JP2004012343A - Method for making a plurality of parallel inspections and mixed liquid for making a plurality of parallel inspections - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method or the like for carrying out efficient treatment timewise, spatially under the strict same conditions by performing a plurality of kinds of inspections in parallel for a plurality of inspection parallel method and a mixed liquid for a plurality of inspection parallel. <P>SOLUTION: The method includes a machining process for machining a group of objects to be examined of a plurality of kinds of inspections; a generation process for generating a group of labeled detection bodies that has a known specific structure of the plurality of kinds of inspections that are or are not connected to the targets to be examined for each kind of inspection, and is labeled by connecting with a labeled substance so that mutual identification can be made among respective kinds of inspections; a reaction process for mixing the group of objects to be examined, a carrier, and the group of labeled detection bodies in the liquid for reacting; a liberation process for separating the carrier connecting with the group of labeled detection bodies via the targets to be examined and liberating the labeled substance from the separated carrier; and a detection process for detecting the presence or absence or degree of the retention of the labeled detection bodies due to the carrier for each kind of inspection based on the liberated labeled substance. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a multiple test parallel method and a multiple test parallel liquid mixture. The present invention is particularly applicable to fields requiring inspection, analysis, and analysis of biopolymers such as genes, immune systems, proteins, amino acids, and sugars, for example, the fields of engineering, food, agriculture, agriculture such as fishery processing, and pharmacy. It is related to all fields such as fields, medical fields such as sanitation, health, immunity, disease, and genetics, and fields of science such as chemistry or biology.
The present invention particularly relates to a multiple test parallel method and a multiple test parallel solution suitable for gene mutation analysis, polymorphism analysis, mapping, nucleotide sequence analysis, expression analysis and the like.
[0002]
[Prior art]
Conventionally, in order to analyze a gene probe (labeled specific DNA or RNA sequence), a target gene sequence is amplified using a gene amplification technique (for example, PCR), and a hybridization / ligation detection method is used. By allowing the sequence of the probe hybridized to the target sequence to be separated and detected (eg, the probe contains a combination of magnetic particles and acridinium S), the presence or absence of a particular sequence is determined. There was something to be decided (Tokuheihei 9-510878).
[0003]
The method is, in particular, a method for identifying a target polynucleic acid sequence,
(A) when the two probes are ligated, such that the probes are complementary to some or all of the predicted sequence of the target polynucleic acid, one of the probes being the one from the reaction mixture The other probe is attached to a moiety that allows it to be easily separated, and the other probe is attached to a label, and (b) mixing the probe with the target polynucleic acid so that the probes (C) adding a reagent for ligation, and (d) denaturing the reaction mixture so that the probe is separated from the target polynucleic acid. The probe is separated using the portion, and (f) the separated probe is analyzed and the label is bound to the probe. It is to determine whether it is.
[0004]
[Problems to be solved by the invention]
By the way, in the above gene probe analysis method, the analysis is always limited to the inspection of one type of base sequence. Therefore, in order to perform a plurality of analyses, it is necessary to perform the above method separately for each type. Therefore, for each type, it is necessary to prepare a large number of containers, reactants, and various types of reagents, it is necessary to wash the containers and the like for each test, and equipment such as a thermostat is required. There is a problem that it is necessary to sequentially perform processing for each type, a long processing time is required, and a large processing space and processing equipment are required.
[0005]
In addition, various types of work are required for each type of inspection, so that the processing is complicated, and there is also a problem that the user takes a great deal of time.
[0006]
Furthermore, since it is necessary to prepare different mixed liquids, reagents, and the like for each process so that cross-contamination does not occur between types, there is a problem that it takes a great deal of time to manage the processes. .
[0007]
Therefore, the present invention has been made to solve the above problems, and a first object is to perform not only the processing time but also the work time by performing a plurality of types of tests collectively and in parallel. An object of the present invention is to provide a multiple inspection parallel method and a multiple inspection parallel liquid mixture that can save space and perform processing efficiently.
[0008]
The second purpose is to collectively perform a plurality of types of inspections in parallel, so that even if the amount is minute for each inspection, they are collectively handled as a bulk amount, thereby making it easier to handle and easier to use. It is an object of the present invention to provide a good multiple test parallel method and a multiple test parallel mixture.
[0009]
The third object is to perform a plurality of types of tests collectively and in parallel, thereby saving goods and equipment such as reagents, environment such as temperature, etc., which are required in common in each test, and reducing test costs. It is an object of the present invention to provide a multiple test parallel method and a multiple test parallel mixture that can be reduced.
[0010]
A fourth object is to perform a plurality of types of tests collectively and in parallel, thereby making it possible to set exactly the same conditions for each type of test, and to compare the test results between the types under the same conditions. To provide a multiple inspection parallel method and a multiple inspection parallel liquid mixture capable of finding essential differences between types and obtaining a reliable and accurate inspection result. It is.
[0011]
The fifth object is to provide a large number of information, such as determination of the base sequence of genetic material, so that a large number of simple processes need to be repeated. It is to provide a parallel method and a mixed liquid for multiple tests parallel.
[0012]
[Means for Solving the Problems]
In order to solve the above technical problems, a first invention is a processing for fixing a group of inspection objects of a plurality of inspection types to a carrier at a predetermined portion of each inspection object, or processing to be able to be fixed. And generating a group of labeled detectors having a known predetermined structure of a plurality of test types that are combined or not combined with the test object for each test type, and that are labeled so as to be mutually identifiable among the test types. And a reaction step of mixing at least the test object group, the carrier, and the labeled detection body group in a liquid to perform a reaction, and binding to the carrier via the test object and the detection body. Separating the carrier, and releasing the labeling substance from the separated carrier, based on the released labeling substance, for each test type, the labeling detection by the carrier through the test object by the carrier Body retention Others by having a detection step for detecting a degree that a plurality testing parallel method concurrently performs multiple inspection types of test.
