is. SSC is used at various concentrations, for example rloxsscJ is 1. 5 moles of sodium chloride and 0.15 moles of sodium citrate. EDTA is ethylenediaminetetraacetic acid, SDS is sodium dodecyl sulfate, and DNA is deoxychloride. Silibopolynucleotide, BSA is bovine serum albumin, Tris:HCM is hydrochloric acid Tris-hydroxymethylaminomethane plasmid DNA adjusted to appropriate pH with A: After transformation. Yumata (E. coli) strain LE392 was cultured, and the resulting bacteria were Obtained by lysis treatment with lysozyme, SDS and NaOH. D NA was further purified by centrifugation in CsC1 in the presence of ethidium bromide. . Moiatis, T., E., E., E., F., and Samtebrock.

J., 1982, Molecular Cloning Near Laboratory Manual Cold Spring Harbor Laboratory 1, New York, USA Reference Spring Barber) Reference 0M13 Bacteriophage, from strain Low The replication form molecule (RF) DNA of E12 strain 71.18 was detected at the time of contamination Single-stranded virion DNA from 0M13 clone mpB1017 obtained in the same manner. is a virus polypeptide purified from pillions in the growth medium of contaminated E. coli, 71.18. Precipitation with ethylene glycol.

CsC! ; centrifugation in L, extraction with SDS, phenol and chloroform Obtained by. Salmontesetes DNA was obtained from Sigma Chemical Company (Missouri, USA). Obtained from St. Louis.

Double-stranded DNA was prepared by Leary et al., Proceedings of the National Academy of Sciences. Demi Obsc. of the Ninis A. 80:4045, 1983 (Rigby et al., Journal of Molecular ・Biology 113=237.1977). Plasmid PH BC6 was derived from plasmid pBR322 (Tsukumaki et al., Se282585, 1982). ) contains 5400 base pairs of the human beta-globin gene cloned in are doing. M13 clone mpB1017 is 131 from plasmid PHBC6 0M13, strain containing 1310 bases of the human globin gene homologous to base 0 10w is a bacteriophage homologous to the 75oO base of clone mpB1017. Contains the same number of bases of the gene. Homologous to PHBC6 and M131 strain 10w Regions in certain mpB1017 are non-overlapping.

A solid phase for harvesting solution sandwich products is described by Ball et al., Journal of Olga. Microcrystalline cellulose using the same method as Nick Chemistry 27:2094, 1962. After activation with carbonyldiimidazole, the protein avidin and Prepared by conjugation. The binding ratio is 1g of activated cellulose to 1g of activated cellulose. The weight was 1 mg. Prepared 1:1 avidin:cellulose solid phase slurry - contains about 1 g of cellulose per 6 ml of buffer.

Solution hybridization was performed using 5X 5SC1 each 0.02% (W/V) polyvinyl Nilpyrrolidone-40, Ficoll-400, and BSA, pH 6.5, 25m M NaPO4! buffering agent, and 250 g g/rdl of sonicated Salmontestes The process was carried out in a solution that ultimately contained the DNA. All DNA (Probe 1. Pro -b2. The target and carrier Salmonestes) initiate the hybridization reaction. Denaturation was performed at 100° C. for 3 to 5 minutes before starting.

A more detailed sample assay method is described below.

Tasks: Testing the feasibility of the assay method of the present invention and identifying target DNAs for which this assay can be effectively used. In order to determine the concentration range of A, the following assay was performed. Plasmid p HB C6 Biotin-11-deoxyuridine triphosphate (Becesta Research) Laboratories, Gaithersburg, Maryland, USA) It was labeled with the ligand biotin. This constitutes probe 1 of the assay. Ba The replicative molecular DNA of cterophage M13 Low is alpha 322-deo. Xycytidine triphosphate (Amersham, USA).

It was labeled with the radiological indicator 32P by cutting and self-transfer using the following methods (Chicago, Illinois).

