JP2001183370A - Test piece and analytical method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent informaiton about a test piece from disagreeing with detection information in a gene expression analysis which uses the test piece on which an object to be detected is plotted in an array shape. SOLUTION: Identification information about the test piece 1 is given, by using a labeling substance, to a prescribed position of a test piece 1 on and to which many kinds of samples 4, to be detected, which are coupled with a sample labeled wit the labeling substance are arranged and fixed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analysis method using test pieces such as microarrays, macroarrays, and DNA chips. More specifically, various types of biomolecules are arranged and fixed on a substrate as detectors. Labeling substances (radioisotopes,
The present invention relates to a test strip that is hybridized with a sample containing a biomolecule labeled with a fluorescent dye or the like and specifies a hybridized detector, and an analysis method using the test strip.

[0002]

2. Description of the Related Art A paper entitled "Gene Expression Analysis Using a Microarray" is published in the January 17 issue of Vol. 17 (1999) of the Experimental Medicine Series (published by Yodosha Co., Ltd.). Here, a method for performing gene expression analysis using a microarray is described in detail.

[0003] This micro array, macro array, DN
A gene expression analysis technique using a test piece such as an A chip has recently been widely known and practiced. As shown in FIG. 8, various types of techniques are provided on the surface of a substrate 40 such as a membrane, glass, a slide glass, or a silicon substrate. Biomolecules (cDNA, oligo D
NA, other DNA, PNA, EST, etc.) are used as a detection object, and a test piece arranged and fixed in a matrix by a spotter device or the like is used. The test piece is referred to as a macroarray, a microarray, a DNA chip, or the like according to the type, size, number of spots, spot size, types of a detection body (probe) and a specimen (target) of the substrate 40, and the like.

On the other hand, cDNA and genomic DN labeled with radioisotopes or fluorescent dyes
A, RNA such as mRNA, biomolecule such as dNTP or PNA is prepared as a specimen.

[0005] Then, the detection body fixed in a matrix and the sample labeled with a radioisotope or the like are hybridized.

[0006] Here, if biomolecules that hybridize (bind) to each other are contained in the detection body and the specimen, the biomolecules are hybridized on the substrate, and radioactivity is applied to the detection body having the hybridized biomolecules. A labeling substance such as an isotope or a fluorescent dye is immobilized via a biomolecule. On the other hand, radioisotopes, fluorescent dyes, and the like are not fixed to the detector that has not been hybridized. The double circles in FIG. 8 schematically show the positions where the hybridized detectors are present on the substrate and the positions where labeling substances such as radioisotopes and fluorescent dyes are fixed. FIG. 8 is a schematic description, in which each of the dots arranged in a matrix is shown in a distinguishable manner. Cannot be identified.

[0007] Then, by detecting where a labeling substance such as a radioisotope or a fluorescent dye is present on the test piece, the location of the hybridized analyte on the substrate is specified from the detected location, and the presence of the label is determined. The type of the hybridized detection object is specified from the position. At the same time, the amount of the hybridized sample is detected.

[0008]

In order to specify the type of the hybridized detector, information on the detection position of the radioisotope or the fluorescent dye and the type and the position of the detector fixed on the test piece are required. Must be compared with information about

However, conventionally, information on the type and position of a detection object arranged and fixed on a test piece has been stored in a spotter device or the like used when manufacturing the test piece. Therefore, when identifying the type of hybridized detector from information on the detection position of radioisotopes and fluorescent dyes,
Researchers had to manually input this information into a computer. Therefore, when experiments are performed on many types of test specimens by changing the type of detector to be placed and fixed on the test specimen, an input error occurs in which researchers mistakenly enter information on different types of test specimens. There is a problem that the information about the type and position of the detection object arranged and fixed on the test piece does not correspond to the information about the position of the detected labeling substance because it is easy to perform an experiment on a different type of test piece. .

According to the present invention, there is provided a test piece and a test piece which can prevent the correspondence between the information on the type and position of a large number of detection objects arranged and fixed on the test piece and the information on the position of the detected labeling substance. An object of the present invention is to provide an analysis method using a test piece.

[0011] In the present invention, the use of the test strip is used for gene expression analysis, base sequence determination, mutation analysis, polymorphism analysis, and the like.
Not limited to those used for gene analysis, but also broadly defined as those that can be applied to the analysis of analytes that selectively bind to a detection object fixedly arranged in a spot on a substrate by some kind of reaction. I do.

[0012]

The test piece according to the present invention comprises:
A test strip in which a large number of detectors that bind to a sample labeled with a labeling substance are arranged and fixed, and identification information on the test strip is arranged at a predetermined position.

It is preferable that the identification information comprises the labeling substance or a labeling substance similar thereto.

