JPH07505760A - Restriction amplification assay - 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] foundation I respect Five times μ y standing 1 The present invention provides methods, reagents, and kits for use in quantifying the abundance of target nucleic acid sequences. (Measurement set)

In particular, the present invention provides a method for detecting a specific target nucleic acid sequence in a sample containing an unknown abundance of a specific target nucleic acid sequence. Regarding the methods used to assay for the presence of specific nucleic acid sequences, related to the probe used in the configuration.

Nine beans and ■ Prisoner's feet Genetic engineering is a technology that evaluates the genetic characteristics of living organisms. A specific gene or gene in an organism or an extract of extracellular genetic material obtained from that organism. It is often desirable to determine whether a portion of a gene is present. Ru. In short, all genes and gene parts are all polynucleotide molecules, or a specific sequence to a nucleic acid that forms part of the polynucleotide in the sample. Test directly to see if the specific sequence of nucleobases that make up the gene is present can be found.

2.3 Examples of the need to know specific nucleobase sequences include, for example, pathogens. Detecting the presence of alleles, determining the presence of alleles, and identifying foci in the host genome. These include detection of presence, detection of specific mRNA, and detection of cell post-modification.

Huntington's chorea, muscular dystrophy, phenylketonuria, thalassemia, Genetic diseases such as sickle cell anemia and sickle cell anemia can be diagnosed by analyzing the NA of the individual. Can be diagnosed. Furthermore, hybridization using nucleic acid probes Viruses, virusoids, bacteria, fungi, and protozoa It is possible to diagnose or identify objects or any other plant or animal organism.

Assays for the detection of various nucleic acids are described in the literature. Such testing methods, In addition, methods requiring the detection of polynucleotides include DNA-DNA or DNA A - purine-pyridine of complementary nucleic acid strand in heavy chain (tenibrex) to RN8 It is based on mid-base pairing. This type of base pairing is based on athenosine chimney. (A-T) and ryanosine-cytosine (GC) are two-stranded DNA. This is most often caused by pairing through elementary bonds, but DNA-RNA hybridization In addition, the molecule has 6 hydrogen bonds formed by the athenosine-upfyl base pair. come. A given nuclease in which the analyte sequence and probe nucleotide sequence are related. The base pairing of nucleic acid strands used to determine the presence or absence of octide sequences is usually performed in the form of nucleic acid hybrids. formation, simply called hybridization.

A powerful tool in molecular biology is the ability to divide nucleic acids into one DNA or another. is the ability to determine whether an RNA molecule has a sequence that is complementary to that of an RNA molecule. Saza In the plot method, DNA separated by electrophoresis is transferred from a gel matrix to a nitrofluorophore. a--Transfer by passive diffusion to the immobilized support phase, followed by labeling. This is a well-known method of hybridizing fibers. By modifying this slightly, RNA There are similar methods used to detect This is called the ``face-fill'' method. Those who use dried akarose gel as the stationary phase The law is said to be An7'l+7) law. All these testing methods are mRN A, 9a-con, gene, fragment, adjacent (7-ranked sequence) sequence, repeating element, etc. It is a valuable tool for the analysis of

U.S. Pat. No. 4,358,535 describes a method for detecting pathogens using target nucleic acid sequences. is listed. In this method, hybridization is performed using radiolabeled nucleotide probes. The target nucleic acid sequence is immobilized on an inert support prior to formation of the compound. The target nucleic acid sequence is This is then confirmed by detecting the presence or absence of the label on the inert support.

European Patent Application No. Of+7/140 describes non-radioactive drug-labeled polynucleotides. A probe and method of using the probe are disclosed. Target nucleic acid sequence The columns are also fixed to fixed supports.

U.S. Patents 4.767.699 and 4.795.701 describe nucleic acid replacement assays. Disclosed. These methods use two types of polynucleotides, one of which is Label the nucleotides and use the remaining nucleotides to connect the labeled probe to the target sequence. , thereby detecting the target molecule. In these tests th, ^T P is used to detect whether hybridization has occurred for the target molecule. .

Many assay methods using nucleotide probes have problems with their detection methods. sign pro Due to the sensitivity of the probe and the background generated during the assay, erroneous results are often produced. become.

In order to more effectively determine the nucleic acid sequence from a given DNA or RNA sequence. Various target amplification methods can be used.

This method is known as modulus +1 CI' and is described in U.S. Pat. No. 4.683.195. However, this method uses a set of primers and DNA polymerase. , extend and amplify the nucleic acid sequence of the target nucleotide and later use it for probe detection . By amplifying the DNA sequence, target nucleotides can be detected using nucleotide probes. can be detected effectively. One of the problems encountered with this probe-based assay is that This is contamination of the reaction medium.

Class II restriction enzymes produce 91 folds of double strands at specific sites in the DNA molecule. It is knowledge. These enzymes are abundant in bacteria, and the 1fiIi subunit Contains Mg! for DNA cleavage. It only requires °. cleavage to DN, Alternatively, cleavage occurs at a specific location within or adjacent to the enzyme's recognition site. Until today As many as 500 types of restriction enzymes have been isolated from Cuteria strains. These restriction enzymes are Its properties have been elucidated, mainly in terms of its recognition sequence and cleavage specificity. most of Restriction enzymes, or endonucleases, recognize sequences 4-6 nucleotides long. However, some have been found to have a recognition site of 7-8 bases. In all However, most recognition sites contain a two-fold axis of symmetry, and in most cases all within the site All bases have been identified as unique.

Hybridized sequences or palindromic symmetrical sequences are Recognized by donuclease. Endonucleas with symmetrical recognition sites zes generally make symmetrical cuts.

The use of restriction enzymes with specific cleavage sites is known in the art. General Restriction enzymes are used for specific mapping, cloning, and identification of DNA sequences. However, these are used in assays using various nucleotide probes. came. For example, U.S. Pat. No. 4,683.194 describes specific restriction sites in a nucleic acid sequence. disclosed a method for detecting the presence or absence of a nucleic acid covering a restriction site. The resulting cleaved and uncut oligos are cleaved by restriction enzymes. mer separation and detection based on probe label type.

A similar idea was developed in the US to detect target nucleotides with half restriction sites. 4.725.537. The patent is based on a substitution type detection method. , the use of endonucleases has been disclosed.

