JP2003021639A - Method for deciding reaction on base material and base material usable therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for facilitating the rear and decision of results visualized in numerous reaction regions integrated on a base material and a base material usable for the method and, simultaneously, a method for confirming the base material position and a base material usable for the method. SOLUTION: A pluraltity of reaction region are combined and arranged on the base material so that letters or graphics may be formed as specified on the base material.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to determination of a reaction, which is carried out on a support such as a multi-well plate or a DNA chip, for use in an immunological reaction screening test, gene expression analysis, etc., and the result of the reaction is visualized. The present invention relates to a method and a support such as a plate and a DNA chip used therefor.

[0002]

2. Description of the Related Art In recent years, the miniaturization or ultraminiaturization has been progressing in all technical fields, and the chemical and biochemical fields are no exception. Especially in the field of genes and proteins, as the word of nanotechnology shows, the units of reagents and samples are rapidly changing from micro (10 −6) liters and micrograms to nano (10 −9) liters and nanograms. It's starting.

However, miniaturization in the fields of chemistry and biochemistry is not so easy. Hundreds of independent reaction areas (matrix) on a 10x5 cm plate
A multi-hole plate with a DN or a DN with a higher degree of integration
In the A chip (= DNA array; DNA in which a large number of DNAs are aligned and attached or bound on a support), each reaction region becomes finer as the number of matrices increases, resulting in a slight difference in the surrounding environment. , Is likely to appear as non-uniformity in each region. For example, differences in reaction time due to uneven temperature and ambient humidity, differences in reaction time due to reagent injection, etc., which were very small when the matrix size was large, have an effect on the entire reaction within the minute reaction region. It is often given. Further, in the spotting of DNA and the like, a slight deviation in the spotting angle may cause nonuniformity.

Although these nonuniformities are generally devised so as to prevent them from occurring during the plate manufacturing process and the reaction process, the final result can be obtained by placing a considerable number of positive and negative controls on the plate. It is common to check.
Controls have been very common in previous reaction systems, but as mentioned above, when individual reactions are carried out in a minute matrix, the control check is most suitable. It is especially important as a simple final check method.

As a condition for making the check by the control reliable, it is needless to say that it is treated the same as other samples, but it is preferable to disperse a certain number of samples. Multi-well plates and DNA with a high degree of integration
Since the reaction conditions may change depending on the position on the support in the array, it is desirable to place a control as much as possible in a position where the surrounding environment is likely to be different.

On the other hand, as the number of controls increases and they are placed in a dispersed manner as described above, determination of the result of the control is becoming a complicated task. As a measurer, before reading the result of the sample, first determine the result of the control and confirm that the reaction of the whole matrix was performed without problems, and if there is a problem, adopt the result of the whole support again. The most efficient procedure is to consider whether or not. In particular, in a qualitative reaction or a semi-quantitative reaction, a control is judged by the naked eye, so that it is possible to judge only a control, unlike a quantitative judgment in which both a sample and a control are measured at once using an instrument. However, as mentioned above, a certain number of controls are dispersed and set for the original purpose of the controls, and further, since each matrix is not numbered in the DNA array like the commercially available multi-well plate, It is not easy to visually confirm the control of.

Therefore, in most cases, the reading of the control and the reading of the control are also performed blindly at the same time.
After that, only the control data is taken out to judge whether the whole data is valid or not. However, in this method, the data of the sample read carefully may be wasted depending on the result of the control.

Further, the comparison result judgment is generally carried out based on the color density, but if the color of a certain specific matrix is judged with the naked eye, it is inevitably influenced by the surrounding colors. That is, when the periphery is light or colorless, it looks dark, and when the periphery is surrounded by a dark color, it looks lighter than it actually is, which causes an error.

Further, as another problem different from the confirmation of the contrast, there is also a problem of confirmation of the directions of the plate and the support, that is, the vertical and horizontal positions. In the case of a commercially available plate, each hole is numbered or marked in order to solve this problem, but in the case of a DNA array, DNA is generally pasted on a slide glass, so the creator arbitrarily writes a pencil. Etc. must be marked. Moreover, these marks use alcohol and organic solvents for subsequent work,
It is often experienced during experiments that the positional relationship is often lost when the final result is judged.