The test piece according to the present invention may be of any type as long as it can arrange a large number of detection objects, such as a microarray, a macroarray, a DNA chip, and the like.

[0015] As a detector, cDNA, oligo DN
A, any other DNA, PNA or EST may be used as long as they are used for the array method. That is, the detector to be arranged and fixed on the test strip according to the present invention is not limited to a biomolecule, and may be of various types as long as it selectively binds to a specimen by some kind of reaction.

Samples include cDNA, genomic DNA,
RNA such as mRNA, biomolecules such as dNTP or PNA are usually used, but these are examples and are not limited thereto.

The "predetermined position" may be any position as long as the detection object is not disposed and fixed, and there is no particular limitation.

The "identification information" is information for identifying a test piece, and is information necessary at least for specifying the type and position of a detection object arranged and fixed on the test piece. An identification number or the like assigned to each test piece may be used. Further, the identification information may include information such as the type of the substrate used for the test piece, the date on which the test piece was created, the serial number, the lot number, and the format relating to the arrangement of the detectors. Further, the information may include information on the specimen as well as the detection body. The identification information is placed at a predetermined position on the test piece, but is preferably coded and placed from the viewpoint of reducing the placement space.

As the labeling substance, radioisotopes, fluorescent dyes and the like are usually used, but these are only examples and are not limited to these.

The identification information relating to the test piece may be arranged and fixed on the test piece using a spotter device, or may be arranged and fixed on the test piece using an ink jet printer.

[0021] The sample analysis method according to the present invention comprises the steps of: bringing a sample labeled with a labeling substance into contact with a test strip on which a large number of detectors are arranged and fixed, and bonding the sample to the detector; In the sample analysis method, comprising: an acquisition step of acquiring detection information about a detection object combined with, before the acquisition step, an application step of providing identification information about the test piece to a predetermined position of the test piece, A reading step of reading identification information.

Examples of the "binding" between the analyte and the detection body include, in addition to hybridization forming a stable double strand between complementary base sequences, for example, binding by specific binding. It should be noted that the case where the sample and the detection body are bound by some kind of reaction may be, for example, various kinds of affinity (affinity).

The "detection information" means that after the detection substance and the sample are hybridized, the labeling substance is fixed to the hybridized detection substance, and the hybridization is obtained by detecting the labeling substance. This refers to information that indicates the location of the detected object.

In the sample analysis method according to the present invention, it is preferable that the identification information comprises the labeling substance or a labeling substance similar thereto.

Here, the "similar labeling substance" refers to a substance which emits the same radiation as the labeling substance, though different from the labeling substance when the labeling substance is a radioisotope. When the labeling substance is a fluorescent substance, it is different from the labeling substance, but is irradiated with the excitation light in the same wavelength range as the excitation light applied to the labeling substance, so that the fluorescence in the same wavelength range as the labeling substance is emitted. A substance that emits

The time when the identification information is arranged at a predetermined position on the test piece may be any time before the detection step, and may be performed, for example, when the detector is arranged and fixed on the test piece, It may be performed after the detection body and the analyte are bound.

[0027]

According to the method of the present invention, since the identification information on the test piece is arranged on the test piece, the information on the test piece can be detected when detecting the labeling substance on the test piece. In addition, the detection information and the identification information can be made to correspond to each other, whereby it is possible to prevent the correspondence between the detection information and the information on the type and the position of the detection object arranged and fixed on the test piece from being inconsistent.

Further, by making the identification information consist of a labeling substance or a labeling substance similar thereto, the identification information can be detected at the same time as detecting the labeling substance on the test piece. The obtaining step and the reading step of the identification information can be performed at the same time, whereby the processing can be performed without increasing extra steps.

[0029]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A method for analyzing biomolecules using a microarray according to one embodiment of the present invention will be described with reference to FIGS.

First, various types of biomolecules (cDNA) are arranged in a matrix on a substrate (membrane) using a spotter device. That is, as shown in FIG. 1, various types of biomolecules 4 are arranged in a matrix on a membrane 2 to form a test piece 1. Note that the cDNA arranged on the membrane 2 is used as a probe (detector).

Next, identification information on the test piece 1 is given to the position indicated by 20 in FIG. Specifically, in the present embodiment, the radioactive isotope ( ¹⁴ C, ³² P, ³³
P etc.) are arranged on the surface of the test piece 1. When there is time from the formation of the test piece 1 to the hybridization, it is preferable to use ¹⁴ C which is a radioactive isotope having a long half-life. Further, the radioisotope forming the identification information may be the same as that for labeling the target described later, and is different from this, but emits the same radiation as the radioisotope for labeling the target. Is also good. FIG. 3 shows the arrangement of the radioactive isotopes arranged on the surface of the test piece 1. As shown in FIG. 3, the identification information on the test piece 1 is encoded, and the encoded identification information is arranged on the surface of the test piece 1. Specifically, the date of preparation of the test piece 1, the preparation number, the type of the test piece 1, the test piece 1
The identification information includes information on the type of the probe arranged and fixed in the data format, the format representing the arrangement form of the probe, and the like.
As a method of coding the identification information, various conventionally known methods can be used.