This constitutes probe 2 of the assay. Biotin nucleotide with probe Xi approximately 6% Probe 2 has a radioactivity of 0.7 to 7 x 1107 cp/pg. Ta. 20pg ~ 1. using target DNALtM13mpB1017. Scope of OJLg Samples containing target DNA of 30% were tested. All assays were performed using probe l (biotin 100 ng of Probe 2 (32P) and 50 ng of Probe 2 (32P). probe and Sample DNA was treated with Tris at pH 7.5: HCJLomM.

EDTAl, mixed in a solution of OmM, heat denatured, and the solution shown in the method described above. Under liquid hybridization conditions, at 68°C in a total volume of 0.2 d. It was damp at night. This incubation time is excessive for reasons of convenience and for complete incubation. It was used solely for the purpose of providing a response. Probe 1 as a control assay ．． or probe 2 and 20 nH of target or probe 1 and no target. Row 9 is for the case containing probe 2. Generating sandwiches from hybridized probes To separate the reaction products, add 2 ml of a 1=1 slurry of hairvidin and cellulose. , moisten at 22℃ for 30 minutes, and filter the cellulose through a 13mm glass fiber filter. This was done by separating.

Wash container once with Tris at pH 7.5: HC4Q, O, ld, 1mM EDTA. The mixture was filtered through the same filter to obtain the first reaction. The filtered solid support is further 2 x SSC and 0.1% (v/v) SDS, O-2 x SSC and 0.1% Washed twice with SDS and twice with o, 1xssc and 0.1% SDS. most The latter two washes were carried out at 50°C and the others at room temperature. All washes are 0.5ml A solution of These washes were performed by Leary et al., P.N.A.S., 80-+404 5 (1983) Southern plot used for hybridization analysis This is an application of what was available. Probe 2 (32P) bound to solid support ) is the amount of resin and Beckman Readysolve in 0.5ml of the solution in the mini bottle. T, M: Performed by liquid scintillation counting using 4.5 ml of scintillation liquid. was measured. All washings were collected and counted simultaneously.

Table 1 shows the results of one test. Here, the amount of bound probe 2 is assayed. It was directly related to the amount of target DNA in the solution. Data are averaged for duplicate samples. It is the average value.

Table 1 0, 0 0.2 0,021 0.2 0,065 0.16 0, 185 0.2 0,556 0.38 1,67 0.72 5.01°67 15, 0 2.26 45.0 2.19 Results from several tests showed that the assay performed in this example yielded no yield confidence at approximately 30 ng of target DNA. The signal value reaches the highest point, and the harvest signal value decreases logarithmically at higher amounts. did. Below 30 ng of target DNA, the assay is performed using the solid support of probe 2. Although limited by background binding to and the radioactive nature of the probe, approximately 0. Exponential increase from 5 ng to 10 ng. The nonlinearity of the signal value with respect to the target amount is 3, and FIG. 4 is an enlarged view of FIG. 3 near the highest signal value.

The non-linear relationship is thought to be due to the self-transfer reaction used in the labeling material of the probe. It will be done. Probes labeled with this method are known to exhibit some elements of cooperative binding. (Maincos and Whirl, Analytical Biochemistry 138 267.1984), this example shows avidin-cellulose solid carrier and biotin This shows the binding properties of the labeled prologue.

Transfer the biotin-labeled probe from the hybridization buffer to avidin-cellulose. biotin-11-dUTP and DNA labeled with both 3H-DATP and 3E-DATP or only 3E-DATP. The test was conducted using the same DNA (control). The used DNA breaks and moves on its own. plasmid pHBC6 (Rigby et al., Journal of Medicine) Physical Biology 133=237 (1977)).

