JP2001116755A - Examination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an examination method that can determine the examination of an organic constituent of a plurality of DNA fragments, RNA, cDNA, peptide, oligonucleoide, or the like, and a substance such as medicines resulting from the organic constituent or the like in parallel at a time. SOLUTION: The examination method includes a process that supplies a desired amount of first samples to a spot being positioned at desired position on a substrate where a specific number of spots are formed, and fixes the first samples onto the surface of the spot for preparing a reaction chip where the desired sample is fixed to a desired place, a process that uses a jet nozzle, and adds the first sample itself or a second one with characteristics for connecting with a specific constituent in the sample to the reaction chip, a process that connects the first sample to the second one under desired conditions, and forms a substance being labeled while the substance can be optically detected, and a process that applies light with desired wavelength to the substance being labeled while the substance can be optically detected, and measures the amount of light emission.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a test method using a reactive material used for analysis of DNA function, analysis of gene mutation and expression, and diagnosis of various diseases such as cancer. More specifically, the present invention relates to a method for testing biological components such as DNA fragments, cDNAs, proteins, and oligonucleotides and pharmaceuticals such as interferons derived from biological components, which are used for various diagnoses and treatments.

[0002]

2. Description of the Related Art Inspection methods using a DNA sequencer or the like are actually used for diagnosis of genetic diseases caused by congenital abnormalities of genes or diseases caused by mutation of DNA, for example, various cancers. . On the other hand, the decoding of genomic base sequences of various organisms has been advanced, and research and development aimed at functional analysis of genes based on the knowledge has been further activated.
Meanwhile, for functional analysis of a gene, there is a need for the development of a technique for efficiently measuring and examining individual differences and mutations of the gene, the frequency of expression in a cell, and the like. In such technology,
As one of the testing methods for cancer and the like, a DNA fragment containing a predetermined probe is added in advance with a test sample consisting of a fluorescently labeled DNA fragment on a DNA chip on which a DNA fragment containing a predetermined probe has been added in order to test for a mutation in a gene. A fluorescently labeled DNA fragment having a sequence complementary to a predetermined probe on the DNA chip is bound to the probe by hybridization, and immobilized on the same chip. When this is irradiated with ultraviolet light, the DNA is immobilized by hybridization. Since only the sample emits fluorescence, there is known a method of examining whether or not a specific DNA fragment is contained in a test sample by identifying the sample.

In the inspection method using the DNA chip, precision, sensitivity, reproducibility,
It is known that cost is a problem. For example, in the case of a DNA chip in which thousands to tens of thousands of types of DNA are integrated and immobilized, the sample is added by adding it to the entire chip surface. Therefore, a volume of about 20 μl is required at present. Therefore, there is a problem that only one kind of sample can be examined with one chip, and there is also a problem that the cost of the reagents necessary for the test, particularly the fluorescent substance, is high, so that the test cost is also high.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide multiple DNA fragments, RNAs, cDNAs, peptides,
It is an object of the present invention to provide a method capable of measuring a biological component such as an oligonucleotide and a substance such as a pharmaceutical derived from the biological component in parallel. More specifically, DN
Prepare a reactive chip in which A fragment etc. are integrated at a high degree of integration,
It is an object of the present invention to provide a method which can measure a large number of specimens at once by using this method.

[0005]

Means for Solving the Problems The present invention provides a step of preparing a reactive chip in which a desired first sample is fixed at a desired position on a substrate; Adding a second sample having the property of binding to a specific component in the sample using a nozzle such as an inkjet, and bonding the first sample and the second sample under desired conditions, An inspection method comprising the steps of: forming a detectably labeled substance; and irradiating the optically detectably labeled substance with light of a desired wavelength and measuring the amount of luminescence is described above. The present invention has been accomplished by finding that the object has been achieved.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a DNA fragment, R
Biological components such as NAs, cDNAs, peptides, oligonucleotides, enzymes, antigens, antibodies, epitopes, proteins, and pharmaceuticals such as interferons derived from biological components can be tested with high precision. According to this method, not only a qualitative test but also a quantitative test is possible. In this method, the first sample refers to a DNA fragment, RNA, c
DNA, peptide, oligonucleotide, enzyme, antigen,
Antibodies, epitopes, biological components such as proteins and sample samples such as interferon derived from biological components may be used,
Further, a substance capable of optically detectably labeling these sample samples may be used. The second sample means that the first sample is a biological sample such as a DNA fragment, RNA, cDNA, peptide, oligonucleotide, enzyme, antigen, antibody, epitope, or protein, or a sample sample such as interferon derived from the biological component. Is a substance capable of optically detectably labeling these sample samples, wherein the first sample is a DNA fragment,
RNA, cDNA, peptides, oligonucleotides, enzymes, antigens, antibodies, epitopes, biological components such as proteins and interferons derived from biological components and the like can be optically detectably labeled, if the biological components described above, It is a substance derived from biological components. Substances that can be optically detectably labeled include biological components such as DNA fragments, RNA, cDNA, peptides, oligonucleotides, enzymes, antigens, antibodies, epitopes, proteins, and other biological components such as probes such as interferons derived from biological components. It is a substance that can bind to components and substances derived from biological components. It is known that each substance has a specific probe. For the probe and its labeling procedure, see, for example, DNA probe technology and application published by CMC Corporation (Toyozo Takahashi, published February 1988).