Ike's assay method, which is based on the idea of cutting nucleic acid sequences with nucleotide probes, is now available in the United States. It is disclosed in patent 4.876,187. - This method requires at least one point Lobe nucleic acid sequences are identified and cleaved, which then binds to complementary target DNA sequences. By removing missing reporter molecules, it is possible to detect sequences in DNA and RN. It is used. This assay has improved sensitivity and improved signal-to-noise ratio of the detection method. It is a highly accurate testing method.

n II Assay Method provides a method for detecting a nucleic acid sequence in a target molecule. It has high sensitivity, easy detection, less contamination of assay medium, and easy operation. Moreover, there is a need for an assay method that prevents erroneously obtaining positive results.

The present invention provides an extremely sensitive assay that uses a novel restriction amplification method to detect nucleic acid sequences. The present invention provides a method to overcome the shortcomings of the above methods. Re-inject the desired cleaved target sequence. A second oligonucleotide can also be used in this assay, which cycles The cleaved probe oligonucleotide is amplified.

eco 4 Therefore, it is an object of the present invention to provide a highly sensitive, recirculating, young nucleic acid sequence for detecting nucleic acid sequences. Alternatively, an assay method based on probe amplification is provided.

Another object of the present invention is to recirculate the target sequence of interest to remove labeled probes. It is an object of the present invention to provide an assay method for amplification.

The present invention provides cleavable bonds, i.e., cleavable bonds that can be cleaved by a cleavage enzyme, in a biological specimen. provided is a method for detecting the presence of a nucleic acid sequence comprising a binding that comprises the steps of: Consisting of

(a) consists of a nucleic acid sequence and a cleavable linkage, and substantially comprises a nucleic acid target sequence; A complementary oligonucleotide is used, and the oligonucleotide has a detectable marker. car is connected.

(b) When the target sequence and the oligonucleotide form a hybrid, the cleavage adding to the reaction mixture a cleaving enzyme capable of cleaving the scissile bond; (c) hybridize the reaction mixture and then (d) apply a detectable marker. detectable cleaved marker in the presence of bound uncleaved oligonucleotide A nucleic acid detection method consisting of detecting.

In a pond aspect of the invention, a nucleic acid sequence comprising a cleavable compound that is cleavable with a cleavage enzyme. A method for detecting the presence of a column in a sample, comprising the steps of: (a) a target nucleic acid sequence comprising a tjlJ cleavable bond; Denaturation in the presence of oligonucleotides that are substantially complementary to the nucleic acid sequence of the target molecule A reaction mixture is formed by:

(b) adding a cutting Vr mixture to the mixture; (c) hybridizing the reaction mixture and cleaving the oligonucleotide with a cleaving enzyme; A marker that can be detected from the sequence is r1: (d) Cleavage in the presence of an uncleaved oligonucleotide coupled to a detectable marker. A method comprising detecting a detectable marker that has been detected.

Furthermore, in the pond embodiment of the invention, a nucleus containing a νJ cleavable bond that can be cleaved with a cleavage enzyme is provided. A method for detecting the presence of an acid sequence in a biological specimen, comprising the steps of: (a) A target molecule containing a scissile bond is detected with a sequence complementary to the target. hybridize to a labeled oligonucleotide probe with a marker that comes Lobe: forms a labeled double strand; (b) cleaving the duplex at the cleavable bond;

(c) recycling the target molecule;

(d) reconstituting a cleavable binding site in the oligonucleotide; (e) in the presence of an uncleaved oligonucleotide coupled to a detectable marker; A method comprising detecting a cleaved oligonucleotide probe.

Yet another aspect of the invention is a method of cleaving a nucleic acid sequence, the method comprising: An oligonucleotide with a target double-stranded recognition and cleavage site is prepared, and the oligonucleotide is cleotide in a solvent that changes the dielectric constant of the reaction mixture and/or promotes cleavage. A method consisting of adding a sufficient amount of class II restriction enzyme.

The purpose of the present invention is to use class 1 and class IIS restriction enzymes. In the assay, partial hairpin oligonucleotides are used as labeled oligonucleotides. It is to use a tide sequence.

Furthermore, another object of the present invention is to provide a kit for diagnosing various diseases by the above method. It is to be.

Ll・　1 FIG. 1 is a schematic representation of the method of the invention used with complementary target sequences.

Figure 2 schematically represents the method of the invention used for sequences that are neither complementary nor targeted. It is something.

Figure 3 shows a few samples conducted using the second oligonucleotide according to the present invention. This is an autoradiograph for the reaction.

Figure 4 shows a partial hairpin oligonucleotide as the first oligonucleotide. This is a schematic representation of the method of the present invention using class IIS restriction enzymes. Ru.

FIG. 5 schematically illustrates the method of the present invention using a labeled first oligonucleotide. It is expressed in terms of

FIG. 6 shows a few results obtained according to the present invention without using a second oligonucleotide. This is an autoradiograph of the reaction of a specimen.

Figure 7 shows the use of partial hairpin oligonucleotides and class II restriction enzymes. This is a schematic representation of the method of the present invention when

In particular, the present invention provides a method for targeting target molecules in Relates to methods of detecting nucleic acid sequences. The second oligonucleotide is the nX target Although the scissile bonds of the molecule can be reconstituted and the first oligo A second oligonucleotide may or may not be used. or do not use . Reconstitution of scissile bonds without the use of a second oligonucleotide , labeled oligonucleotides can be used to recycle target sequences. cost point Since it is more efficient, it is better to perform this method without using the second oligonucleotide. It's advantageous.

The term "oligonucleotide" as used herein refers to a shortened number of nucleotides. means a compound formed by the combination of The exact oligonucleotide size is It depends on many factors, including the final function of the Creogate when used.

As used herein, the term "cleavable bond" refers to a restriction enzyme or restriction end refers to a site-specific recognition nucleotide sequence that can be cleaved with the use of a nuclease .

As used herein, the terms “restriction endonuclease” and “restriction enzyme enzyme” refer to each An enzyme that cuts double-stranded DNA at or near a specific recognition nucleotide site means.

As used herein, the term "partial duplex recognition and cleavage site" refers to a cleavable linkage. Refers to a nucleotide sequence that is not completely double-stranded at the joining site.

The term “partial hairpin array °°” used here refers to the internal nucleotide sequences complementary to the target sequence to be detected. Refers to an oligonucleotide sequence with an extra column.

Nucleic acid samples used here may be derived from any source, including living organisms. However, when using class II restriction enzymes, the target contain a specific restriction site, or when using class IIS restriction enzymes, downstream ψ must be able to be cleaved; therefore, this method Either pure NA or cDNA or a phosphoric acid mixture can be used. The source is pI Blasmi such as IR322, cloned DNA and any source Genomes containing ONΔ can be obtained from bacteria, yeast, etc. , biological specimens containing viruses and higher organisms such as plants, birds, reptiles, and mammals. obtained from.