Tris: Suspended in a solution containing HCMlomM and EDTAI-OmM (pH 7.5) 30 bottles each of turbid avidin-cellulose (compressed volume 0, 05 rrtl) into a microcentrifuge tube. Biotin labeling material DNA or control DN A 10 ng, carrier Salmonestes DNA 50 JLg, yeast RNA 20ILg-sodium citrate O = 075M, sodium chloride 0.75M and Bovine serum albumin in sodium biphosphate 0.025M (pH 6,5) Coal (fico[I) and 0.02% each of polyvinyl and lolidone (W/V-) was added to the appropriate test tube. containing the above solid phase and hybridization mixture. The test tube was placed on a shaking table and kept in a wet layer at room temperature for a period of time ranging from 1 minute to 300 minutes. Onshu end After completion, the solid phase was collected by filtration on a Teflon membrane filter. Amount of bound DNA The radioactivity value in the filtrate and washing solution was subtracted from the total radioactivity value added to each sample. . The data are shown in Figure 5. Approximately 92% of the biotin-labeling material DNA was attached within 30 minutes. binds to vizin-cellulose, while on average less than 2% of the control DNA is immobilized. Binds to body carrier. Binding is rapid, effective, and the biotin label on the probe It is unique to knowledge.

Biotin-labeling material: imparting desired properties to the avidin component of the solid phase that binds to the DNA. ji! which was subjected to various pretreatments using the same DNA as above! tested on one carrier . Free biotin competes with biotin-DNA for avidin sites on the solid phase. The pretreatment of the solid phase with 20 172-capacity free biotin should Chin-DNA binding was reduced by 50%. Plus 10x more free biotin pretreatment Biotin-DNA binding was further reduced by 30% by treatment. li! ；One-body type lonase, 1% (w/v) sodium dodecyl sulfate and 0.1% (w/v) v) Pretreatment with betamercaptoethanol inhibits biotin-DNA binding. After 50% reduction and further treatment of free biotin with this digestive resin, biotin -DNA binding disappeared. According to slightly different experiments, biotin-DNA solids Most of the binding to the carrier is not only biotin-dependent but also avidin-dependent It was shown that Control solid carrier combines cellulose with avidin Using the same method as above, but using TrisniH (,fL) instead of adipine in the reaction. It was prepared by treating with This control resin is used in the kinetic studies mentioned above. under the same conditions as for 5-6% control DNA probe and 15-16% was combined with a biotin-labeled DNA probe. In this way, biotin-labeled The majority (more than 85%) of the binding of the probe to the solid carrier is due to the special ligand avidi This is due to the interaction with the

flood This example shows M1 containing a MstII fragment adjacent to a sootycytic mutant cell. 3 shows the structure of the vector.

The assay method of the present invention is used to assay for the major genotypes of sickle cell anemia. A special structure of M2S is always required - basically the 6th cell of each calcaneus cell. DN from either side of the MstII restriction site that is the same as the number codon error. The Allr piece was inserted into M13 phage. The two types of fragments used for this purpose were Derived from plasmid pHBC6 (Tsukumaki et al., 2B=585 (1982)) , purified by acrylamide gel electrophoresis. Characteristic M s t U site 200 base pairs of So1 (MstlI isoschizomer) approximately 10n located directly downstream Fill the M s t II site with J dXTP3 in the presence of 250% M. Treated with Renow DNA polymerase. pt this “insensitive JDNA fragment” It was inserted into the HCII site of yc18.

24pUC: Analysis of 18-B-200 monomer showed two types of isolates.

200 bases inserted into pUclB-B-200-6 and pUclB-B-200-9 showed the existence of Following CsC1 preparation, PUClB-B-200-9 approximately 200 ug was digested with EcoRl and Hdm.

This EcoRl and HdII purified by gel electrophoresis [terminal 200 base pair truncated] The piece was inserted into M13mp18 to obtain single-stranded DNA. This 200 base break Add structure using fragments to easily produce large quantities of special RNA sequences. Ta. In particular, this fragment was used in transcription vectors p"r'y-i and PT7-2 (Unite Hard States Biochemical Corporation, 44122 Ohio, Kentucky -Brand, p, o, box 22406). This structure allows wood to In vitro synthesis of labeling material RNA used as a probe for Akatsuki's sooty red blood cell assay I can do it. Other recombinant vectors required for assaying the compositional cell signature are MstlI- 800 base pairs located directly downstream of the Tl restriction site Using the Hpal fragment 1, this fragment covers the 5' overhang found at the end of Mstn. Clone in the same manner again using Flenow DNA polymerase to "round" the ends. It turned on.

This 800 base pair fragment was inserted into four different vectors to obtain single-stranded RNA and DNA. inserted into. They are as follows.