In the present specification, a specific component in a sample means, for example, when the probe-nucleotide is a probe of the same substance, one part of a cDNA corresponding to a specific mRNA sequence, one part of a genomic clone, a known part. Refers to a synthetic oligonucleotide having sufficient sequence homology to the target sequence, and refers to a target substance from which a probe or the like of the same substance can be detected. In addition, as the substrate, a slide glass made of silicon or glass can be used, of course, as long as a sample can be loaded thereon, regardless of the material or the like. Of course, the surface of the substrate may be subjected to an appropriate surface treatment such as poly-L-lysine or silanization for the purpose of imparting an affinity to the above-mentioned substance contained in the specimen. Usually,
On the substrate, at least 100 spots each having a spot volume of at least about 5 picoliters, preferably, depending on the type of the specimen and the purpose of the test,
The number is 1,000 or more, more preferably 10,000 or more.

In the inspection method according to the present invention, in fixing the first sample to the substrate, the patent application “Method for producing a reactive material and reactivity that can be produced by the method”, filed on the same date as the present application. Materials ”(reference number WP0304
The method described in the specification of 3) may be followed. That is, the step of fixing the first sample at a desired position on the substrate includes preparing a substrate on which a desired number of spots, for example, at least 100 spots of at least 5 picoliters are formed,
A solution containing the first sample is discharged from a jet nozzle at a spot located at a desired position on the substrate in an amount of 5 picoliters or more per spot, and a spot located at a predetermined position is made desired. This discharging operation may be repeated until the first sample is loaded (or carried).
In this method, the first sample is fixed to the substrate using a needle pin or a reactive chip manufactured by using a method such as applying photolithography technology. Is also good. More specifically, from organs and tissues in which cancer cell development is observed and / or organs and tissues in which cancer cell development is suspected, and a plurality of biological samples from normal organs and tissues, respectively, collected from postoperative cancer patients A prepared solution containing a DNA fragment or the like is prepared, supplied from a jet nozzle to spots located at predetermined locations, and fixed, whereby a sample can be arranged as designed. That is, by arranging in this way, it is possible to determine the presence or absence of cancer cell proliferation in various organs and tissues of the same patient after surgery by a single test.

The term “jet nozzle” as used herein refers to a nozzle utilizing a piezoelectric / electrostrictive element, and is provided in a predetermined number of nozzles, for example, a cartridge type nozzle provided with at least 10, preferably 1000 nozzles. Are mounted. More specifically, an ink jet head described in Japanese Patent Application Laid-Open No. 6-40030, which is an application of the present applicant, is suitably used as such a nozzle. The adjustment of the discharge amount or the like may be performed by a CPU or the like incorporated in advance. Regarding the method of filling the jet head with the sample, the operating conditions, and the like, the patent application “Reactive chip manufacturing method, reactive chips and reactive substances that can be manufactured by the same method” filed on the same date as the present application ( What is necessary is just to follow the description of the specification number WP03043).

Next, the solution containing the second sample is supplied without error onto the spot on which the first sample is mounted. When the second sample to be supplied is a substance capable of optically detectably labeling biological components such as DNA fragments, cDNAs, peptides, oligonucleotides, enzymes, antigens, antibodies, epitopes, proteins, and interferons derived from biological components. In this method, at least three types of optically detectably labelable substances prepared by the method disclosed in JP-A-8-501632 may be supplied onto a desired spot using a jet nozzle. . This supply can be completed by one discharge operation unless there is a special situation. Of course, a probe not previously labeled with the labeling material is first supplied, the probe and the analyte are bound by hybridization or the like, and then a conjugate in which the labeling material is bound to an antibody or the like that recognizes both substances is added thereto. The analyte may be provided to optically detectably label the analyte. In addition, the characteristics of the specimen,
Depending on the purpose of the test or the like, a biological sample such as DNA fragments, RNA, cDNA, peptides, oligonucleotides, enzymes, antigens, antibodies, epitopes, proteins, and interferons derived from biological components, etc. Using a substance that can be detected and labeled, as a second sample, a biological component such as a DNA fragment, RNA, cDNA, peptide, oligonucleotide, enzyme, antigen, antibody, epitope, or protein, or a sample such as interferon derived from the biological component May be used.