[0010]

DISCLOSURE OF THE INVENTION An object of the present invention is to perform a visualized reaction in a matrix integrated on a support, and to facilitate reading and determination of the visualized reaction result, and a method thereof. It is to provide a support using the method. Another object of the present invention is to provide a method for confirming the position of a support and a support using the method at the same time as providing the method and the support.

[0011]

The inventors of the present application have studied and devised these problems, and as a result, as a result, the reaction area on a support such as a plate, a slide glass, a membrane, or the like is made into a concrete character or figure. Choosing to shape makes it easier to locate the controls and identify the results, and moreover allows the naked eye to recognize them as'groups', making the semi-quantitative decisions made with the naked eye more accurate. Found. By further shaping these letters and figures,
It was found that the positional relationship of the support, that is, the front, back, top, bottom, left and right can be confirmed in an instant.

Most existing macro techniques not only require a large number of control settings for measurement, but also limit the number of matrices that can be included on a particular area. It is hard to say that it is practical. Therefore, it can be said that the present invention is a means for solving a problem newly emerged with the miniaturization of technology.

That is, according to the present invention, a large number of reaction regions are defined on a support, a reaction in which the result of the reaction is visualized is performed in each reaction region, and the reaction in each reaction region is performed based on the visualized result. A method of determining the presence and / or degree, wherein a plurality of reaction regions among the reaction regions are control regions for performing a control reaction, and the plurality of control regions are
Provided is a method for determining a reaction on a support, which is arranged so as to draw a predetermined character or figure. Further, the present invention defines a large number of reaction regions on a support, performs a reaction in which the result of the reaction is visualized in each reaction region, and based on the visualized result, the presence / absence of a reaction in each reaction region and / or Or a method of determining the degree, wherein a plurality of reaction areas among the reaction areas are used as test areas for performing a reaction with respect to a sample, and the plurality of test areas are arranged so as to draw predetermined characters or figures. And a method for determining a reaction on a support. Furthermore, in the present invention, the reaction region defined on the support carries a reagent to be subjected to the reaction,
There is provided a support for carrying out the method of the invention as described above.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a plurality of controls are combined in a biochemical reaction or a chemical reaction occurring in a large number of independent reaction regions (matrix) arranged on a support such as a plate, a glass slide, and a membrane. To make letters or figures. The support has a flat plate shape as a whole, but includes a multi-hole plate, a plate having minute projections on the plate, and a matrix having a three-dimensional structure.

The shape of the character or graphic to be created is arbitrary, but it is completely symmetrical in the matrix vertically and horizontally,
An asymmetrical shape is desirable, for example, a cross shaped in the center cannot be used to confirm the position of the support. Also,
The character or figure may be provided in one place, or may be divided into several places (FIGS. 1 and 2). For example, it is also possible to disperse characters or figures in several places by changing the concentration of the positive control and to make a semi-quantitative monitor. In addition, in the present specification, the “predetermined figure” is a figure which is intentionally formed with some regularity and is recognized as a figure having regularity by a person who judges the reaction, and the regularity is not defined. Randomly drawn diagrams etc. do not correspond to "predetermined figures". Further, if the product name of the support or the name of the manufacturing company is used as the character, an advertising effect can be expected.

Further, in the present specification, "a large number" of "a large number of reaction regions" means a number capable of forming at least a predetermined character or graphic, usually 100 or more, preferably 5 or more.
It is 00 or more. In the present invention, there is theoretically no upper limit of "a large number", but at the present technical level, in a DNA chip, about 30,000 (Affymetri) per support is used.
(Company x), multi-well plates allow up to about 1,500 per support (plate) (Nalge Nunc, etc.). However, these upper limits are increasing rapidly with the miniaturization of technology.

The control may be negative or positive, but it is necessary to be visualized by color development or the like since both are finally judged by the naked eye. However, in the process of visualization, reading or data processing may be performed using a device. The present invention is also effective in the case of highly integrated and difficult to read with the naked eye, dyes and coloring reagents that are difficult to identify with the naked eye, read using a device, and visually judge an image created mechanically. Function. Further, in the current technology, it is also possible to use two different kinds of dyes to perform color classification into negative and positive, depending on the object and means to be detected.

Further, the creation of these characters or figures can be applied not only to the control but also to the sample (measurement object). This is because, in the ultratrace measurement, for the same reason as setting a plurality of controls, it is often the case that a sample having the same concentration or a change in concentration is reacted in a plurality of regions.