M2S B-19-1-800β-globin message J-shaped strand M13 B-18-1-800β-globin non-message pT7-1-800 non-message Sage-like RNApT7-2-800 Message-like RNA The present invention relates to certain aspects. Although described in considerable detail, other embodiments are possible. However, it was , the spirit and scope of the appended claims is not limited to the above description of the embodiments. stomach.

4) βJλfv\f℃ρ for improved nucleic acid hybridization b. Solution sandwich hybridization. -Probe 1 + Probe 2 + Possesses modified target first binding group X Possesses standard mL improved sickle cell Mourning” Mouth begging Z See Figure 4 Label detection relative signal value vs. target DNA concentration Figure 2 Enlarged view around the peak value international search report -1-1-ml&men---1-1 P cT/υS 8fu 1025481 proboscis IM 1mlJ-Mu@@,,,Naka,,,,PCT/US 87102548 International Survey report international search report

Claims (1)

[Claims] 1. 1. An assay method for detecting a single-stranded target polynucleotide in a liquid sample, comprising: (a) combining the sample with at least two different probes to form a reaction mixture; The probes are a first probe and a second probe, and each probe is a first probe and a second probe. The probes comprise a single-stranded polynucleotide containing a nucleotide sequence that is complementary to one segment of the target polynucleotide, and the probes form a hybrid molecule by complementary base pairing with the target polynucleotide. The probe is also fixed on a solid carrier. (b) the reaction mixture is then bound to the first probe, but the probes do not hybridize with each other, and the second probe has a detectable label; contacting a solid carrier that binds neither the probe nor the target polynucleotide; (c) then determining whether any label is bound to the solid carrier. 2. 2. The method according to claim 1, wherein the first probe is an RNA or DNA fragment. 3. 2. The method according to claim 1, wherein the second probe is an RNA or DNA fragment. 4. Solid carriers include agarose, cellulose, glass, latex, polyacrylic polyamide, polyamide, polycarbonate, polyethylene, polypropylene, polyamide, 2. The method of claim 1, wherein the material is selected from the group consisting of listyrene, silica gel, silica, and derivatives thereof. 5. Solid carriers can be used as microparticles, macroparticles, sheets, beads, tubes, pipette chips, etc. The method according to claim 1, which has a shape selected from the group consisting of a cup, a plate, and a filter. Law. 6. If the label is a radioisotope, chemiluminescent compound, bioluminescent compound, or fluorescent compound substances, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, The method according to claim 1, wherein the method is selected from the group consisting of otin, carbohydrates, lectins, metal chelators, and derivatives thereof. 7. Claim 1: The first probe has a first binding group, the solid carrier has a second binding group, the two types of binding groups are capable of binding to each other, and the two types of binding groups constitute one binding pair. Method described. 8. The binding pair is avidin-biotin, hapten-antibody, one antibody-antigen, one carbohydrate pair. cutin, riboflavin-riboflavin binding protein, metal ion-metal ion binding 8. The method according to claim 7, wherein the method is selected from the group consisting of a synthetic substance, an enzyme-substrate, a boronatesis diol, a Staph A protein-antibody, an enzyme-inhibitor, and a derivative thereof. 9. 9. The method of claim 8, wherein the solid carrier is avidin-cellulose and the binding pair is biotin-avidin. 10. Each probe is 15 to about 50,000 nucleotides in length, and the first and second probes are each complementary to a different portion of the target polynucleotide, with the shortest distance between the two portions being about 300 nucleotides in length. Claim 1 is not longer than How to put it on. 11. 11. The method of claim 10, wherein the probe and the complementary portion are directly adjacent. 12. The first probe is an RNA or DNA fragment and the second probe is an RNA or DNA fragment; the solid carrier is agarose, cellulose, glass, latex. polyacrylamide, polyamide, polycarbonate, polyethylene, polyester Formed from a material selected from the group consisting of propylene, polystyrene, silica gel, silica, and derivatives thereof; selected from the group consisting of pipettes, beads, tubes, pipette tips, plates, and filters. the label is a chemiluminescent compound, bioluminescent compound, fluorescent compound, Selected from the group consisting of fluorescent compounds, enzymes, enzyme cofactors, heptene, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof. the first probe has a first binding group; the solid carrier has a second binding group; The two types of bonding groups can be bonded to each other; the two types of bonding groups form a bonding pair. The binding pairs are avidin-biotin, heptene-antibody, antibody-antigen, carbohydrate-lectin, riboflavin-riboflavin-binding protein, metal ion-metal ion. 11. The method according to claim 10, wherein the method is selected from a protein binding substance, an enzyme substrate, a boronate cis diol, a Staph A protein antibody, an enzyme inhibitor, and a derivative thereof. 