Genome I) NA can be extracted from tissues such as blood, urine, chorion, and amniotic cells using various methods. It can be prepared by

(For example, the method reported by Mannytheis et al., I) 11 280-281 (1982) [Maniatis et al, kl o I c c t + l a r 7, pp280-281 (198 2)], if there is a need or desire to reduce viscosity, human DN prepared for analysis. A sample can be physically sheared or digested using a specific restriction endonuclease. This can be achieved with

The first oligonucleotide used in the present invention is a single-stranded oligonucleotide. , has a structure that is one complement to the nucleic acid sequence to be detected. Probe is normal DN In A, there is no limit to the number of bases/groups, but as a probe, less than about 100 bases, especially Preferably it has 10-40 bases. Is the probe Metsenger RNA? complementary cD obtained by reverse transcription to metsenger RN with reverse transcriptase. obtained from the NA strand, or the genome can be easily digested with endonucleases. The genes or gene fragments can then be "corned" using known methods. It can be obtained by See, for example, the following literature: Kornberg, DNA Re 1i cation.

W, Il, r’reeman and Co,, San Francisco, (1980) pp670-07’l: So etal, Infect, I mmun, , 21:pp, 405-411 (+978), aim After isolating and characterizing a gene or DNA fragment, Fragments can be used for probe preparation. The probe is also myridan O ( Chemically synthesized using an automatic synthesizer such as a registered trademark) You can also do that. Probe oligonucleotide probes are mostly atomic or inorganic Radicals, radioactive nucleotides, heavy metals, antibodies, enzymes, biotin, immunobiotin, etc. Labeled with a detectable marker. The use of radioactive labels is convenient.

Radioactive labels include l1l), 11(, +1() with adequate signal and sufficient half-life. Any radioactive label can be used, provided there is a suitable period. Other signs include A ligand that can serve as one member of a specific binding pair for an antibody (d), as a member of a specific binding pair for fluorophores, chemiluminescent substances, enzymes, and labeled ligands. Antibodies, etc. that can act as Various formats have been used in immunoassays However, these can be used for the verification of this application. For indicator selection, the indicator is The rate of hybridization and binding of probe to genetic DNA is not affected. Defined by considering the impact. Label detects amount of DNA available for hybridization It is necessary to provide sufficient sensitivity to The important thing to consider is the synthesis of the probe. These include the ease of use, automation ability, and ease of use. mark The method of attaching the label to the probe will depend on the nature of the label. The radioactive label is Seed methods can be used. Commonly used methods include γ-”P-NTP and T End labeling is performed with 4 polynucleotide kinase. Another method is to combine nucleotides. by replacing one or more elements with radioactive isotopes, e.g. hydrogen This is a method of substituting tritium.

When using other radionucleotide labels, a variety of linking groups can be used. Wear. The terminal hydroxyl group can be esterified with an inorganic acid. For example, 3! P phosphate or +4 0 Organic acids can be esterified with a terminal hydroxyl group, or esterification can improve the labeling properties. It is possible to generate a bonding group that Furthermore, the intermediate base can be converted into an activatable binding group. It is also possible to substitute and then attach to a label.

A wide variety of ligands and antiligands can be used. If the ligand is Places with natural receptors such as thyroxine, thyroxine and cortisol In this case, a labeled natural receptor can be accompanied by a ligand. hapten or antigen All chemical compounds can be used with antibodies.

Enzymes that can be used as labels are mainly hydrolytic enzymes, especially nisterase and glycosylation enzymes. dase, phosphatase or oxidoreductase, especially peroxidase. fluorescence The body compounds are fluorescin and its derivatives, rhodamine and its derivatives, dansyl, Umbelliferone, etc. Chemiluminescent substances are luciferin and 2,3-dihydre Contains loftalazinedione, or luminol.

Additionally, other methods for labeling and detecting nucleotide probes are described in U.S. Pat. , 868.103.

According to this method, energy transfer causes bathochromic movement and/or delayed fluorescence emission. bring about. The fluorescent radiation emitted from the first energy emitter (IEI) is transferred to the second energy emitter (IEI). is absorbed by the energy emitter (El). The second emitter is the first energy emitter emit longer wavelength fluorescent radiation. The energy released by (El) is In order for (El) to be absorbed by (El) and (El) to emit fluorescence energy with a long wavelength, , (IEI) and (El) must be located close to each other.

In the present invention, labeled probes may be detected by any means. However, the sign The oligonuclease is attached at a location remote from the bond to be cut, and is attached to the first oligonucleotide. Preferably, it is at the 3' or 5' end of the leotide.

The first oligonucleotide not only has a label, but also has a label. must also contain a cleavable bond that is complementary to the intended target molecule. do not have. This cleavable bond can be formed in a hybrid form when class II restriction enzymes are used. Once grown, it must be easily cleavable with restriction enzymes or other methods. But The use of the ras IIS restriction enzyme requires a label sequence; Wake up in the flow.

In a preferred embodiment of Kinoe1ff+, the first oligonucleotide is an IV constant support. fixed to the body. In the present invention, any immobilization method can be used as long as the oligonucleotide can be attached. Supports can also be used. Examples of supports are glass, test tubes, microtitration plates, nylon , beads, agarose beads, magnetic beads, glass beads, Teflon, polyethylene These include Ren beads and optically detectable chips. Oligonucleotide as a fixed support When attaching, either end of the oligonucleotide is attached to a carboxyl or The method of derivatizing with an amino group is known in the art. the An example of another attachment method is to use avidin-conjugated beads and attach oligonucleotides through biotin. It is to make the cleotide l-t,v. When using a fixed support or carrier, support Labeling the end opposite to the end group of the oligonucleotide to which the body is attached It's advantageous.

Restriction enzymes are known and have specific recognition sites.

8l+al11.　8lu　1. Aoc I, 8 pa 1. 8 paL 1.　 Aval, 8va II, Roal I, Ham Ill, '[1cl' I, [ Igl I, IjgI+I, l1ssll 11. B5tlE II, C1a 'I, Dra I, Eco52 1゜1Eco I, IEco RII, R co　(V, r’sp　I, 1lae　If, 1lha1, l1ind　II I, l1pa I, l1pall, Kpn I,' Ksp I.

Nci I, Mst II, Nae I, Pst I, Pvu I and and Xba I etc. There are over 500 types of restriction endonucleases that can be used. Any of these enzymes can be used in the present invention. The standard for selecting restriction enzymes is a recognition site for a target nucleotide molecule, a first oligonucleotide probe and an enzyme Determined based on elementary properties. a first oligonucleotide block containing a cleavable bond; The lobe must be complementary to the recognition or cleavage binding site of the target nucleotide. In this case, it is necessary to add restriction enzymes after hybridization of these nucleotides. The double strands are cleaved.

