JP2005083942A - Method for detecting cyanin series organic coloring matter deterioration environment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting environment for deteriorating a specified cyanin-based organic coloring matter in advance. <P>SOLUTION: A Cy5 solution is exposed into an inspection environment for four to 24 hours. When the absorbance of the Cy5 solution after the exposure decreases at least by 25% as compared with the absorbance of the non-exposed Cy5 solution, the environment is determined to be a Cy5 deterioration environment. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The invention of this application relates to a method for detecting an environment in which a specific cyanine organic pigment deteriorates. More specifically, the invention of this application detects in advance a test environment that causes an error in detection accuracy in a DNA microarray or the like due to deterioration of a specific cyanine organic dye frequently used as a fluorescent labeling substance in a DNA microarray or the like. The present invention relates to a method and a method for converting a cyanine-based organic dye-degraded environment detected by this method into a non-degraded environment.

  A DNA microarray is a device in which thousands to tens of thousands of gene DNA (capture probes) are arranged at high density on a glass substrate, and gene expression profiles and gene polymorphisms are analyzed on a genome scale by hybridization with sample DNA. It is possible to do.

  Hybridization of the capture probe and sample DNA in the DNA microarray employs a method in which the sample DNA is fluorescently labeled and the fluorescence signal of the sample DNA remaining on the microarray is detected by a confocal laser scanner or the like by hybridization with the capture probe. Has been.

In addition, methods that use control DNA together with sample DNA, or methods that perform competitive hybridization using two types of DNA obtained from different specimens (tissues and cells) are widely used. Different fluorescent labels are used for different types of sample DNA and control DNA (two fluorescent labeling method). In this two-fluorescent labeling method, a plurality of types of cyanine organic dyes (Patent Document 1; Cy3, Cy5 or Cy7: registered trademarks of Amersham Pharmacia Biotech) having different fluorescence signal intensities are most often used (see above). For example, refer nonpatent literatures 1 and 2).
Special table 2003-522333 Edited by Mark Schena (translated by Tomoyuki Kato), "DNA microarray", TaKaRa, 2000 Supervised by Masaaki Matsumura and Hiroyuki Nami, separate volume of cell engineering genome science series "DNA microarray and latest PCR method", Shujunsha, 2000

  When testing with a DNA microarray in a normal laboratory environment, dust in the room adheres to the microarray, and the dust emits light by the laser, so the fluorescence signal of the sample DNA cannot be measured accurately. There is. Therefore, implementation in a dust-free room (clean room) is preferably employed to eliminate an error signal due to dust.

  However, when the inventors of this application performed a DNA microarray test by a two-fluorescence method or the like in a clean room, a specific cyanine organic dye deteriorates, and the original signal intensity cannot be obtained. A problem was found that it was impossible to make a judgment by comparison with a cyanine organic dye signal without deterioration.

  If the inspection is performed while ignoring such deterioration of the cyanine organic pigment, an accurate result cannot be obtained. In addition, the measurement of fluorescent signals in DNA microarray inspection is the final stage in the inspection process. Prior to that, much labor, time, and cost were required for the preparation process and hybridization process of labeled sample DNA. If the measurement fails, all of the effort, time, and money that has been spent up to that point will be lost. Therefore, it is extremely important to detect in advance whether or not the inspection environment causes deterioration of the cyanine organic pigment.

  The invention of this application has been made in view of the circumstances as described above, and provides a means for detecting in advance a specific cyanine-based organic dye degradation environment in a DNA microarray inspection by a two-fluorescence labeling method. Is an issue.

  Another object of the invention of this application is to provide a means for converting a detected cyanine-based organic pigment deterioration environment into a non-deterioration environment.

  The invention of this application provides the following formula as a first invention for solving the above-mentioned problems:

Wherein X is the same or different and represents CR ³ R ⁴ , Y is the same or different and is selected from a functional group having a hydrogen atom, a hydrocarbon group, or a hetero atom, and R ¹ or R ² is Any one represents an active ester group, the other represents a hydrocarbon group which may have a functional group, R ³ and R ⁴ are a hydrogen atom or a hydrocarbon group, at least one is a hydrocarbon group, and n Is 2 or 3)
A test solution containing the above-mentioned cyanine organic dye is exposed to the test environment, and the absorbance of the test solution is determined. A method is provided in which the test environment is determined as a cyanine-based organic pigment degradation environment when the water content decreases.