For example, in a DNA array, a gene having a high expression amount or a housekeeping gene having a relatively constant expression amount is used as a control, and a gene of interest is used as a sample. There is no difference between the sample and. Therefore, when a character or a figure is created with a sample, the same effect as when the character or the figure is created with a contrast can be expected, for example, it becomes easy to confirm the spot position and to compare and determine the density. Also, with current technology, 1
It is possible to create thousands to tens of thousands of DNA spots on one slide glass, and by setting in advance the spotter where and what gene fragment is spotted, a relatively complicated character can be created. You can easily design the design.

The reaction itself to which the determination method of the present invention can be applied is well known in this field, and examples thereof include an antigen-antibody reaction and hybridization between nucleic acids. For example, like the sandwich method in the immunoreaction, after binding the antibody of the protein of interest to each hole of a multi-well plate and injecting a sample into it to bind and wash the protein of interest, the secondary protein of the protein is detected. Examples include a method in which an antibody labeled with a dye is reacted and detected. By this method, for example, a protein contained in human serum or an antibody produced against the protein can be detected, so that an infectious disease, an allergic reaction, a transplant compatibility test (HLA), and a disease-specific expression protein can be detected. Etc., and can be widely applied to clinical examinations and drug research.

As the nucleic acid, for example, a DNA array, that is, a part of the sequence of a gene to be detected (dsDNA or ssDNA consisting of several tens to several hundred bases) adhered to a slide glass, the cDNA of the sample to be measured There is a method of detecting a dye-labeled product by carrying out a reverse transcription reaction and then carrying out a hybridization reaction. DNA array
It is a technology that makes it possible to detect the expression of individual genes, which had been investigated one by one by Northern hybridization, in units of hundreds or thousands at once.
It is used to search for genes that are expressed in a tissue-specific manner and to compare gene expression between tissues (for example, a difference between cancer tissue and normal tissue). Also, the sample is not necessarily human tissue,
It can be used for all living things including bacteria and fungi,
Experiments on gene expression, detection of gene mutation,
It is widely applicable to identification of pathogenic bacteria, classification method of plants and animals, etc.

The present invention also provides a support for use in the above-mentioned method of the present invention, wherein the reaction region defined on the support carries a reagent to be subjected to the reaction. provide. Here, as is apparent from the above description, as the reagent carried on the support, the antigen or antibody (particularly protein) used for the above-described antigen-antibody reaction, or the one used for hybridization between nucleic acids Nucleic acid and the like. Specific examples of such a support include a nucleic acid array such as a DNA array in which DNA is immobilized in the reaction region, a protein array for immunoassay in which an antigen or an antibody is immobilized in the reaction region, and an immunoassay. An ELISA plate etc. can be mentioned.

[0023]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

1. Experimental method Preparation and spotting of dsDNA fragments Genes shown in the table below (part of genes, about 200-20
00bp) 1 to 8 clones were amplified by PCR using ExTaq (manufactured by Takara Shuzo) by PCR. As a positive control, GAPDH (glyceraldehyde)
-3-phosphate dehydrogenas
e) A part of the gene (550 bp) was similarly amplified. These dsDNAs were purified by ethanol precipitation and after drying,
0.5 μg of DNA fragment 1 to 8 after adding 3xSSC
/ Μl, the control gene (G) was adjusted to 0.1 μg / μl. Add these solutions to the layer (Geneeqs Arr).
ayer 2000; manufactured by Kaken Genex Co., Ltd., Matsudo City) and moved from position a-1 to the right on the slide glass coated with poly-L-lysine GGS12GP3GSS4.
Spotted 8 cycles in the order of 5P6SGPS7PS8S. In addition, the control gene GAPDH was
8, m9, m13, m14, m15, n5, n7, n1
2, o5, o7, o9, o10, o13, o14, p
2, p5, p7, p10, p15, q3, q4, q8,
JGS letters were made by spotting on q9, q12, q13, q14, and q17 (FIG. 1).

[Table 1]

(2) Pretreatment for Hybridization The above spotted slides were baked for 2 hours at a constant temperature of 80 ° C., and then UV crosslinker (SPEC) was used.
The DNA was fixed by irradiating UV with TRO LINKER (manufactured by Spectronic).