The coded identification information may be arranged as a bar code as shown in FIG.
As shown in FIG. 3B, they may be arranged in a dot shape. The arranged identification information is composed of a part indicating the creation date, a part indicating the type of the test piece 1 and a part indicating the type of the probe, and these are integrated into one piece of code information. May be represented. As described above, in the present embodiment, since the identification information on the test piece 1 is encoded, the identification information can be arranged in a narrow space on the test piece 1.

Then, the membrane 2 is formed by the above-described procedure.
After arranging the probe on the surface of the sample, the probe is fixed to the membrane 2 by irradiating ultraviolet rays (UV) to form the test piece 1.

Next, using poly (A) RNA prepared using RNA extracted from cells to be analyzed as a template, a cDNA labeled with a radioisotope is synthesized by a reverse transcription reaction.
This labeled cDNA is used as a target (sample).

When information on the target is known in advance, it is preferable that such information is also coded and arranged on the test piece 1.

Then, the test piece 1 is immersed in the prepared solution to hybridize the target (FIG. 4). Then, by cleaning the surface of the test piece 1,
The unhybridized target 6 is removed, leaving only the hybridized radioisotope-labeled target 8 on the test strip 1, as schematically shown in FIG.

Next, in order to detect the position of the target 8 remaining on the test piece 1, the stimulable phosphor sheet 30 is brought into close contact with the test piece 1 (FIG. 6). The stimulable phosphor sheet 30 is left in contact with the stimulable phosphor sheet 30, and the stimulable phosphor sheet 30 is exposed to radiation from a radioisotope. Before the exposure to the radiation, the stimulable phosphor sheet 30
Is irradiated with visible light to erase unnecessary information stored in the stimulable phosphor sheet 30. Then, by leaving the test piece 1 for a certain period of time after the close contact, the radiation energy from the target 8 remaining on the test piece 1 and the radiation energy from the radioactive isotope arranged at the position indicated by 20 in FIG. The stimulable phosphor sheet 30 is accumulated.

Here, the stimulable phosphor sheet absorbs radiation energy when irradiated with radiation, stores and records the radiation energy, and thereafter, when excited with a laser or the like in a specific wavelength range, is irradiated with the radiation. A material having the property of emitting stimulated emission light in an amount corresponding to the energy amount of the emitted radiation. Typically, BaFX (where X is a halogen) phosphor particles are densely filled in a binder on a support. A coated panel is known, which is also known as a radiation conversion panel using a stimulable phosphor.

Next, the test piece 1 and the stimulable phosphor sheet 30 are separated, and the radiation energy stored in the stimulable phosphor sheet 30 is detected. This operation will be briefly described with reference to FIG. 7. By using a half mirror or a dichroic mirror 12, the reading laser 10 is reflected to scan the entire surface of the stimulable phosphor sheet 30, and the stimulable phosphor sheet 30 is scanned. The stimulated emission light 14 emitted at 30 passes through the mirror 12 and is detected by a photomultiplier (PMT). That is, a portion where radiation energy is accumulated by being exposed to radiation from a radioisotope which is a label of a labeled target 8 hybridized to a dot 4 of cDNA on the test piece 1 in a hybridization process; Since the photostimulable emission light is emitted from a portion where radiation energy from the radioisotope is exposed to radiation from the radioisotope indicating information on the test piece 1 arranged and fixed on the photoreceptor 1, the radioisotope is detected by the PMT to detect the radioisotope. Can be specified.
The stimulated emission light detected by the PMT is converted into an electric signal, input to the computer C, and information indicating the light emission position is stored in the computer C.

Here, the information indicating the light emission position stored in the computer C includes the test piece 1 at the position indicated by reference numeral 20 in FIG.
Identification information on the test piece 1 based on the identification information.
(In the present embodiment, information such as the creation date of the test piece 1, the membrane type, the probe type, and the format indicating the probe arrangement) can be read.

Therefore, in the present embodiment, when analyzing the information indicating the light emission position stored in the computer C, first, the information indicating the light emission position of the portion corresponding to the position indicated by 20 in FIG. Read the encoded information.
Thereafter, the date of preparation of the test piece 1 used in the experiment, the type of the test piece, the type of the probe, and the like are specified from this information.
After such identification, the researcher inputs information on the position and type of the cDNA placed and fixed on the test piece 1 to the computer C, and compares this information with the information indicating the light emission position stored in the computer C. Thus, cDNA that has not hybridized with cDNA that has hybridized with RNA extracted from cells is specified.