13. 2. The method of claim 1, wherein the detection of the target polynucleotide is qualitative. 14. Detection of target polynucleotides determines the presence of a label bound on a solid carrier. 2. The method according to claim 1, wherein the method is determined quantitatively. 15. 2. The method of claim 1, wherein the probes are sequentially combined with the sample. 16. 2. The method of claim 1, wherein the sample is combined with all probes simultaneously. 17. 2. The method of claim 1, wherein the single-stranded target polynucleotide is formed by denaturation of a double-stranded polynucleotide. 18. A further step of determining whether any labels are bound on the solid carrier includes (a) separating from the solid carrier any second probe that is not incorporated into the hybrid molecule; and (b) subsequently adding the second probe to the sample. 2. The method of claim 1, comprising the step of detecting the presence or absence of a label on the solid carrier that indicates the presence or absence of the target polynucleotide. 19. A further step of determining whether any label is bound on the national carrier is to (a) measure the amount of unbound second probe in the solution, and (b) add that measurement to the reaction mixture. The presence of target polynucleotide in the sample compared to the total amount of second probe added to the sample. 2. The method of claim 1, comprising the step of determining whether or not. 20. 1. An assay method for detecting a plurality of double-stranded target nucleotides in a plurality of liquid samples, the method comprising: (a) at least a portion of at least one liquid sample suspected of containing a target nucleic acid; is also combined with two different probes to form a reaction mixture; The probes are a first probe and a second probe, each probe targeting a target polynucleotide. A single-stranded polynucleotide containing a complementary nucleotide sequence to a portion of a polynucleotide. The first probe and the second probe are complementary salts to the target polynucleotide. A hybrid molecule is formed by group pairing, and each probe is immobilized on a solid carrier. (b) the reaction mixture is then combined with the first probe, but not with the second probe, and the second probe is a probe with a detectable label; (c) then determining whether any of the labels are bound on the solid carrier, provided that the solid carrier does not bind to the plurality of polynucleotides. against that A method of binding each specific first probe. 21. each first probe has a first binding group, the solid carrier has a second binding group, the second binding group is a first binding group of the first probe specific for the plurality of target polynucleotides; 21. The method of claim 20, wherein: 22. Each probe is about 15 to about 50,000 nucleotides in length, and each In each pair of probes that are specific for a target polynucleotide, the first and second probes are complementary to different portions of the respective target polynucleotide, such that the shortest distance between the portions is less than about 300 nucleotides. Claim 20 that is not long Law. 23. 23. The method of claim 22, wherein the complementary portions are directly adjacent. 24. 21. The method of claim 20, wherein the at least one single-stranded target polynucleotide is formed by denaturation of a double-stranded polynucleotide. 25. 21. The method of claim 20, wherein at least some of the probes are combined with the sample at different times. 26. An assay method for detecting multiple single-stranded target polynucleotides in a liquid sample. (a) combining a sample with a plurality of different probes to form a reaction mixture, wherein the probes are at least two specific for the target polynucleotide; 2 probes, the first probe and the second probe each containing a nucleotide complementary to a portion of the respective target polynucleotide. The first probe and the second probe form a hybrid molecule by complementary base pairing with the target polynucleotide. the second probe has a detectable label; any of the plurality of probes is probes are not fixed on the rear, multiple probes do not hybridize with each other, and multiple target polynucleotides Each second cleotide-specific probe is a probe with a different label. (b) then contacting the reaction mixture with a solid carrier that binds the first probe but not the second probe or the target probe; (c) then any of the different labels are placed on the solid carrier; A method comprising the step of determining whether or not they are combined. 