In a more preferred embodiment of the invention, the partial hairpin oligonucleotide is Can be used as a nucleotide component, in which case the oligonucleotide is labeled. must be maintained. A partial hairpin oligonucleotide sequence is defined as , within which hybridization can occur, thus creating a loop, and the desired target Contains an extra single-stranded oligonucleotide sequence capable of hybridizing to the sequence.

The hairpin loop can be present at either the 5° or 3° end of the oligonucleotide. may be labeled as described above.

1. Class I'lS Restrictions in Connection with the Use of Partial Hairpin Oligonucleotide Sequences The endonuclease cuts the double-stranded DNA at a precise distance from the recognition site. Can be used to cut.

Examples of class IIS restriction endonucleases are lobvl, lobvII, orinI , FokI, llgaI, l1phI, MboII, Mn1I, S[aNI, Includes TaqlI, TtbllEI, and 1 (inGuI, etc.).

Regarding this in particular, see 5zybalski, Gene, 40:169- 173 (1985); and Podhajska etal Gene, 40 : 175-182 (1985), Reference 0 In the practice of this invention, the Class IIS system The limiting enzyme can also be used without the partial hairpin oligonucleotide sequence. − Figures 4 and 7 show how hairpin oligonucleotides are used in the method of the invention. It explains what can be done.

Restriction enzymes and endonucleases are fairly stable proteins. Usually homogeneous There is no need to make it pure. All restriction enzymes cut Mg! ' or M Requires cofactors such as n"" and is most active between pH 7.2 and 7.6. is high.

Usually, an appropriate buffer is used in enzymatic cleavage.

These buffers fl12 are capable of cleaving the hybridized double strands by 1. ・Varies depending on the restriction enzyme used. Therefore, the DNA substrate and the control illumination;(・; In addition to ゛(, the reaction solution is usually Tris, i.e. 1 min., 1 min. Tc i ill<　, u　2°, NaCl, 2-mercaptoeta Noor, 1. Various restrictions, including Bovine 111L Clear Rubano, (IIsA) The difference between enzymes is their dependence on ionic strength. Cutting efficiency up to 1:・ Different restriction enzymes may be mixed with different buffers. For example, l When using 1ind III restriction enzyme for assay, how to maximize the cutting effect of 1ind III restriction enzyme , use the following buffer: 50m^I'NaCl, 25mM) Lis-HCl ( pH 7,7), 10 + nlI odor CI, 10mM p-meliabutoethano and B5A 100μmH/ml, when using Ideal enzyme. uses the following buffers: 50 m & l NiCl, room M) Lith-salt Acid (pH 7,5), C1 in 54 nkl, and usAIoo1tg/ml. subordinate Therefore, in the present invention, different 111 buffers are used depending on the restriction enzyme used in the assay. I am planning on using it.

The +1J enzymes also have different optimum temperatures. Cutting is usually done at 37" (:, but inside Although the number is not very large, Sma I-like/C endonuclease is low in culture. (Incubation) Humidity = 1', <, U<, especially for thermophilic organisms. For Taq I, r 4e 133, a significantly higher temperature is required. The reaction 1QIX according to the invention is based on the assay i2t+: considering the +++'+tu enzyme used. and interpret.

After forming a high frit, the double-stranded molecule is subjected to restriction enzyme tJ]1vi. All available methods can be used for the cutting process, e.g. using chemicals. 2 pieces f (cut the complex at a specific site 11) 1 can be made.

According to the present invention, the second oligonucleotide is reconstituted with the desired cleavage target molecule. nucleotides can also be used, thereby allowing hybridization with nucleotides Rod formation occurred at assay 11°I, ζ. The second oligonucleotide is a target of interest. It plays the role of 1', l which makes the array of The width of the IJ-b that was created will be 't'1. This second oligonucleotide 1゛1''; first probe oligonucleotide such as 1° end or 5° end is a single-tree nucleotide complementary to the end of The second oligonucleotide is the 1 can be derived by a method similar to the synthesis of oligonucleotides.

complementary to the 3° or 5° end of the first oligonucleotide, as well as The oligonucleotide reconstitutes the target molecule at the site cleaved by the restriction enzyme. It must contain a base such as a 3' or 5' base. target molecule After reconstitution with the truncated end, hybridization is resumed with the first labeled oligonucleotide. The process is also “circulated” and the labeled probe is added to the reaction medium. emit light. The second oligonucleotide is less than 100, especially 10-20@ salt groups, especially 14 bases. Figures 1 and 2 show the implementation status of the present invention. This is an explanatory example showing the following.

In a preferred embodiment of the present invention, since the second oligonucleotide is not used, the target molecule No reconstitution of the truncated end occurs. Therefore, the desired positive sequence can be easily detected. can.

Contrary to the known prior art, hybridization was performed by the inventors under various chemical conditions. 1. When using class II restriction enzymes, It has been found that a double-stranded recognition and 9J cleavage site is sufficient. The present invention various chemicals used to promote cleavage at partial double-stranded recognition cleavage sites. Examples (usually used diluted in a suitable solvent) include cobalt, magnesium, and zinc. divalent cation; glycerin, dimethyl sulfoxide (DMSO), Things like Chilporumamide (DMF), Formamide, Ethylene Glycol including. Reference 11: George al, Journal of B iological chemistry, 255゜pp, 6521-6524 (1980), used to change the dielectric constant or promote cleavage of restriction sites. The concentration of diluted chemical used will vary depending on the assay conditions. Usually, 10- 50% by volume is used, preferably 10-30% by volume.

Figures 5 and 6 show that the present invention works effectively without using the second oligonucleotide. This is an example to explain.

The assay begins by denaturing the analyte target molecule to produce a single-stranded molecule.

Denaturation of the target molecule or substrate is common:! :I: Perform by boiling. Furthermore, a substrate, the first Nucleotide, second oligonucleotide, j! ! Mix the equilibrated liquid and distilled water with each other in the fr ratio , boil for 5-10 minutes. The mixture is cooled to room temperature. After cooling, remove the enzyme to start the reaction.

Class I I 1 lil restriction enzyme with partial hairpin loop oligonucleotide and J ( The method used is different from the method described above.

Basically, this reaction requires that a class II restriction enzyme be labeled first. Figure 7 A description of the use of class If restriction enzymes using partial oligonucleotides. It is a diagram.