[0013]
Here, the “inspection type” refers to a plurality of inspection types to be performed in parallel. The inspection type can be distinguished by, for example, the type of the inspection target and the type of the inspection site in the same inspection target.
[0014]
Further, the “labeled detector” is a labeled detector, and the “detector” is a solid used for detection, and a large number is contained in the mixture for each test type. “Labeling” is performed, for example, by binding to a labeling substance. The labeling substance may be performed not only by an optical substance but also by a substance having various other physical quantities and stoichiometric quantities which can be quantified instantaneously.
[0015]
The term "degree of retention" means the amount of retention, that is, the degree of labeling at the time of detection, and when labeling is performed optically, for example, the intensity of light. Examples of the “fixing the test object” include a case where the test object is bonded, the test object is attached, or the test object is adsorbed, and the test object is coated on the surface of the carrier, or other This includes cases where the substance to be inspected is mediated by a substance. The “test target” includes biomolecules such as genetic material, immune system, proteins, amino acids, and sugars, and small biomolecules.
[0016]
The “carrier” is capable of holding one or more molecules of a labeled detector, preferably capable of holding a large number of labeled detectors, and capable of being separated and collected from a solution by magnetic force, centrifugation, or the like. There is a need. The size of the carrier is not particularly limited as long as it can be accommodated in a container used for inspection, and is preferably a spherical particle having a particle size of 0.1 μm or more and 100 μm or less. As a result, it is possible to obtain an appropriate separation / collection performance and a sufficient amount of the labeled detector. In the present invention, when determining the structure of the substance held by the carrier, it is not necessary to have any correspondence between the binding position of the substance on the carrier and the structure of the substance, or It is not necessary to have as detailed a correspondence as necessary to determine the structure of the substance.
[0017]
"Release of labeling substance" includes not only the cleavage of genetic material such as DNA, peptides, carbohydrates, lipids and other biological macromolecules or biological low molecules, but also the modification of pH or salt concentration, And denaturation by heating or the like.
[0018]
In the second invention, in the generation step, for each test type, a large number of detectors and a labeling substance of each test type in which a predetermined type is included in a predetermined quantitative ratio are mixed in a liquid and bound. The method is a multiple-inspection parallel method that includes a step, wherein the types or the quantitative ratios thereof are different from each other for each inspection type so as to be distinguishable from each other. Here, the “labeling substance” includes, for example, a luminescent substance such as a fluorescent substance, a substance that emits an electromagnetic wave, a substance that emits a magnetic field, and a substance having a charge.
[0019]
In a third aspect of the present invention, in the generation step, the entirety of the labeling substance of each test type is distributed to substantially all of the labeled detectors of each test type, and one labeled detector is one or more types. Is a multiple test parallel method including a step of binding to a labeling substance.
[0020]
A fourth invention is a multiple inspection parallel method in which the separation of the carrier in the releasing step is performed by applying a magnetic force to the carrier. Here, the carrier is a magnetic substance, preferably a superparamagnetic substance in a particle form, and more preferably a particle diameter of 0.1 μm to 100 μm.
[0021]
According to a fifth aspect of the present invention, in the processing step, a plurality of test object groups are processed so as to be fixed to a carrier at a predetermined portion of each test object. This is a parallel multiple test method in which a group of substances and a group of labeled detectors are mixed and reacted in a liquid, and then a carrier is mixed and reacted in the mixed liquid.
[0022]
Here, first, after the test object group and the labeled detection object group were mixed and reacted in the liquid, the carrier was mixed in the liquid and reacted, and the test object group was used. Further increase the possibility of reactions that occur at an overwhelmingly high probability among the labeled detector groups, and further increase the possibility of reactions including reactions between other test object groups or labeled detection pairs. To lower it. On the other hand, when a reaction between a carrier and a test object group is performed first, generally, in a reaction involving a biological macromolecule such as a molecular biological reaction or a biochemical reaction, the test object fixed to the carrier is used. This is because the reaction probability of each other is low but not 0, and the possibility of the combination cannot be ignored. For example, in genetic material such as DNA, the probability of a ligation reaction between certain base sequences is lower than the reaction between base sequences having a complementary relationship but is not zero. In particular, it is possible to prevent a ligation reaction between molecules immobilized on a carrier, which is likely to occur when the protruding end becomes palindromic.
[0023]
According to a sixth aspect of the present invention, in the reaction step, each of the labeled detectors of the group of labeled detectors is prepared without reacting and binding to each other, and the number thereof exceeds the number of the test objects excessively. In this manner, the method is a multiple-test parallel method in which a group of labeled detectors is mixed.
[0024]
In the generating step, the number of the labeled detectors in the group of labeled detectors is set to be larger than the number of the test objects and is used for the reaction, whereby the probability of the reaction is low but not 0. The presence of binding between the labeled detectors caused can be statistically ignored by the presence of the binding with the highest probability of reaction. For example, the presence of an excess of all labeled detectors containing a sequence complementary to the protruding end (single-stranded site) enables a competitive ligation reaction, and enables two or more types of labeled detection. Only the ligation of the labeled detector with the highest sequence complementarity in the body occurs with high probability. As a result, highly reliable measurement is possible even under conditions where the sequence of the protruding end is, for example, only a single base difference and a non-specific ligation reaction is likely to occur due to non-complementarity of the protruding end site.