27. Each probe is about 15 to about 50,000 nucleotides in length, and a first probe and a second probe specific for each target polynucleotide are used for each target polynucleotide. complementary to different parts of the target polynucleotide, and the shortest distance between the two parts is 27. The method of claim 26, wherein the method is no longer than about 300 leotides. 28. 28. The method of claim 27, wherein the complementary portions are directly adjacent. 29. 27. The method of claim 26, wherein the at least one single-stranded target polynucleotide is formed by denaturation of a double-stranded polynucleotide. 30. At least some of the probes may be combined with the sample at different times. The method according to claim 26. 31. 27. The method of claim 26, wherein all probes are combined with the sample at the same time. 32. A kit for detecting a single-stranded target polynucleotide in a sample, comprising: (a) a polynucleotide reagent comprising at least two different probes, the probes comprising a first probe and a second probe; probes, each probe consisting of a single-stranded polynucleotide containing a nucleotide sequence complementary to a portion of the target polynucleotide; the probes form a hybrid molecule by complementary base pairing with the target polynucleotide; (b) the probes are not immobilized on a solid carrier and do not hybridize with each other; the second probe has a detectable label; (b) it binds to the first probe, but the second probe and the target polynucleo A kit comprising a solid carrier that does not bind to TiD. 33. 33. The kit according to claim 32, wherein the first probe is an RNA or DNA fragment. 34. 33. The kit according to claim 32, wherein the second probe is an RNA or DNA fragment. 35. Solid carriers include agarose, cellulose, glass, latex, polyacrylic selected from the group consisting of lylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silica gel, silica, and derivatives thereof. 33. The kit of claim 32, wherein the kit is formed of a material comprising: 36. Solid carriers include microparticles, macroparticles, sheets, beads, tubes, pipettes 33. The kit of claim 32, selected from the group consisting of tips, plates, and filters. 37. If the label is a radioisotope, chemiluminescent compound, bioluminescent compound, or fluorescent compound 33. The kit of claim 32, which is selected from the group consisting of compounds, phosphorescent compounds, enzymes, enzyme cofactors, heptene, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof. 38. the first prog has a first binding group; the solid carrier has a second binding group; 33. The kit of claim 32, wherein the two binding groups are capable of binding to each other and the two binding grooves form a binding pair. 39. The binding pair is avidin-biotin, hapten-antibody, antibody-antigen, carbohydrate-lectin, riboflavin-riboflavin-binding protein, metal ion-metal ion binding substance, enzyme-substrate, boronatesis diol, Staph A protein-antibody. 39. The kit of claim 38, wherein the kit is selected from the group consisting of , enzyme inhibitors, and derivatives thereof. 40. The solid carrier is avidin-cellulose and the binding pair is biotin-avidin. 40. The kit according to claim 39, which is a kit. 41. each probe is about 15 to about 50,000 nucleotides in length, the first and second probes are each complementary to a different portion of the target polynucleotide, and the shortest distance between the two portions is about 15 to about 50,000 nucleotides in length; 33. The kit of claim 32, having a length of no more than 300 mm. 42. 42. The kit of claim 41, wherein the probe and the complementary portion are directly adjacent. 43. 33. A kit according to claim 32, comprising means for qualitatively detecting the label. 44. 33. The kit according to claim 32, comprising means for quantitatively detecting the label. 45. 33. The kit of claim 32, wherein at least one of the polynucleotide reagents contains one or more probes. 46. 33. The kit of claim 32, wherein all probes are contained in a single reagent. 47. A kit for detecting a plurality of single-strand target polynucleotides, the kit comprising at least two different probes for each target polynucleotide. a polynucleotide reagent comprising a first probe and a second probe; lobes, each probe containing a nucleotide complementary to a portion of its target polynucleotide. It consists of a single-stranded polynucleotide containing a leotide sequence, and both the first probe and the second probe undergo hybridization by complementary base pairing with their target polynucleotide. the second probe is a probe with a detectable label; none of the probes is immobilized on a solid carrier; the probes cannot hybridize with each other; the second probe is a probe with a detectable label; A kit comprising a solid carrier capable of binding to a second probe, but not to any second probe or to any target nucleotide. 