The temperature during the assay reaction depends on the restriction enzyme used and the length of the oligonucleotide probe. , and the presence of G+C in the oligonucleotide present at the time of assay. Suggs etc. are oligonucleotides 1) Based on the dissociation temperature of the NA complex, an appropriate hybrid An empirical formula to determine the sodo formation temperature was developed.

(SugBs at al Developmental Biology Jsing1’urif’ird (iencs, “”(D, D, Ilr own, ad, + ) + L 683° 8 academic I’rcss, New York, 1981° The derived formula is as follows: 1', = 2° (to + number of T residues) + 4° (number of G + C residues i). mentioned above As explained above, the restriction enzymes that are active at 37'C are determined to be multiplied.

The reaction time depends on the desired sequence concentration, the severity of the reaction conditions, and the complementary first oligo. The length of the nucleotide prosequence varies depending on the restriction enzyme used. intended target It takes some time to re-arrange the sequence, but 2 hours is particularly preferable.

After the reaction, add the reaction solution to the polyacrylamide gel loading buffer. llI'σ month to stop the reaction. Electrophores the sample, and after the gel (auto) traradiograph). If necessary, measure probe production using a scanning densitometer. It will be quantified.

In order to illustrate the present invention and its features, the following specific practical examples are included, but are not intended to explain the invention. It is understood that this is for the sake of clarity and is not intended to limit the invention in any way. Good morning.

81゛ of the first gonucleotide Two types of oligonucleotides can be used in restriction amplification methods. '°8 CA TG GCT GAT CCTGCA GGTΔCCAAT Contains the sequence of GIo The first oligonucleotide (28mar [ffi body]) and the sequence “GGA TC A. The second oligonucleotide (18mer) containing “GCCATGGT” is an amide It was prepared by phosphorylation chemistry and purified by anion exchange II PLC. Amidrine The oligonucleotide synthesized by the acid method contained a free 5°01 (group).

Partial Yabin oligonucleotides can be synthesized in a manner similar to that described above.

,]Identification of the first gonucleotide Since the first oligonucleotide contains a 5°-O1+ group, the polynucleotide Use a kinase to convert [γ-tp] to 8TP! II] Due to Boss Fight transfer Labeled. The labeling reaction was carried out by dissolving each oligonucleotide in distilled water. .

The concentration of oligonucleotide dissolved in water was 0.5xlO-5 molar. reaction is 1 μl of the first oligonucleotide (0.5 x 10-” molar concentration), 0.5 M 1-Lis-hydrochloride (P117.5), 1mM spermidine, 50mM 0mM but Thiothreitol MEDTΔ and 09 mouths + MgCl, 30.5 μl distilled water, 1 2.5 μl of 12p-8 l'l' (0,125 milliquites, i.e. 3000 cuyu) 1 µm of this product (1 μm) and 1 μm of this product (10 units) 5μm Kinner 7゛Light including? ai　i [M was used. Mix the above reaction raw materials The cells were incubated at 37°C for 1 hour. The reaction was carried out by heating the reaction mixture for 80°CIO minutes. It was stopped.

The partial Yabin oligonucleotide is ti Can be converted into n.

By this method, the labeled first oligonucleotide and the partial oligonucleotide Creotide is stable for at least one week when stored at -20'C.

comparison 11r'V16-DNA virus was isolated from the uterine lesion by known methods and labeled as cloned into pBR322 using standard techniques.

Colonized l)N8 was isolated by a method modified from the following author's report. (If , C, [lirnboim and J, Daly, Nucleic 8ci ds to Rcs, 7.1513 (1979).

Cells were incubated at 37°C in 5 ml of Lll broth containing 100 μg/ml ampicillin. Propagated with vigorous shaking. Isolate 1.5ml of culture medium into 1.5+n1ii hearts. The mixture was transferred to a tube and centrifuged at 10,000×g for 1 minute. A residual beret is formed. Carefully remove the supernatant and allow the cells to dry once again in 50mM glucose (triple). lJTAIOmM, 1 to 25mM of Lis-HCl in 100 μl water-cooled solution The mixture was suspended by shaking with p118. The resuspended cells were incubated for 5 minutes at room temperature.

Add 0.2N NaOH and 1% dodecyl sulfate (SDS) to the cells. Then, 20 μm of the freshly prepared solution was added and mixed by IW. cells further Incubate for 5 minutes at o'c. After culturing, water-cooled potassium acetate was added to p114-8 to 150 μm ( Add 11.5 ml of glacial acetic acid and 28.5 ml of water to 5M potassium acetate G On+1. (prepared in addition) and the mixture was inverted for 10 minutes and incubated at 0°C for 5 minutes.

The 8 mixture was centrifuged at 10,000 x g for 5 minutes, and the supernatant was transferred to a separate test tube. . The supernatant was further centrifuged at 10.0OOX g for 5 minutes, and the supernatant was then freshly centrifuged. Transferred to a new test tube. Add 1 (NascA) to the supernatant to give a final concentration of 20 μg/m It was set as l. The reaction solution was incubated at 37°C for 20 minutes.

After incubation, an equal volume of phenol-chloroform (11, 50 mM Tris, pH 8) was added. - Add hydrochloride, 100mM NaCl and 1mM [saturated with EDTA] and mix. The mixture was shaken for 30 seconds and centrifuged at IQ, 000 x g for 30 seconds. aqueous phase was transferred to a new test tube.

25 volumes of primary ethanol were added to the aqueous phase and mixed. Incubate the mixture at -70°C for 5 minutes. fed. Centrifuge the mixture for 5 minutes at 101,000x and remove the supernatant. Ta. The remaining pellet was washed by adding 1 ml of pre-chilled ethanol and briefly Mixed. This mixture was centrifuged for 1 minute. Remove supernatant and vacuum pellet Dry.

The DNA obtained by this method was dissolved in 20 μl of deionized water.

21 μJ of oligonucleotide 1;・8 method This restriction amplification sample method has 11 Mix Pv-16 substrate, first labeled probe, Pst I buffer, and deionized water It started with that. In this R to MP test, two control samples (controls) are tested. It reacted simultaneously with the body. In this example, two different temperatures, 32°C and The tests were carried out at 37°C.

First, a stock solution of various reaction components was prepared. Dilute oligonucleotide 0.5x The second oligonucleotide was similarly diluted to a concentration of 0.5 A x 10-s stock solution was prepared. lIr’V-16 stock solution was diluted with distilled water. So, looμg/mlt? I made it into a stock liquid. Pst I restriction enzyme stock solution is 50 units / μl.

Aliquots of melons were taken from these stocks for each assay.