In the method of the first invention, in Formula 2, R ¹ or R ² is dUTP (5-Amino-propargyl-2′-deoxyuridine5′-triphosphate) or dCTP (5-Amino-propargyl-2′-deoxycytidine5 ′). -triphosphate) is a preferred embodiment.

  In the method of the first invention, when the exposure time is 4 to 24 hours and the decrease rate of the absorbance of the exposed Cy5 solution is 25% or more, the test environment is determined to be a cyanine-based organic dye deterioration environment. This is a preferred embodiment.

  This application is, as a second invention, a method for converting a cyanine-based organic dye environment detected by any of the above-mentioned methods into a cyanine-based organic dye non-degraded environment, wherein air in the environment is adsorbed or washed with water. A filtering method is provided.

  In the method of the second aspect of the invention, the adsorbent is activated carbon.

  In the present invention, “deterioration of cyanine-based organic dye” means a state (functional deterioration) in which a fluorescent signal having an intensity inherent in the dye cannot be emitted. The “degraded environment” refers to an environment containing a non-specific substance that functionally degrades a specific cyanine-based organic dye (for example, Cy5, Cy7).

  In the following description, among the cyanine-based organic dyes represented by the following formula (Formula 3), n = 1 dye (for example, Cy3) is represented by N1 dye, and n = 2 dye (for example, Cy5) is represented by N2. A dye having n = 3 (for example, Cy7) may be referred to as an N3 dye. N2 dye and N3 dye may be referred to as N2 / 3 dye.

Wherein X is the same or different and represents CR ³ R ⁴ , Y is the same or different and is selected from a functional group having a hydrogen atom, a hydrocarbon group, or a hetero atom, and R ¹ or R ² is Any one represents an active ester group, the other represents a hydrocarbon group which may have a functional group, R ³ and R ⁴ are a hydrogen atom or a hydrocarbon group, at least one is a hydrocarbon group, and n Is 1, 2 or 3.)

  According to the present invention, it is possible to detect in advance an environment in which the N2 / 3 dye (for example, Cy5, Cy7) deteriorates, and further convert the N2 / 3 dye deteriorated environment to a non-degraded environment. As a result, DNA microarray inspection can be performed in a favorable environment, and accurate inspection results can be obtained. Moreover, a great amount of labor, time and cost for re-inspection are not required. Furthermore, the method of the present invention also provides a standard in setting up a preferred clean room environment for DNA microarray testing.

  In the method of the first invention, when the test solution containing N2 dye or N3 dye is exposed to the test environment and the absorbance of the test solution after this exposure decreases, the test environment is determined to be the N2 / 3 dye deteriorated environment. To do.

  N2 / 3 dye is a compound represented by the following formula (Formula 4) or a derivative thereof.

Wherein X is the same or different and represents CR ³ R ⁴ , Y is the same or different and is selected from a functional group having a hydrogen atom, a hydrocarbon group, or a hetero atom, and R ¹ or R ² is Any one represents an active ester group, the other represents a hydrocarbon group which may have a functional group, R ³ and R ⁴ are a hydrogen atom or a hydrocarbon group, at least one is a hydrocarbon group, and n Is 2 or 3.)
Specifically, X (CR ³ R ⁴ ) in the formula is C (CH ₃ ) when R ³ is CH ₃ and R ⁴ is H, R ³ is CH ₃ , and R ⁴ is CH ₃ . Is C (CH ₃ ) ₂ , R ³ is CH ₃ , and R ⁴ is C ₂ H ₅ , it is C (CH ₃ ) (C ₂ H ₅ ). Y is a hydrogen atom, an alkyl group such as methyl, ethyl, propyl or butyl group, an aryl group, a hydroxyl group, a thiol group, an amino group, a cyano group, a nitro group, a sulfone group, a carboxyl group or an alkylcarbonyl group. Furthermore, the active ester group of R ¹ or R ² is, for example, a group having NHS (N-hydroxysuccinimide) at the terminal.