As described in detail above, in the detection system according to the present embodiment, the identification information associated with the management information (the date of creation, the type of the test piece, the probe type, etc.) of the test piece 1 in the test piece 1. Since the information is given using the labeling substance (radioisotope), the stimulable phosphor sheet 30 can also store the identification information of the test piece 1. Therefore, when detecting the position of the hybridized detector from the stimulable phosphor sheet, the identification information of the test piece 1 can be detected at the same time.

Further, even if the number of types of the detection objects arranged on the test piece 1 increases and the types of the test pieces 1 to be experimented increase, if the identification information is read from the test piece 1, the detection information can be obtained correspondingly. Accordingly, it is possible to prevent the identification information and the detection information of the test piece 1 from being inconsistent.

Further, since the identification information of the test piece 1 is arranged using the same or similar substance as the labeling substance, no special reader is required for reading the identification information, and the identification information is high. Since information on the test piece 1 can be detected simultaneously when detecting the position of the labeling substance of the hybridized detection body, the processing can be performed without increasing an extra step.

In the above embodiment, the membrane 2
Although a spotter device was used when arranging the radioactive isotopes, an ink jet printer device could be used instead. In this case, a radioactive isotope is printed (placed) on the surface of the membrane 2 instead of the ink.

Further, in the above embodiment, the identification information of the test piece 1 is arranged on the test piece 1 before the hybridization, but the present invention is not limited to this, and the identification information may be arranged after the hybridization. In addition, when a radioisotope is arranged as identification information after hybridization, it is not necessary to fix the radioisotope arranged on the test piece 1. This is because the radioisotope disposed during the hybridization step or after washing away the target that has not hybridized may be stripped off, but the radioisotope is not stripped off thereafter.

Further, in the above embodiment, a radioactive isotope is used for labeling a target, but a fluorescent dye (Cy5, Cy3, etc.) may be used. In this case, the test piece 1 may be directly irradiated with excitation light for exciting the fluorescent dye without using the stimulable phosphor sheet, and the fluorescence emitted from the fluorescent dye may be detected by the PMT.

In the above embodiment, the information on the test piece 1 is arranged using the same or similar substance as the labeling substance. However, the present invention is not limited to this. May be arranged. Further, information on the test piece 1 may be coded and arranged on the test piece 1 by a method such as forming unevenness or forming a hole in the membrane 2.

[Brief description of the drawings]

FIG. 1 is a perspective view showing an example of a test piece used in an embodiment of the present invention.

FIG. 2 is a perspective view schematically showing a state in which biomolecules are arranged and fixed in a matrix on the surface of a test piece.

FIG. 3 is a diagram showing an example of coded information arranged and fixed on a test piece;

FIG. 4 is a perspective view schematically showing a hybridization step.

FIG. 5 is a perspective view schematically showing a state after hybridization.

FIG. 6 is a perspective view schematically showing a process of superposing a stimulable phosphor sheet on a test piece and exposing the stimulable phosphor sheet to radiation.

FIG. 7 is a perspective view schematically showing a state in which a reading laser is emitted to emit light.

FIG. 8 is a perspective view schematically showing a conventional microarray substrate.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Test piece 2 Membrane 4 cDNA matrix 6 Target 10 Laser for reading PMT Photomultiplier

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 G01N 35/02 C 35/02 C12N 15/00 A F-term (Reference) 2G042 BD19 CB03 DA08 FB07 FC01 2G058 BA08 CC09 EA11 FB01 GA02 GC05 4B024 AA11 AA19 CA01 CA09 CA11 HA13 HA14 4B029 AA07 AA21 BB20 CC03 CC11 FA15 4B063 QA01 QA13 QQ42 QQ52 QR32 QR35 QR38 QR55 QR66 QR84 QS34 QS39 QX02 Q

Claims (5)

[Claims] 1. A test piece comprising a plurality of fixed test pieces arranged and fixed to a specimen labeled with a labeling substance, wherein identification information on the test piece is arranged at a predetermined position. Pieces. 2. The method according to claim 1, wherein the identification information comprises the labeling substance or a labeling substance similar thereto.
Test specimen as described. 3. The test piece according to claim 1, wherein the identification information is arranged using a spotter device or an ink jet printer device. 4. A test piece on which a large number of detectors are arranged and fixed,
A sample analysis method comprising: contacting a sample labeled with a labeling substance, and binding the sample and the detection body; andan obtaining step of obtaining detection information on the detection body bound to the sample. A sample analysis method, further comprising, before an obtaining step, a providing step of providing identification information on the test piece to a predetermined position of the test piece, and a reading step of reading the identification information. 5. The apparatus according to claim 4, wherein the identification information comprises the labeling substance or a labeling substance similar thereto.
The sample analysis method according to the above.