(3) Preparation of fluorescent DNA To 5 μg of total RNA prepared from cultured cells HepG2 (obtained from ATCC), 4 μg of oligo dT primer was added to make 10 μl of the whole, and the mixture was heat-denatured at 70 ° C. for 10 minutes and then rapidly cooled. Reverse transcriptase (Superscri)
6 μl and 3 μl of 5 × buffer and 0.1 M DTT attached to pt II, GibcoBRL, respectively, and further 25 mM dATP, dGTP, dCTP and 15 mM dTTP (all Amershampharmacia).
Each) 0.6 μl, 1 mM Cy3-dUTP
(Amershampharmacia) 3 μl was added. Finally, reverse transcriptase (Superscript I
2 μl of I) was added, the whole was adjusted to 30 μl with ddH ₂ O, and the mixture was reacted at 42 ° C. for 1 hour. The reaction was stopped by adding 0.5 M EDTA, and the product was purified by ethanol precipitation. Dry the labeled DNA and add TE buffer to 20μ
It was set to l. To this, add 4 μl of 1N NaOH and incubate at 70 ° C. for 1 hour to denature RNA to
4 μl of Cl was added to neutralize. It was again ethanol precipitated and dried.

(4) Hybridization The above labeled DNA was dissolved in 12 μl of TE buffer, and further 2.55 μl of 20 × SSC (pH 7.0), 10%
0.45 μl of SDS was added. After incubating at 100 ° C. for 3 minutes, the mixture was allowed to stand at room temperature for 15 minutes, dropped on a spotted slide, and a cover glass was placed on the slide. This is 6
It was put in a high temperature bath at 5 ° C. and hybridized for 14 to 18 hours. The cover glass was removed, and the plate was washed with 1 × SSC solution for 5 minutes and then 0.2 × SSC. Water on the microarray is removed and the scanner (Scan Arra
y5000, manufactured by GSI Lumonics) to capture the signal.

2. The result of the signal read by the judgment scanner is shown in FIG. The positive control (GAPDH) is spotted on a portion labeled (G) in FIG. The positive controls distributed above the array have more DNA than other genes, so you can quickly see some of them as controls, but at the top of the array it is difficult to distinguish them from other samples,
It is necessary to refer to each schematic diagram (Fig. 1). Also, it is difficult to attach the intensity of fluorescence.

On the other hand, "JG" located at the bottom of FIG.
At a glance, the letters "S" indicate that these are control spots. Regarding the intensity of fluorescence, there is not much difference between the characters, but it is immediately apparent that the size of the spot is slightly smaller for the character J. Further, the presence of the letters "JGS" makes the positional relationship between the top, bottom, left and right of the array clear.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of characters by a positive control (G) in a DNA array.

FIG. 2 is a diagram showing an example in which a graphic of a positive control (G) is dispersed.

FIG. 3 is a diagram showing a result of actual color development of the schematic diagram of FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/566 G01N 33/566 37/00 102 37/00 102

Claims (8)

[Claims] 1. A large number of reaction regions are defined on a support, a reaction in which the result of the reaction is visualized is carried out in each reaction region, and the presence or absence of the reaction in each reaction region and / or the reaction based on the visualized result. A method of determining a degree, wherein a plurality of reaction regions among the reaction regions are used as a control region for performing a control reaction, and the plurality of control regions are arranged so as to draw predetermined characters or figures. Method for judging reaction on support. 2. A plurality of reaction regions are defined on a support, a reaction in which the result of the reaction is visualized is carried out in each reaction region, and the presence or absence of the reaction in each reaction region and / or the reaction based on the visualized result. A method for determining a degree, wherein a plurality of reaction regions among the reaction regions are used as test regions for performing a reaction with respect to a sample, and the plurality of test regions are arranged so as to draw predetermined characters or figures. A method for determining a reaction on a support. 3. The method according to claim 1, wherein the reaction is an antigen-antibody reaction or hybridization between nucleic acids. 4. The method according to claim 1, wherein the detection target is a nucleic acid or a protein. 5. The method according to claim 1, wherein the reaction region defined on the support carries a reagent to be used in the reaction. 6. The support used for the method according to claim 5, which supports the reagent. 7. The support according to claim 6, wherein the reagent is an antigen or an antibody used for an antigen-antibody reaction, or one nucleic acid used for hybridization between nucleic acids. 8. The support according to claim 7, which is a protein array or a nucleic acid array.