[0025]
In order to make “each labeled detector in the group of labeled detectors not react with each other and bind”, for example, when DNA is used as the detector, the 5 ′ end existing at the linking site may be used. It is possible to remove the phosphate group of.
[0026]
According to a seventh aspect of the present invention, in the processing step, when inspecting the conformity of the structure of a predetermined test site of a plurality of test types of genetic material, the carrier is provided with one of a pair of specific binding substances, As a single-stranded test site is included for each test type, the test site is cut so as to have a protruding end, and, at the other end of each test object, of the pair of specific binding substances Processed to provide the other, the labeled detection body of a plurality of test types in the generation step, the test object of each test type, depending on its structure, binds to the test site of the genetic material or This is a multiple test parallel method in which genetic material has a single-stranded base sequence that does not bind.
[0027]
In an eighth aspect of the present invention, in the processing step, a double-stranded DNA having a plurality of test types of test sites, and a plurality of test type primer groups each having the specific binding substance bound to one end of each test type test site. And a pair of primers having a cleavage site in which a test site downstream of the 3 ′ end of the primer has a cleavage site as a protruding end, and a recognition site for a restriction enzyme different from the recognition site and the cleavage site. And amplifying the double-stranded DNA fragment by PCR and treating the amplified DNA fragment with the restriction enzyme to produce a DNA fragment having a protruding end of the test site at the other end. The step of enzymatic reaction, the group of test objects of the plurality of test types is fixed to a carrier or processed so as to be fixable. A label having a protruding end serving as a base sequence complementary to each test site of the test object group of the test type, and the other end labeled with a labeling substance so as to be mutually identifiable for each test type; A double-stranded DNA fragment as a labeled detector, a processed test object group, a carrier on which the test target is fixed or the test target can be fixed, and the labeled detector This is a multiple test parallel method for detecting the presence or absence or the degree of the retention of the labeled detector via the test object by the carrier by mixing the groups in a liquid in a predetermined order and performing a ligation reaction.
[0028]
Here, the “restriction enzyme having a different recognition site and a different cleavage site” is preferably Type III or Type IIS restriction enzyme. The “predetermined order” is, for example, the order described in the fifth invention.
[0029]
In a ninth aspect of the present invention, in the processing step, the restriction enzyme comprises a base at a recognition site and a cleavage site of a double-stranded DNA having a plurality of test sites in the test object, for each test site; This is a multiple-test parallel method in which the restriction enzyme is not overlapped and is separated so as not to affect the PCR primer, and the recognition site of the restriction enzyme is provided on the upstream side of the 3 ′ end of the primer.
[0030]
According to a tenth aspect of the present invention, in the case where the correctness of the structure of a predetermined test site of a plurality of test types of proteins, the presence or absence of the structure, or the degree of the presence thereof is tested, the test object in the processing step includes a plurality of types. A predetermined site other than the test site of the protein is fixed to the carrier, or is processed so as to be fixable, and the labeled detector in the production step is specific to the test site. A multiple test parallel method in which a substance selected so as to bind or not bind is labeled with the labeling substance so as to be mutually identifiable for each of a plurality of test types.
[0031]
An eleventh invention is characterized in that, when performing a plurality of types of tests for genetic material in the releasing step, release of the labeling substance from the carrier bound to the group of labeled detectors cleaves DNA at an arbitrary position. This is a multiple test parallel method using possible enzymes.
As the enzyme, DNase I is preferred.
[0032]
According to a twelfth aspect, in the release step, when a plurality of types of tests are performed on the protein, the release of the labeling substance from the carrier bound to the labeled detector group can be performed by arbitrarily removing the polypeptide chain of the protein. This is a multiple test parallel method that uses an enzyme that can be cleaved at a position.
[0033]
A thirteenth invention is directed to a test object group of a plurality of test types fixed to a carrier at a predetermined portion of each test object or processed so as to be fixable, and combined with the test object for each test type. Having a known structure of a plurality of test types that do not bind to each other, a labeled detector group that is labeled so that they can be distinguished from each other, and a carrier on which the test object group is fixed or can be fixed. A mixed solution comprising: separating the carrier bound to the labeled detector group through the test object, releasing the labeling substance from the separated carrier, based on the released labeling substance, For each test type, by detecting the presence or absence or the degree of the retention of the labeled detection body by the carrier, the test for multiple test types is performed in parallel using the mixed solution in the multiple test parallel liquid mixture. is there.
[0034]
In a fourteenth aspect, the labeled detector of each test type is labeled by binding only to a labeling substance of each test type, and the entirety of the labeling substance for each test type has a predetermined type. , And the type or the ratio is a mixed liquid for multiple tests in parallel so as to be mutually identifiable for each test type.
[0035]
In a fifteenth invention, the entirety of the labeling substance for each test type is distributed to substantially all of the labeled detectors for each test type, and one labeled detector binds to one or more labeling substances. This is a mixed solution for multiple inspections.
[0036]
In a sixteenth aspect, the carrier is a mixed liquid for multiple inspections parallel which can be separated by magnetic force.
[0037]
A seventeenth invention is a multiple-test parallel liquid mixture in which the test object group and the labeled detector group are mixed and reacted in a liquid, and then the carrier is mixed.
[0038]
In an eighteenth aspect, each of the labeled detectors of the group of labeled detectors is prepared without reacting and binding to each other, and is mixed so that the number thereof exceeds the number of the test objects. It is a mixed solution for multiple inspections.
[0039]
According to a nineteenth aspect, when inspecting the conformity of the structure of a predetermined test site of a plurality of test types of genetic material, each of the test objects includes a single-stranded test site for each test type. It is a genetic material such as a cut DNA fragment, and the labeled detector of each test type has a single-stranded base sequence that binds or does not bind to the test site of the genetic material, depending on its structure. It is labeled genetic material.