48. 48. The kit of claim 47, wherein each first probe has a first binding group and the solid carrier has a second binding group, the second binding group being capable of binding to the first binding group on the first probe. . 49. 48. The kit of claim 47, wherein each second probe specific for each target polynucleotide comprises a different label. 50. 50. The kit of claim 49, comprising means for detecting a label on the second probe. to. 51. Each probe is about 15 to about 50,000 nucleotides in length, each pair of probes is specific for one target nucleotide, and each first probe and second pair of probes are specific for one target nucleotide. 49. The kit of claim 48, wherein the lobes are complementary to different portions of the target polynucleotide, and the shortest distance between the portions is no greater than about 300 nucleotides. 52. The probe must be complementary to an immediately adjacent location on the target polynucleotide. The kit according to claim 51. 53. 48. The kit of claim 47, wherein at least one of the polynucleotide reagents contains one or more probes. 54. All probes complementary to each target polynucleotide are contained in a single reagent. 48. The kit of claim 47, wherein the number of polynucleotide reagents is equal to the number of target polynucleotides. 55. A kit for detecting a plurality of target polynucleotides in a sample, the kit comprising: (a) a polynucleotide reagent for each target polynucleotide, the polynucleotide reagent comprising at least two different probes for each target polynucleotide; The probe is the first probe and and a second probe, each probe comprising a single-stranded polynucleotide containing a nucleotide sequence complementary to a portion of its target polynucleotide; Both the lobe and the second probe are activated by complementary base pairing with their target nucleotide. to form a hybridizing molecule, none of the probes is immobilized on a solid carrier, the second probe has a detectable label, and all probes for multiple target polynucleotides cannot hybridize with each other; against multiple target polynucleotides (b) each second probe that binds to the first probe is a probe with a different detectable label; A kit comprising a solid carrier that does not bind to ocide. 56. 56. The kit of claim 55, comprising means for detecting a different label on the second probe. 57. Each probe is a single-stranded polymer with a length of about 15 to about 50,000 nucleotides. each pair of probes is specific for each target nucleotide, and each first and second probe is complementary to a different portion of its target polynucleotide, such that the shortest distance between the portions is Longer than about 300 nucleotides 57. The kit of claim 56. 58. The probe must be complementary to an immediately adjacent location on the target polynucleotide. The method according to claim 57. 59. 56. The kit of claim 55, wherein at least one of the polynucleo reagents contains one or more probes. 60. 56. The kit of claim 55, wherein all probes are contained in a single reagent. 61. Samples containing standard polynucleotides along with target polynucleotides, all that is, the nucleotide sequences of both polynucleotides are substantially the same and the standard polynucleotide leotide has an A portion and a B portion, the target polynucleotide has an A′ portion and a B′ portion, and the standard and target polynucleotides have at least one nucleotide located between the A portion and the B portion of the standard polynucleotide. are different from the nucleotide located between the A' and B' parts of the target polynucleotide ▽ 1. An assay method for detecting a target polynucleotide in a sample containing both a standard polynucleotide and a target polynucleotide, the method comprising: In the case of cleotides, there is no segment containing both A and B moieties; In the case of polynucleotides, at least one segment contains both A' and B' parts. (b) The sample treated in (a) is combined with at least two probes, each of which contains a first probe and a first probe. second probe, each probe is a single-stranded polynucleotide. cleotide, the probes cannot hybridize with each other, the first probe is in complementary base pairing with the A part and the A' part, and the second probe is in complementary base pairing with the B part and the B' part. (c) both probes form a hybrid molecule by complementary base pairing with a single-stranded segment of the target polynucleotide, both containing both A' and B' moieties; A method comprising the step of determining the presence of a hybridizing molecule containing a probe. 62. 62. The method of claim 61, wherein the cleaving step comprises restriction enzyme treatment. 63. 