The total reaction time was 50 μJ. The sample for each reaction was 1 μm (100 ng). Liver V-16, 2 μl labeled first oligonucleotide, 2 μl unlabeled first oligo nucleotide and 4 μl of second oligonucleotide, 100 mM NaCl, 10mM Tris-HCl, pIf 7.7. l0mM MgC12,1m M DT, 10 (containing 5 μm PstI buffer containing 1 μg/ml ll5A) I had it. Pst was used at 5 μm. Add varying amounts of distilled water to adjust the final volume of each test tube. The product was set to 50 μm.

The assay was performed in 5 separate tubes. The first tube (#l) is labeled with 32] 2 μm of oligonucleotide, 2 μm of non-radioactive unlabeled first oligonucleotide, Second oligonucleotide 71μ1. Pst I buffer 5 μl and distilled water 3 2 μl was included. No 1IPV16 was added to the first tube.

The second tube (#2) contains IIPV-16 at tμ+ (100μg)” [) for 1 vote. 2 μl of labeled first oligonucleotide, non-radioactive unlabeled first oligonucleotide 2 μl, 4 μl of second oligonucleotide, 5 μl of Pstl buffer and It contained 31 μm of distilled water.

The third tube (#3) contains 1 μl (looμg) of IIPV-16 in co2P. 2μl of labeled first oligonucleotide, 2μl of non-radioactive first oligonucleotide It contained 5 μm of Pst1 buffer, and 35 μl of distilled water.

The 4th (7) W (#4) is 1 μl (100 pg) of HPV-16, 3! Marked with P 2 μl of labeled first oligonucleotide, non-radioactive unlabeled first oligonucleotide 2 μm, 5 μl of Pst 1* buffer, and 35 μm of distilled water.

The fifth (#5) (7) tube contains 1 pi (100 μg) of IIPV-1621) 2 μl of the first oligonucleotide labeled with non-radioactive, non-labeled first oligonucleotide 2 μl of chloride, 4 μl of second oligonucleotide, 5 μl of PsL I buffer, and 31 μl of distilled water.

Before adding Pst 1, boil the 5 test tubes above for 5-10 minutes and leave them at room temperature for about 10 minutes. It was cooled to

After cooling the sample, place the sample in a constant temperature bath and was cultured at 37'C. Assay tube 2.3 was incubated at 320C. I started the test For this purpose, 5 μl of Pst I enzyme was added to each tube. The assay was allowed to react for 2 hours.

After incubation, each tube was filled with 15 mλ1 Tris-80% formamide, pH 7.6. Hydrochloride, 1mM EDTA, 0.1% v/v fluoromophenol, and IO μm of a solution containing 0.1% W/V Kijirenzi 1 No # FF was added. . The 6 tubes were then heated to 65°C for 5 minutes and quickly cooled.

The polyacrylamide gel contains 30% Acrylamide stock solution (acrylamide). 19 [+ acrylamide] Add deionized water to N, N-methylene hydroxide, A diluted stock solution was prepared and formed with a body volume of 67 ml. Rich T [The buffer solution of llE consists of 108 g of l.lis ll54A, 55 g of boric acid, 9. It was prepared by diluting 3 g of disodium ITΔ, 2+10 to 1 liter. 'Rich It's 131E! X! The buffer solution should preferably be adjusted to a pl+ of 8.3. .

Purity of 48g↑! j　XJi's urea (8 questions) containing 30% acrylamide Added to the stock solution. Add 10 ml of THE buffer solution and dissolve urea in this solution. Ta. N, N, N″% l-tetramethylene ethylenediamine (TEM [ED) 50 μl (per 100 m of 7 claramide solution) 10% ammonium persulfate 1 ml (per 100 ml) was added and mixed well.

Pour the cell between the two force glass plates of the electrophoresis device, and quickly insert the comb. did.

20 μl of each sample was added to the polymerized gel and 0.089M pH 8.3 tras- Running buffer containing borate and 0.025M disodium EDTA added. The specimen was electrophoresed for 4 hours at 80 bottles.

The cable is then wrapped in a plastic folder and wrapped in a It was placed in close contact with the TAR-film and exposed for 10 minutes.

FIG. 3 is an explanatory diagram of the auto-tracing operator 7 of the present invention.

28-mer oligonucleotide and 18-mer oligonucleotide Substrate production can be easily confirmed by the presence of a oligonucleotide band. Leh The presence of doublet spots in points 2 and 5 indicates that two different substrates are present. It is shown that it is present in the reaction medium at a certain temperature. Recirculate substrate from lanes 3 and 4 There is no second oligonucleotide used to label the labeled first oligonucleotide. It shows that only Chido exists. Lane 1 is the first and second oligonucleotides were both present in the assay mixture and the substrate (e.g. HPV16) was absent from the reaction. It shows the pattern obtained when

Was there one type of oligonucleotide? , ``'''' restriction amplification specimen method is IIPV- 16 substrate, first oligonucleotide labeled probe, Pstl buffer, deionized The R In the AMI) assay, the control reaction was carried out at 5% concentration.

First, a stock solution of various reaction components was prepared. Dilute oligonucleotide 0.5x The I(PV-16 stock solution was diluted with distilled water to make a 10" molar solution. 00 μg/ml stock solution.

The 1'st 1 restriction enzyme stock solution contained 50 units/μl.

Aliquots were taken from these stocks for each assay.

1 (100 ng) of IIPV-16, 2 μl of labeled first oligonucleotide, 2 μl of unlabeled first oligonucleotide, NaCl 1100 mM.

1) Tris-hydrochloride salt of H7,7 10mM, lomM MgCl2, lm M I) T, 5 μl of Pstl containing 100 μg/ml of BS8 ! Contains a buffer solution. 5 μm Pst I was used. Different amounts of glue”9+9n After adding 100 μl of distilled water, add different amounts of distilled water until the final volume of each tube is 50 μl. It was to so.

Six assays were performed in separate tubes.

The first tube (#1) contains 2 μl of P-labeled first oligonucleotide, non-radioactive. 2 μm of unlabeled first oligonucleotide, 5 μm of r’st I buffer, 1 μl HI’V113. 2.5 μL of phosphorus (5% by volume) '9'e9' ) and 32.51 tl of distilled water.

Tube #1 was used as a control.

The second tube (#2) contained lμl (100μg) of HPV-16 labeled with 32P. 2 μm of first oligonucleotide, non-radioactive non-labeled first oligonucleotide 21 Add 5 μl of t1, P, stI buffer, 5 μl and 30 μl of distilled water.