[0040]
The twentieth invention is directed to a method for testing the correctness of the structure of a predetermined test site of a plurality of types of proteins, the presence or absence of the structure, or the degree of the presence thereof, wherein the test object is fixed to a carrier at the predetermined site. Or a protein processed so as to be immobilizable, wherein the labeled detector has, for each test type, a known predetermined structure of a plurality of test types that binds to or does not bind to the test object. , Is a mixed liquid for multiple tests in parallel, which is labeled so as to be mutually identifiable among the test types.
[0041]
According to a twenty-first aspect, in the labeling detector, a double-stranded DNA having a plurality of test sites is used in each of the test sites so that bases in a recognition site and a cleavage site do not overlap with each other, thereby affecting PCR primers. A primer group in which a recognition site of a restriction enzyme that is different from a recognition site and a cleavage site that are so far away from each other is provided on the upstream side of the 3 ′ end of the primer, and a labeled group of a plurality of test types that are paired with the primer group And amplifying the double-stranded DNA fragment by PCR and treating the amplified DNA fragment with the restriction enzyme, whereby one end is labeled and the other end has a protruding end of the test site at the other end. It is a fragment.
[0042]
Here, "the bases in the recognition site and the cleavage site in each test site do not overlap and are separated to the extent that they do not affect the PCR primer" preferably refer to a case where they are separated by 10 bases or more. . Ideally, they should be separated by about 20 to 30 bases. However, currently known Type IIS restriction enzymes and the like have a maximum of about 10 to 20 bases.
[0043]
A twenty-second invention is a multiple-test parallel mixture in which an enzyme capable of cleaving DNA at any position is mixed in order to release a labeling substance from a carrier bound to the labeled detector group. .
[0044]
A twenty-third aspect of the present invention is directed to a multi-test parallel test in which an enzyme capable of cleaving a polypeptide chain of a protein at an arbitrary position is mixed in order to release a labeling substance from a carrier bound to the labeled detector group. It is a mixture.
[0045]
BEST MODE FOR CARRYING OUT THE INVENTION
The multiple inspection parallel method and the multiple inspection parallel mixture according to the embodiment of the present invention will be described below with reference to the drawings. This embodiment should not be construed as limiting the present invention unless otherwise specified.
[0046]
FIG. 1 shows a multiple test parallel method and a multiple test parallel solution according to an embodiment of the present invention.
[0047]
As shown in FIG. 1 (a), in the present embodiment, the test sites 13, 14 in each of two specific double-stranded sample DNAs 11 and 12, which are genetic materials extracted from one patient, are used. Whether or not there is a mutation in the structure of each test object having a certain base or base sequence, that is, a polymorphism test is performed. As such sample DNAs 11 and 12, for example, exon 6 and exon 8 of human ATase (amidophosphoribosyltransferase; amidophosphoribosyltransferase) are used. It is considered that the mutation on the ATase gene causes excessive purine production which causes gout.
[0048]
Here, the “test type” is two types of tests at the test site 13 in the sample DNA 11 and the test site 14 in the sample DNA 12. In this example, two types of two bases are shown for simplicity of explanation, but the number of types and the number of bases are not limited.
[0049]
FIG. 1A shows primers 16 and 17 having one end bound to biotin 15 in addition to the sample DNAs 11 and 12. In addition, a type IIS restriction paired with the primer, in which the recognition site and the base at the cleavage site are separated by 10 or more bases so that the inspection sites 13 and 14 have cleavage sites provided upstream of the 3 ′ end. A primer 19 having an enzyme recognition sequence 18 is shown.
[0050]
Here, the test sites 13 and 14 are type A sequences whose base sequences are “AG” and “AC”, respectively. The case where the base sequences are "GG" and "AT" respectively is defined as a type B sequence. Bse RI is used as the Type IIS restriction enzyme. The recognition site of the restriction enzyme is “GAGGAG (N) (10/8)”, where “N” represents any of A, C, G or T.
[0051]
Next, in FIG. 1 (b), the sample DNAs 11, 12 and the primers 16, 17, 19 are mixed and PCR (polymerase chain reaction) is performed.
Then, as shown in FIG. 1 (b), two types of double-stranded DNA fragment groups 20 and 22 (type A) having biotin 15 at one end and a Type IIS restriction enzyme recognition site near the end are in parallel. Amplified. Note that the double-stranded DNA fragment groups 21 and 23 shown in FIG. 1B have a case where the test sites 13 and 14 of the type B base sequence are present.
[0052]
Next, as shown in FIG. 1 (c), the amplified DNA fragment 20 and the amplified DNA fragment 21 and the amplified DNA fragment 22 and the amplified DNA fragment 23 amplified by the PCR method are treated with the Type IIS restriction enzyme. A number of DNA fragments 24 and DNA fragments 28 each having biotin 15 at one end and “AG” as the protruding end 25 of the test site 13 and “GG” as the protruding end 29 at the other end. A large number of DNA fragments 26 and 30 each having “AC” as the terminal 27 and “AT” as the protruding terminal 31 are generated. The DNA fragment 24 and the DNA fragment 28 and the DNA fragment 26 and the DNA fragment 30 correspond to the two types of test objects.
[0053]
Next, as shown in FIG. ₁ , F ₂ , F ₃ , F ₄ , And labeled at the other end, with four types of labeled detectors 33, 35, 37, having protruding ends 32, 34, 36, 38 of base sequences complementary to the test sites 13, 14. 39 is generated.