63. The method of claim 62, wherein the target and standard polynucleotides are double-stranded and the cleavage step further comprises denaturation of the target and standard polynucleotides. 64. 62. The method of claim 61, wherein portion A is the same as portion A' and portion B is the same as portion B'. 65. 62. The shortest distance between the A and B moieties on the standard polynucleotide and the shortest distance between the A' and B' moieties on the target polynucleotide is not greater than about 300 nucleotides. Method. 66. 66. The method of claim 65, wherein the shortest distance between the A and B moieties on the standard polynucleotide and the shortest distance between the A' and B' moieties on the target polynucleotide is not greater than about 10 nucleotides. . 67. The target polynucleotide is a genetic variant of the standard polynucleotide; 62. The method of claim 61, wherein the target polynucleotide is the result of a genetic mutation that is indicative of a genetic disease. 68. 68. The method of claim 67, wherein the genetic disease is sickle cell negative blood. 69. Neither probe is fixed to a solid carrier, and the second probe is (a) combining the reaction mixture with the first probe, but only the second probe and the β or B' portion; 62. The method of claim 61, comprising the steps of: (b) subsequently determining whether any labels are bound on the solid carrier. 70. Solid carriers include agarose, cellulose, glass, latex, polyacrylic selected from the group consisting of lylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silica gel, silica, and derivatives thereof. 70. The method of claim 69, wherein the method is formed of a material comprising: 71. Solid carriers include microparticles, macroparticles, sheets, beads, tubes, pipettes 70. The method of claim 69, wherein the shape is selected from the group consisting of a chip, a plate, and a filter. 72. If the label is a radioisotope, chemiluminescent compound, bioluminescent compound, or fluorescent compound 70. The method of claim 69, which is selected from the group consisting of compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof. 73. the first probe has a first binding group; the solid carrier has a second binding group; 70. The method according to claim 69, wherein the two types of bonding groups are capable of bonding to each other, and the two types of bonding groups constitute one bonding pair. 74. The binding pair is avidin-biotin, hapten-antibody, antibody-antigen, carbohydrate-lectin, riboflavin-riboflavin-binding protein, metal ion-metal ion binding substance, enzyme-substrate, boronatesis diol, Staph A protein-antibody. 74. The method of claim 73, wherein the method is selected from the group consisting of enzyme inhibitors, enzyme inhibitors, and derivatives thereof. 75. The solid carrier is avidin-cellulose and the binding pair is biotin-avidin. 77. The method of claim 76. 76. The first probe is an RNA or DNA fragment and the second probe is an RNA or DNA fragment; the solid carrier is agarose, cellulose, glass, latex. polyacrylamide, polyamide, polycarbonate, polyethylene, polyester Formed from a material selected from the group consisting of propylene, polystyrene, silica gel, silica, and their derivatives; selected from the group consisting of pipettes, beads, tubes, pipette tips, plates, and filters. the label is a chemiluminescent compound, bioluminescent compound, fluorescent compound, Consisting of fluorescent compounds, enzymes, enzyme cofactors, heptene, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof; the lobe has a first binding group, the solid carrier has a second binding group, the two binding groups are capable of binding to each other; the two binding groups are a binding pair; The binding pair is avidin-biotin, heptene-antibody, antibody-antigen, carbohydrate-lectin, riboflavin-riboflavin-binding protein, metal ion-metal ion-binding substance, enzyme-substrate, boronate cis diol, staff A protein-antibody, 77. The method according to claim 76, wherein the method is selected from enzyme inhibitors and derivatives thereof. 79. Determining whether any labels are bound on the solid carrier further comprises the steps of: (a) separating from the solid carrier any second probe that is not incorporated into the hybridizing molecule; and (b) baking the sample into the sample. 70. The method of claim 69, further comprising the step of detecting the presence or absence of a label on the solid carrier indicating the presence or absence of the target polynucleotide. 80. Determining whether any label is bound on the solid carrier further comprises: (a) measuring the amount of unbound second probe in solution; and (b) adding that measurement to the reaction mixture. target polynucleotide in the sample compared to the total amount of second probe 70. The method of claim 69, comprising the step of determining the presence or absence of tide.