The third tube (#3) contains 1 μl (100 μg) of IIPV-16, labeled with 32P. 2 μl of the first oligonucleotide, non-radioactive, non-labeled first oligonucleotide 2 μl, 5 μl of PstI buffer, Riyarin (20 volume% glue, 9t9) 10 μm and 25 Itl of distilled water.

Part 'l)'f &(#4) contains 1 μl (loOpg) of IIPV-16, 2P-labeled first oligonucleotide 2 μm, non-radioactive unlabeled first oligonucleotide 2 μm of leotide, 5 μl of PstI buffer, 30% by volume of 9'9t 9) contained 15 μm of water, and 20 μm of distilled water.

The fifth tube (#5) contained IIPV-161 μl (loOpg), labeled with 32P. 2 μm of first oligonucleotide, non-radioactive, non-labeled first oligonucleotide 2 μl, PstI buffer 5 μm, 5 μm of resin (40 volume% glue) 2 0 μl, and 15 μl of distilled water.

The sixth tube (#6) contained 1 μm (100 μg) of IIPV-16, labeled with 32P. 2 μl of the first oligonucleotide, non-radioactive, non-labeled first oligonucleotide 2 μm, Pstl buffer 5μm, Riyarin (50 volume% glue “9t9”) 25 μl, and 10 μl of distilled water.

Before adding r’st I, boil the 6 wooden test tubes mentioned above for 5-10 minutes and leave them in the room for about 10 minutes. Cooled to warm temperature.

After cooling the sample, the sample was placed in a constant temperature bath and the sample was incubated at 3700 °C. inspection To start the assay, 5 μl of 1'st I enzyme was added to each tube. The test is 2 Allowed time to react.

After incubation, each tube was filled with 80% formamide, 15mM Tris-salt, pH 7.6. acid salt, 1mM EDTA, 0.1% v/v fluoromophenol, or Add 10 μm of a solution containing 0.1% W and xylene Schifnow FF to start the reaction. It stopped. The 6 tubes were then heated to 65'C for 5 minutes and quickly cooled.

, polyacrylamide gel is prepared using a 30% acrylamide stock solution (acrylamide (19g of acrylamide, Add deionized water to Ig's N,N-methylene base solution 9# Amibi and adjust the total volume. A diluted stock solution with a volume of 67 ml was prepared and formed. Rich TOH The buffer solution was 5.108g Tris-based, 55g boric acid, 9.3g 2N4 8 g of extra pure urea (8M) is added to a stock solution containing 30% acrylamide. I got it. 10 ml of a buffer solution of T[lE was added and the urea was dissolved in this solution. N, N , N', N'-tetramethylene ethylenediamine (TEMED) 50 pl ( Accumamide solution (per room), 10% ammonium persulfate (per 100ml) 1 ml) was added and mixed well.

Pour the cell between the two force glass plates of the electrophoresis device, and quickly insert the comb. did.

Add 20 μl of each sample to the polymerized gel and add 0.089 M of 1) 118.3 Running supplement containing sulforate and 0.025M disodium EDT A buffer solution was added. The specimen was electrophoresed for 4 hours at 80 bottles.

The cable is then wrapped in a plastic folder and placed in a cloth ゛rΔ1? - Close contact with the film and expose for 10 minutes.

Figure 6 shows the hairpin loop of the autoratio section of the present invention using the method of the present invention. oligonucleotide labeled probe, Pst1 buffer , 2 μl of deionized nucleotides, non-radioactive, unlabeled partial hair oligonucleotides. 2 μm, 5 μl of Pst I buffer, iμl of HPV16, and 35% of distilled water. Contains LL1.

The fourth tube (#4) contains 2 μm of partial hairpin oligonucleotide labeled with 0p; Non-radioactive non-labeled partial hairpin oligonucleotide 21tl, PsL I buffer 5 μL contains 5 μl of HPV16 and 31 LL liters of distilled water.

Add 1'sL I to the above 4-wood test tube, boil for 5-10 minutes, and leave in the room for about 10 minutes. Cool to warm.

After cooling the sample, place the sample in a 37'C thermostat. To start the test At the same time, add 5 μM of Pst I enzyme to each tube. The assay is allowed to react for 2 hours. .

After incubation, add 108ml of 80% formamide, pH 7.6, to each test tube to react. stop. The tube is then heated to 65°C for 5 minutes and quickly cooled.

Polyacrylamide gel is made from 30% acrylamide stock solution (acrylamide Bisacrylamide = 19:1), 19g of acrylamide, Add deionized water to tg of N,N-methylene acetate acrylamide and reduce the total volume. Add 1 ml of ammonium acid (per 100 ml) so that the amount is 67 ml, The 6 cells, which are mixed well, are poured between the two force glass plates of the electrophoresis device, and the 6 cells are mixed well. Insert immediately.

Therefore, the scope of the present invention extends to the following Figure 1, including equivalents thereof. 2nd Ori°゛8ri'Ochido゛　5' GGA TCA GCCATG GT 3 ゜＊・Detectable marker HPV　16　XXX　TG[i　TACC11A　CTA　G[lA　C[i T, CCA Ding GG XXXX ten XXX TGfli TACαiA + CT^G [iA CGT CCA T GG XXX+ Oligo hybridization to target end + CIICGA C revised CCA TGG reed Xt detectable marker + HPV16 XXXTGGTAC’CGACTAGGACGTCCA Ding G [i TT8 CXXX+ XXX, TGG TACCCA CTA C+ A CGT CCA TGG TTA CXXX◆Detectable marker Control %Glyceri＞　10　20　30　40　50　(5%) Mama Mama Mama Mama Ma Figure 6 513' t-tpv 16 xxx TGG TACCGA CTA CGA CGT CCA TGG TTA CXXXXXX TGG Ding^CCGA CTA% A CGT CCA T(i(i τ■＾cx’xxXXX ACCATG [IC Ding GAT CCT GCA GGT ACCAAT G XXXXXXX TGG TACCGA CTA G6 ＾ Cf1iT CCA T[iG TTA　CXXXACTCT　QC^　÷　GGT　ACCAAT　[1+Silkworm( Tgang G4 A CGT CCA TG (i TTA cm8 to TCTGCAGGT8 to CA ^TGI (TG AGA CGT CCA TGG ■A CXXX1 detectable marker Figure 7 Copy and translation of supplementary IE document) Submission (Article 184-8 of the Patent Act) May 12, 1994

Claims (1)