[0054]
Here, the labeling substance F ₁ , F ₂ , F ₃ , F ₄ Are, for example, four types of fluorescent substances of different types. The fluorescent substance is selected from, for example, organic substances such as IRD700, IRD40, Cy5, Cy3, FITC (fluorescein isothiocyanate), rhodamine and isothiocyanate and inorganic substances such as europium complex.
[0055]
Here, for example, one end has a labeling substance F ₁ A labeled detector 33 having a protruding end 32 of a base sequence "TC" complementary to the base sequence "AG" of the test site 13 at the other end, and labeled at one end. F ₃ A labeled detector 35 having a protruding end 34 of a base sequence "TG" complementary to the base sequence "AC" of the test site 14 at the other end, and a labeling substance at one end. F ₂ A labeled detector 37 having a protruding end 36 of a base sequence “CC” complementary to the base sequence “GG” of the test site 13 at the other end, and labeled at one end. F ₄ And labeling by binding to the IRD selected as above, to generate a labeled detector 39 having a protruding end 38 of the base sequence "TA" complementary to the base sequence "AT" of the test site 14 at the other end.
[0056]
Next, as shown in FIG. 1 (e), first, the DNA fragment groups 24, 26, 28, 30 and the labeled detector groups 33, 35, 37, 39 are mixed and a ligation reaction is performed. At that time, the numbers of the labeled detection body groups 33, 35, 37, and 39 are mixed in the liquid so as to be excessively larger than the respective numbers of the DNA fragment groups 24, 26, 28, and 30. Then, depending on whether the test site 13 of the sample DNA 11 and the test site 14 of the sample DNA 12 are a base sequence of type A or a base sequence of type B, one of the DNA fragment groups 24, 26, 28, and 30, Any of the chemical detection object groups 33, 35, 37, and 39 will bind.
[0057]
Next, a large number of magnetic particles 40 are mixed in the mixture as the carrier. Here, since avidin which specifically binds to the biotin 15 is fixed to the magnetic particles 40 in advance, when these are mixed, the biotin of the DNA fragment group 24, 26, 28, 30 and the magnetic particles 40 are mixed. Avidin specifically binds, and the labeled detector groups 33, 35, 37, and 39 are captured by the magnetic particles 40 via the DNA fragment groups 24, 26, 28, and 30.
[0058]
These magnetic particles 40 are separated by a dispenser (not shown) and washed to remove impurities such as labeling substances not captured by the magnetic particles 40. The dispenser can apply a suction / discharge unit for suctioning / discharging a liquid, a pipette tip detachably attached to a nozzle of the suction / discharge unit, and releasably apply a magnetic field to a liquid passage of the pipette tip. Magnetic means. The magnetic particles 40 are adsorbed and separated by applying a magnetic field to the liquid passage of the dispenser.
[0059]
In FIG. 1 (f), the separated magnetic particles 40 are transferred to another container and resuspended by repeating suction and discharge of the liquid contained in the container.
[0060]
In FIG. 1 (g), each of the labeling substances F ₁ , F ₂ , F ₃ , F ₄ And the magnetic particles 40 are released. This release is performed, for example, by cleaving the DNA fragment with DNase I. The separated magnetic particles 40 are adsorbed and removed on the inner wall of the liquid passage by repeating the suction and discharge of the liquid while applying a magnetic field to the liquid passage of the dispenser. By performing an optical measurement on the liquid from which the magnetic particles 40 have been removed, it is possible to eliminate the optical effects due to the presence of the magnetic particles 40, and thus to obtain a clear and reliable type of the labeling substance and a quantitative ratio thereof. A measurement can be made.
[0061]
In the optical measurement, for example, two or more types of inspection sites can be simultaneously detected using a fluorescence measurement device. The detection by the fluorescence measuring device corresponds to the detection step.
[0062]
FIG. 2 shows an example of the measurement result measured in this way. As shown in FIG. ₁ (Eg red fluorescence), F ₂ (For example, yellow fluorescence), an experiment was performed on a large number of specimens. In each case, the polymorphic sequence group 1 (A / A) and group 2 (A / G) ), Group 3 (G / G) belonged to a certain area that does not overlap, and were identified reliably. Similarly, in another examination site 14, F ₃ , F ₄ Can be reliably identified.
[0063]
In the above description of the embodiment, it is only checked whether the structure of the inspection site 13 or the inspection site 14 (portions corresponding to AG and AC, respectively) is type A or type B. If the structure of each of the test sites 13 and the test sites 14 is to be specified, a combination of base sequences corresponding to the structures of all the test sites expected as the above-mentioned structure is used as the labeling detector group. A group of labeled detectors containing at or near the 3 'end is formed, and those which are labeled so as to be mutually identifiable are all labeled differently for each test site (13, 14). It can be performed by using.
[0064]
Further, as the two-base overhang used in the above description, an overhang of three or more bases, such as a four-base overhang with a Type IIS restriction enzyme such as Fok I, can be used. For example, in the case of a 4-base protruding end, the number of cases where recognition is possible as a different sequence is 256, and the number of mutation sites or mutations that can be detected simultaneously can be increased.
[0065]
The above embodiment has been specifically described for better understanding of the present invention, and does not limit another embodiment. Therefore, it can be changed without changing the gist of the invention. For example, in the above description, for convenience of explanation, two types of tests have been described in which tests are performed in parallel using two types of labeled detectors and labeled with two types of labeling substances. However, the present invention is not limited to this case, and the test can be performed using three or more types of tests and three or more types of labeling substances.
[0066]
In the above example, the case where the labeling substance is a luminescent substance has been described. However, the labeling substance is not limited to this example, and may be a substance having various instantaneously quantifiable physical quantities such as a magnetic field and a nuclear spin state. . Further, even in the case of a luminescent substance, not only the luminescence wavelength and the luminescence intensity but also the luminescence polarization degree, the luminescence phase, the luminescence life and the like may be detected.