In the case of the present method, as the first sample, biological components such as DNA fragments, RNA, cDNA, peptides, oligonucleotides, enzymes, antigens, antibodies, epitopes, proteins, and interferons derived from biological components are used. The substrate on which the sample is fixed may be divided into a plurality of sections in advance according to the purpose of the test, and a substance that can be optically detectably labeled as desired for each fraction may be supplied. Only a desired substance that can be optically detectably labeled may be supplied. Of course, the probe is supplied to all spots first, then at least one kind of labeling compound is supplied to all spots, and then only a specific spot is supplied with a labeling compound different from the at least one kind of labeling compound. Thus, only a plurality of sample samples fixed to spots at specific locations may be labeled. That is, the method of loading the probe can be arbitrarily selected according to the purpose. When the present method is used for measuring an activity unit such as interferon, a sample solution that is sequentially diluted at a specific ratio is supplied and fixed so as to be arranged at least in duplicate on a spot. In addition, a desired substance that can be optically detectably labeled can be supplied and optically labeled.

The step of binding the first sample and the second sample under desired conditions to form an optically detectably labeled substance is usually performed by hybridization. Hybridization conditions may vary depending on the type of sample, but since the conditions are well known for each sample, the conditions may be followed. Of course,
The first or second sample not fixed by hybridization or the like is preferably removed from the substrate by washing or the like. The step of irradiating the optically detectable labeled substance with light of a desired wavelength and measuring the amount of luminescence is performed by fluorescence measurement-application to biological science "Spectroscopy Society of Japan Measurement Series 3" Kazuhiko Kinoshita, Makoto Hashihiro, Society Publishing Center (19
(January 1983).

[0013]

EXAMPLES Hereinafter, the present invention will be further described with reference to Examples. (Example) 1. Preparation of Reactive Chip A slide glass 1 cm x 1 cm in size, which was previously surface-treated with poly-L-lysine, was used as a substrate for detection to clarify the relationship between drug metabolism and gene polymorphism. As a DNA fragment, LDL receptor polymorphism was 100
The DNA for detection was arranged so that the test could be performed at once for each person.

[0014] 2. Preparation of Optically Detectable Labelable Substance A small amount of RNA extracted from the cells of each patient is reverse transcribed with reverse transcriptase and amplified as a cDNA by PCR (polymerase chain reaction). The cyanine dye Cy5 (Cy
^TM ).

[0015] 3. Addition of sample sample to reactive chip Prepare a nozzle with 100 ejection holes and store reagents via supply channels so that sample solutions for 100 people can be supplied to each spot as a second sample sample. An apparatus having a piezoelectric / electrostrictive element provided with a tank was prepared, and each type of DNA solution was spotted on the substrate prepared in 1 above in one spot.
Spotting was performed by 0 picoliter. After the spotting, the substance that can be optically detectably labeled prepared in the above 2 was added to a desired spot by operating a discharge nozzle. After the addition, hybridization was performed at 65 ° C. for 2 hours for labeling. After hybridization, S
The chip was washed with an SC-SDS solution, and the amount of luminescence of the hybridized cDNA fixed on each spot was measured by the following method.

[0016] 4. Measurement Each spot was excited at 633 nm by a confocal laser, and the emission intensity at 670 nm was measured. Thus, it was possible to test a large number of test items for 100 samples in a single test. According to this method, the amount of the specimen sample is small, and the gene polymorphism and the fluctuation of the gene expression can be examined in a very short time for a large amount of the specimen at a time.

[0017]

According to the test method of the present invention, simultaneous analysis of multiple samples becomes possible. That is, when diagnosing metastasis or the like, it is possible to simultaneously examine a cDNA fragment prepared from a sample sample taken from a different tissue or organ of a postoperative patient. The amount of sample used for each spot can be significantly reduced compared to conventional methods, and the sensitivity is high, so detection of very small amounts of sample or trace components contained in the sample is possible. Becomes

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Claims (3)

[Claims] A step of preparing a reactive chip in which a desired first sample is fixed at a desired position on a substrate; and a step of preparing a reactive chip containing the first sample itself or a specific component in the sample. Adding a second sample having a binding property using a jet nozzle, binding the first sample and the second sample under desired conditions, and forming an optically detectable labeled substance. An inspection method comprising: a step of forming; and a step of irradiating light having a desired wavelength to a substance optically detectably labeled to measure a light emission amount. 2. The method according to claim 1, wherein the first sample is a DNA fragment, RNA, cD
At least one selected from the group consisting of NA, enzyme, antigen, antibody, epitope, protein, etc., and the second sample is a group consisting of DNA fragments, RNA, cDNA, enzymes, antigens, antibodies, epitopes, proteins, etc. The test method according to claim 1, wherein the test method is a probe of at least one sample selected from the group consisting of: and a substance which is optically detectably labeled. 3. The inspection method according to claim 2, further comprising selectively adding the second sample only to a specific location on the substrate to which the first sample is fixed.