[Claims] 1. (a) contains a nucleic acid sequence and a cleavable bond in the reaction mixture and includes a substantially nuclear An oligonucleotide complementary to the acid target sequence is prepared, and the oligonucleotide is complementary to the acid target sequence. A detectable marker is attached to the code; (b) When the target sequence and the oligonucleotide hybridize, cleavage is possible. adding to the reaction mixture a cleaving enzyme capable of cleaving a possible binding site; (c) hybridize the reaction mixture; and then (d) carry a detectable marker. Detects cleaved and detectable markers in the presence of two uncleaved oligonucleotides The compound sample contains a cleavable bond that can be cleaved by a cleavage enzyme. A method of detecting the presence of a nucleic acid sequence containing 2. 2. The method of claim 1, wherein said oligonucleotide comprises a single-stranded DNA sequence. 3. The cleavage enzyme is selected from the group consisting of [sequence is present] and Xba I. 2. The method according to claim 1. 4. 11. The method of claim 10, wherein said hybridization is carried out at 37<0>C. 5. 5. The method of claim 4, wherein said hybridization is carried out for 1 to 4 hours. 6. The reaction mixture further comprises a buffer system, the buffer system comprising HgCL2, NaCl, Lis(hydroxymethyl)aminomecne-hydrochloric acid (trisul), dithiothlate 2. The method of claim 1, comprising: (DTT) and bovine standard albumin (BSA). . 7. Buffer system is 10mM NACl, Pll 7.7 Tris(hydroxymethyl) Aminomethane-hydrochloric acid (Tris-HCL) 10mM, MgCl210mM, DTT 7. The method according to claim 6, comprising a buffer system containing 1 mM HSA, 100 μg/ml. 8. The detectable marker in the oligonucleotide is A labeling compound selected from the group consisting of photons, enzymes, biotin, and mixtures thereof. The method according to claim 1, wherein the ligand acts as a specific binding pair component for a substance. 9. 9. The oligonucleotide according to claim 8, wherein the oligonucleotide is a 10-40mer. Method 10. 5. The oligonucleotide is a partial hairpin oligonucleotide. 1. Method 11. 11. The method according to claim 10, wherein the cleaving enzyme is Fok I. 12. Additionally, 80% formamide, pH 7.6 tris(hydroxymethyl)amide Nomekune-HCl (Tris-HCl) 15mM, 1mM EDTA, 0.1% ww /v bromophenol blue and 0.1%/v xylene cyanol FF The step of adding a stop solution consisting of 2. The method of claim 1, comprising: 13. (a) A target nucleic acid sequence containing a cleavable bond is attached with a detectable marker. a moiety that contains a cleavable bond and is substantially complementary to the nucleic acid sequence of the target molecule. forming a reaction mixture by denaturing in the presence of an apin oligonucleotide; (b) adding a cleaving enzyme to the denaturation mixture; (c) hybridizing the reaction mixture; , releasing a detectable marker from the oligonucleotide sequence with a cleaving enzyme; ( In the presence of an uncleaved partial hairpin oligonucleotide with a detectable marker of The sample consists of detecting a detectable marker that has been cleaved using a cleavage enzyme. A method of detecting the presence of a nucleic acid sequence comprising a cleavable bond that can be cleaved by. 14. 14. The target sequence consists of a single-stranded or double-stranded DNA sequence. the method of. 15. Part 1, wherein the partial hairpin oligonucleotide is complementary to the target sequence. 14. The method according to claim 13, comprising a double-stranded DNA sequence. 16. The cutting enzyme is BbvI, BbvII, FokI, HgaI, HphI, HboII, MnII, SfaNI, TaqII, TthIII, and Hin 14. The method according to claim 13, wherein the restriction enzyme is selected from the group consisting of GuI. 17. The reaction mixture is further mixed with MgCl2, NaCl, tris(hydroxymethylene). ) aminomecun-hydrochloric acid (Tris-HCl), dithiothreitol (1) TT) and a buffer system consisting of bovine standard albumin (BSA). Law. 18. (a) connecting a target molecule to a sequence complementary to the target with a cleavable bond; hybridize to a labeled oligonucleotide probe with a marker that can be forming a duplex between the lobe and the target; (h) cleaving the duplex at the cleavable linkage; (c) recycling the target molecule; (d) reconstituting a cleavable binding site on the oligonucleotide; (e) detecting the cleaved oligonucleotide probe; Method of detecting the presence of a nucleic acid sequence in a biological enclosure containing a cleavable bond . 19. The oligonucleotide probe is a nuclease that is substantially complementary to the target molecule. Claim 17, wherein the oligonucleotide is a partial hairpin oligonucleotide consisting of a nucleotide sequence. the method of. 20. The cutting step (b) is carried out with a restriction enzyme, and the restriction enzyme is Aat II, A cc I, Acc III, Aha II, AluiI, Eco52 I, Ec o RI, Eco RII, Rco RV, Fsp I, Hae III, Hh a I, Hind III, Hpa I, Kpn I, Ksp I, Nci I, Mst II. Nae I, Pst I. Consists of Puv I and Xba I 18. The method of claim 17, wherein the method is selected from the group. 21. The hybridization is determined by the efficiency of the cleavage enzyme and the amount of oligonucleotide. 18. The method of claim 17, wherein hybridization is carried out at increasing temperatures. 22. Said cutting step (b) is further carried out in a buffer system, said buffer system comprising MgCl2,N aCl, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl), Claims containing dithiothreitol (DTT) and bovine standard albumin (BSA) The method according to item 17. 23. Buffer system is Tris (hydroxymethyl) with NaCl 10mM and Pll 7.7 Aminomecne-hydrochloric acid (Tris-HCl) 10mM, HgCl2mM, DTT1m 22. The method of claim 21, comprising 100 μg/ml of M.RSA. 24. The method according to claim 17, wherein the oligonucleotide is less than 100mer. . 25. Further 80% formamide, pll7.6 tris(hydroxymethyl)a Minomecun-HCl (Tris-HCl) 15mM, 15M, 1mM EDTA, 0. 1% w/v bromophenol blue and 0.1% w/v xylene cyano A step of adding a stop solution consisting of FF to the hybridization reaction mixture. 18. The method of claim 17, comprising the steps of: 26. Prepare oligonucleotides with partially double-stranded recognition and cleavage sites, and The oligonucleotide may promote partial double-stranded recognition and cleavage at the cleavage site. Oligonucleotide cleavage consisting of adding a sufficient amount of restriction enzyme in the presence of a solvent Method. 27. The solvent may be glycerin, glycerol, dimethyl sulfoxide, dimethyl 26. The method of claim 25, wherein the compound is selected from the group consisting of: 28. 26. The method according to claim 25, wherein the restriction enzyme is a class II enzyme.