[0067]
The above tests have been performed for DNA polymorphisms, microbial species tests, and protein mutation tests, but are not limited to these examples and can be used for tests on sugars and amino acids. Needless to say.
[0068]
In addition to the antibodies, substances that specifically bind to the test substance, such as lectins, other proteins, and low molecular substances, can be used. In this case, it is possible to test various substances capable of specific binding, such as sugars, lipids, and other low and high molecular weight substances.
[0069]
In the above example, the mutation site was described only for a mutation of one or two bases. However, the present invention is not limited to this example. For example, a mutation having a base sequence of three or more bases may be deleted. It goes without saying that the present invention can be applied to the case where there is a loss or insertion. Furthermore, the method of analyzing a mutation site is not necessarily limited to the case where a plurality of types of tests are performed in parallel, and can be used when only one type of test is performed.
[0070]
【The invention's effect】
According to the first or thirteenth aspect, in addition to the above-described effects, for example, a structure such as a base sequence of DNA can be determined with high reliability and high accuracy.
[0071]
Further, by detecting and not detecting the state of labeling by the labeled detector held via the test object for each carrier, the type of test to be identified by the label is specified, and the test is performed. Various types of information can be easily obtained for the types.
[0072]
In addition, by multiplexing a plurality of types of tests, the tests can be performed in parallel. Therefore, the processing time can be shortened and the efficiency can be improved, the work area required for the processing can be omitted, and the apparatus to be used can be compact.
[0073]
In addition, even if the amount is very small for each inspection, by handling the bulk amount collectively, it is easier to handle and easier to use. Further, it is possible to save articles such as reagents commonly required in each test, equipment such as environment such as temperature, and manpower, thereby lowering test costs.
[0074]
Furthermore, it is possible to set exactly the same conditions for each type of inspection, and it is possible to compare the inspection results under the same conditions between each type. As a result, it is possible to discover essential differences between the types, and obtain reliable and accurate inspection results. Further, since it is necessary to obtain a large amount of information as in the determination of the base sequence of genetic material, the method is suitable for efficiently performing inspections and processes in which many simple processes need to be repeated.
[0075]
Further, according to the present invention, instead of labeling the test object, a labeling of a detection object having a known structure is used. Labeling is easier than in the case of labeling a test object whose structure is unknown, and a more reliable test can be performed because the structure of the test object is not changed.
[0076]
In addition, by releasing the labeling substance from the carrier, it is possible to eliminate the adverse effect on the measurement of the labeling substance due to the presence of the carrier, and to carry out high-precision and clear identification.
[0077]
Furthermore, according to the present invention, the detection of the presence or absence or the degree of the retention of the labeled detection body is performed based on only the labeling substance, and therefore any of the properties, physical properties, and chemical properties of the detection body itself Detection is easy because it does not depend on Further, since the range of the detectable body that can be adopted is not limited to a specific property or specific, the range of selection of the substance that can be adopted as the detectable body is wide.
[0078]
According to the second invention or the fourteenth invention, labeling is performed by making a labeling substance containing a predetermined type in a predetermined quantitative ratio different so as to be identifiable among the test types. Therefore, in addition to the effects described above, there is an effect that it is possible to distinguish between many types of tests (for example, hundreds, thousands, tens of thousands or more types) using a small number of types of labeling substances. In addition, by mixing a large number of related substances for each test type, accurate and precise labeling within a statistical error can be easily realized.
[0079]
According to the third invention or the fifteenth invention, in addition to the above-described effects, each detection object is bound to one or more labeling substances. Therefore, the quantitative ratio is the detection intensity of the labeling substance specifically bound to each carrier, so that not only the presence or absence but also the degree of presence can be easily detected.
[0080]
According to the fourth or sixteenth aspect, the separation of the carrier is performed by a magnetic force, so that the separation can be performed easily and reliably.
[0081]
According to the fifth invention or the seventeenth invention, by further increasing the possibility of the reaction between the test object and the labeled detector occurring at the highest probability, and further decreasing the possibility of the other reactions, A highly reliable inspection can be performed.
[0082]
According to the sixth invention or the eighteenth invention, the ratio of the existence of the binding due to the reaction between the test object and the labeled detector, which occurs with the highest probability, is further increased, and the existence of the binding due to the other reactions is further increased. By lowering the ratio, a more reliable test can be performed.
[0083]
According to the seventh or nineteenth aspect, even when one DNA contains a plurality of test sites, the test sites are included one by one, and the test sites are connected to the protruding end. By performing the cutting as described above, it is possible to multiplex and inspect the validity of the structure of each inspection site. Therefore, the effects as described in the first invention are obtained.
[0084]
Further, according to the eighth aspect, by using the restriction enzyme recognition site such as Type IIS, the double-stranded DNA can be cleaved at an arbitrary position without adversely affecting the inspection site. Versatile or versatile tests can be performed.
[0085]
According to the ninth aspect, the double-stranded DNA having a plurality of test sites is separated from each other at such a position that the bases at the recognition site and the cleavage site do not overlap with each other and do not affect the PCR primers. Therefore, the cleavage can be reliably performed at a predetermined position by the restriction enzyme such as Type IIS.
[0086]
According to the tenth or twentieth aspect, in addition to the above-described effects, as a labeled detector, a test is performed for each test type to determine whether or not the protein structure is present, whether or not the protein is present, and the degree of its presence. As described above, it is labeled with a labeling substance via a substance such as an antibody group selected so as to bind to or not bind to the test site. As a result, it is possible to quickly and efficiently perform a test for a mutant type of a protein for a plurality of types of protein test sites in parallel.
[0087]
According to the eleventh invention, the twelfth invention, the twenty-second invention or the twenty-third invention, the release of the labeling substance can be carried out using an enzyme capable of cleaving at an arbitrary position of DNA or polypeptide chain. . This is because detection is performed based on the labeling substance, and it does not matter where the detection body is cut. Therefore, there is no need to limit to restriction enzymes that cut at specific sites, and the range of enzyme selection is wide.
[0088]
According to the nineteenth aspect, in addition to the above-described effects, a test object which is cut so as to include a single-stranded test site in which a test of the structure of one type of DNA or the like is performed is used. I have. Therefore, a plurality of mutations in the DNA base sequence can be efficiently and accurately specified in parallel.
[0089]
According to the twentieth aspect, in addition to the above-described effects, a labeled detection object is combined with a test site so as to examine whether or not the structure of one type of protein is present, whether or not it is present, and the degree of its existence. Labeled with a labeling substance via a substance such as an antibody group selected so as not to bind or bind. As a result, it is possible to quickly and efficiently perform a test for a mutant type of a protein for a plurality of types of protein test sites in parallel.
[0090]
According to the twenty-first aspect, by using the restriction enzyme recognition site, double-stranded DNA can be cleaved at an arbitrary position without adversely affecting a test site. Certain tests can be performed.
[0091]
According to the twenty-first aspect, in addition to the effects described above, a double-stranded DNA having a plurality of test sites is used as the detector, and a recognition site and a cleavage site are 10 base pairs for each test site. By using a restriction enzyme recognition site where the recognition site such as Type IIS and the cleavage site separated from each other is different from each other, it is possible to surely cut at a predetermined site, so that highly reliable processing can be performed.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram of a mixed solution and a method according to an embodiment of the present invention.
FIG. 2 is a graph showing measurement results of the present invention.
[Explanation of symbols]
11, 12 ... Sample DNA
13, 14 ... Inspection site
15 ... Biotin
16, 17, 19 ... primer
18 ... Type IIS restriction enzyme recognition sequence
20, 21, 22, 23 ... amplified DNA fragment
24, 26, 28, 30 ... DNA fragments
25, 27, 29, 31 ... protruding end
33, 35, 37, 39 ... labeled detector
32, 34, 36, 38 ... protruding end
40 ... magnetic particles (carrier)

Claims (23)

A processing step of fixing a group of inspection objects of a plurality of inspection types to a carrier at a predetermined portion of each inspection object, or processing so as to be fixable, and bonding or bonding with the inspection object for each inspection type A generation step of generating a group of labeled detectors having a known predetermined structure of a plurality of types of tests that are not mutually distinguishable among the types of tests, and the test object group, the carrier, and the labeling detection. A reaction step of mixing the groups in a liquid to carry out a reaction, and a releasing step of separating the carrier bound to the labeled detector group via the test object and releasing the labeled substance from the separated carrier. And a detection step of detecting, for each test type, the presence or absence or the degree of retention of the labeled detection body via the test object by the carrier, based on the released labeling substance. Characteristic multiple detection Parallel way. The generating step includes, for each test type, a step of mixing a large number of detection objects and a labeling substance of each test type in which a predetermined type is included in a predetermined quantitative ratio in a liquid and binding them. 2. The multiple inspection parallel method according to claim 1, wherein the types or their ratios are different for each inspection type so that they can be distinguished from each other. In the generation step, the entirety of the labeling substance of each test type is distributed to substantially all of the labeled detectors of each test type, and one labeled detector binds to one or more labeling substances. 3. The multiple inspection parallel method according to claim 1, further comprising a step. 4. The method according to claim 1, wherein the separation of the carrier in the releasing step is performed by applying a magnetic force to the carrier. In the processing step, a plurality of inspection target inspection object group, processing so that it can be fixed to the carrier at a predetermined portion of each inspection object,
In the reaction step, the test object group and the labeled detector group are mixed and reacted in a liquid, and then the carrier is mixed and reacted in the mixed liquid. The multiple inspection parallel method according to any one of claims 1 to 4. In the reaction step, the respective labeled detectors of the group of labeled detectors are formed without reacting and binding with each other, and are mixed so that the number thereof exceeds the number of the test objects. 6. The multiple inspection parallel method according to claim 5, wherein: In the processing step, when inspecting the conformity of the structure of a predetermined inspection site of a plurality of inspection types of genetic material, one of a pair of specific binding substances is provided on the carrier, and a single-stranded inspection is performed as the inspection object. A site is included for each test type, cut so as to make the test site a protruding end, and processed so as to provide the other of the pair of specific binding substances at the other end of each test object. Then, the labeled detectors of a plurality of test types in the generation step, the test object of each test type, depending on its structure, a single-stranded single or non-bonded to the test site of the genetic material 7. The method according to claim 1, wherein the genetic material has a base sequence. In the processing step, a double-stranded DNA having a plurality of test types of test sites, a plurality of test type primer groups having the specific binding substance bound at one end to each test type of test sites, and a pair thereof PCR is carried out by mixing a primer having a cleavage site in which a test site downstream of the 3 'end of the primer has a protruding end, and a recognition site and a cleavage site having a different restriction enzyme recognition site inserted therein. An amplification step of amplifying a double-stranded DNA fragment by treating the amplified DNA fragment with the restriction enzyme to generate a DNA fragment having a protruding end of a test site at the other end. By having, the test object group of the plurality of test types is fixed to a carrier, or is processed so that it can be fixed,
In the generation step, one end has a protruding end which is a base sequence complementary to each test site of the test object group of a plurality of test types, and the other end is mutually identifiable for each test type. A double-stranded DNA fragment is produced as a labeled detector that is labeled with a labeling substance,
The processed test object group, the carrier on which the test object is fixed or the test object can be fixed, and the labeled detection body group are mixed in a liquid in a predetermined order to perform a ligation reaction. 8. The method according to claim 7, wherein the presence or absence or the degree of holding of the labeled detection body by the carrier via the test object is detected. In the processing step, the restriction enzyme, a double-stranded DNA having a plurality of test sites of the test object, the base at the recognition site and the cleavage site for each test site, do not overlap, PCR primers 9. The parallel method for multiple tests according to claim 8, wherein the restriction enzyme is separated so as not to affect the restriction enzyme, and the recognition site of the restriction enzyme is provided upstream of the 3 'end of the primer. Whether the structure of a plurality of test types of protein for a predetermined test site, the presence or absence or the degree of its presence, when testing the test object in the processing step, a plurality of types of proteins, A predetermined site other than the test site of the protein is immobilized on the carrier or processed so as to be immobilizable, and the labeled detector in the generation step specifically binds or binds to the test site. 7. The method according to claim 1, wherein a substance selected so as not to be labeled is labeled with the labeling substance so as to be mutually identifiable for each of a plurality of test types. Multiple inspection parallel method. In the release step, when performing a plurality of types of tests for genetic material, release of the labeling substance from the carrier bound to the labeled detector group is performed using an enzyme capable of cleaving DNA at any position. The method according to claim 1, wherein the method is performed. In the case where a plurality of types of tests are performed on the protein in the releasing step, release of the labeling substance from the carrier bound to the labeled detector group is an enzyme capable of cleaving the polypeptide chain of the protein at any position. The method according to claim 10, wherein the method is performed by using: A group of test objects of a plurality of test types fixed to a carrier at a predetermined portion of each test object or processed so as to be fixable, and a plurality of tests that are combined or not combined with the test object for each test type A mixed liquid comprising a group of labeled detectors having a known structure of a known type and labeled so as to be mutually identifiable between the types of tests, and a carrier on which the group of test objects is fixed or can be fixed. hand,
The carrier bound to the group of labeled detectors is separated via the test object, the labeled substance is released from the separated carrier, and based on the released labeled substance, for each test type, the carrier A mixed liquid for parallel use for multiple tests, wherein tests of a plurality of test types are performed in parallel using the mixed liquid by detecting the presence or absence or the degree of holding of the labeled detection body. The labeled detector of each test type is labeled by binding only to the labeling substance of each test type, and the entire labeling substance for each test type contains a predetermined type in a predetermined quantitative ratio. 14. The mixed liquid for parallel use for multiple tests according to claim 13, wherein the types or the quantitative ratios thereof are different for each test type so as to be distinguishable from each other. The entirety of the labeling substance for each test type is distributed to almost all of the labeled detectors for each test type, and one labeled detector is bound to one or more labeling substances. The mixed liquid for parallel use for a plurality of tests according to any one of claims 13 to 14. 16. The liquid mixture for parallel execution of multiple tests according to claim 13, wherein the carrier is separable by applying a magnetic force. 17. The method according to claim 12, wherein the test object group and the labeled detection body group are mixed and reacted in a liquid, and then the carrier is mixed. Mixed solution for multiple tests. The labeled detectors of the group of labeled detectors are produced without reacting and binding to each other, and are mixed so that the number thereof exceeds the number of the test objects. 18. The mixed liquid for multiple inspection parallel according to any one of items 13 to 17. In the case of testing the conformity of the structure of a predetermined test site of a plurality of test types of genetic material, each of the test objects is a DNA fragment or the like cut so as to include one single-stranded test site for each test type. The labeled detector of each test type is a labeled genetic material having a single-stranded base sequence that binds or does not bind to the test site of the genetic material depending on the structure. 19. The mixed liquid for multiple tests in parallel according to any one of claims 13 to 18, wherein: When inspecting the correctness or non-existence of the structure of a predetermined test site of a plurality of test types of proteins, the presence or absence or the degree of its presence, the test object is fixed to a carrier at a predetermined site, or can be fixed. A protein that has been processed to
The labeled detector has, for each test type, a known predetermined structure of a plurality of test types that binds or does not bind to the test object, and is labeled so as to be mutually identifiable among the test types. 19. The mixed liquid for multiple tests in parallel according to any one of claims 13 to 18, wherein: The labeled detector separates a double-stranded DNA having a plurality of test sites from each other so that the bases at the recognition site and the cleavage site do not overlap with each other and do not affect the PCR primers. A primer group in which a recognition site for a restriction enzyme having a different recognition site and a cleavage site is provided on the upstream side of the 3 'end of the primer, and a pair of labeled primer groups of a plurality of test types paired with the primer group are mixed. A double-stranded DNA fragment is amplified by PCR, and the amplified DNA fragment is treated with the restriction enzyme, whereby one end is labeled and the other end has a protruding end of a test site. 20. The mixed liquid for parallel use for multiple tests according to any one of claims 13 to 19. 20. An enzyme capable of cleaving DNA at an arbitrary position is mixed in order to release a labeling substance from a carrier bound to the labeled detector group, wherein the enzyme is mixed. 22. The mixed solution for multiple inspections according to claim 21. An enzyme capable of cleaving a polypeptide chain of a protein at an arbitrary position is mixed in order to release a labeling substance from a carrier bound to the group of labeled detectors, wherein the enzyme is mixed. 21. The mixed solution for multiple inspection parallel according to claim 18 or 20.

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