  Commercially available products (for example, Cy5 and Cy7) can be used for such N2 dye and N3 dye, respectively. Cy2 phosphoramidite (manufactured by Glen Research) as the N2 dye, 1,1 ', 3,3,3', 3'-hexamethylindotricarbocyanine (Kanto Chemical Co., Ltd.), etc. as the N3 dye Can also be used. Furthermore, for example, when targeting a DNA microarray test environment, Cy5-dUTP or Cy7-dUTP (Amersham Pharmacia Biotech) used for the preparation of labeled sample DNA, or Cy5-dCTP (Amersham) as Cy5-dNTP It is preferable to use Cy7-dCTP (Amersham Pharmacia Biotech) as Cy7-dNTP. Further, the dNTP binding to Cy5 or Cy7 may be a mixture of four types of dNTPs (dATP, dTTP, dGTP, dCTP), or one of them.

  As the solvent to be mixed with the N2 dye or the N3 dye, purified water, distilled water, sterilized water, or a buffer (for example, phosphate buffer (pH 7.5) or the like) can be used. The concentration of the dye is 0.5 to 20 μmol / L, preferably about 1 to 10 μmol / L.

  The test solution thus prepared is placed in a transparent container (for example, a glass petri dish, a test tube, a PCR tube, etc.). The volume of the test solution can be appropriately determined according to the type of the container and the like, but 10 to 100 μL, preferably about 40 to 60 μL is sufficient.

  Next, with the container filled with the test solution opened, the container is placed in an examination environment and exposed to the environment. The exposure time of the test solution to the environment is 4 to 24 hours, preferably about 7 to 16 hours. Moreover, it is preferable that the test environment (temperature, humidity, presence / absence of illumination, etc.) is set to the same condition as that for performing DNA microarray test, for example, and is not changed during exposure of the test solution.

  Then, the absorbance of the test solution after exposure to the environment is measured using an absorptiometer. At this time, the amount of solvent evaporated during environmental exposure is replenished to obtain the initial volume. The absorbance is determined by measuring the OD value according to the test dye, for example, OD650 nm when using Cy5 as the N2 dye, and OD740 nm when using Cy7 as the N3 dye.

  The absorbance decrease rate is obtained by comparing the absorbance of the test solution thus measured with the control value. The control value can be, for example, the absorbance of the test solution before exposure to the environment, the test solution not exposed to the environment (for example, the test solution placed in the same environment for the same time as the test solution with the container covered), or under any environment However, a solution of an N1 dye (for example, Cy3) having almost no functional deterioration can be used.

  As a result of such comparison, when the rate of decrease in absorbance of the test solution exposed to the test environment is 25% or more, particularly 30% or more, it is determined that the test environment is an N2 / 3 dye deterioration environment. When the absorbance of the N1 dye solution is used as a control, the test environment is N2 / 3 dye when the change in absorbance of the test solution is 10% or more, especially 30% or more greater than the change in absorbance of the N1 dye solution. It can also be determined that the environment is deteriorated.

  Next, the method of the second invention will be described. This method is characterized in that the N2 / 3 dye-degraded environment is converted to a non-degraded environment by subjecting air in the environment detected as the N2 / 3 dye-degraded environment to a filtration treatment with an adsorbent or water. To do.

  This method is carried out, for example, by installing a locally closed space (for example, a clean booth) in the N2 / 3 dye deterioration environment and filtering the air sucked into the closed space with an adsorbent or water washing. be able to. As the adsorbent, activated carbon, zeolite, silica gel, activated alumina and the like can be adopted, and the use of activated carbon is particularly preferable. The water washing treatment can be carried out by a method such as contact filtration using a filter medium such as porous ceramic, a multilayer disk, or the like.

  Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.

1． Sample preparation
Cy5-dUTP was dissolved in 10 mM phosphate buffer (pH 7.5) to prepare four Cy5 solutions at concentrations of 1 μmol / L, 3.3 μmol / L, 10 μmol / L, and 40 μmol / L. Also, four types of Cy3 solutions having the same concentration as described above were prepared using Cy3-dUTP. Each of these solutions was filled into 50 mL of 0.5 mL PCR tubes.
2. Test Method Each tube was left in the inspection environment for 0 to 16 hours without sealing. The inspection environment was "clean room" and "laboratory without dustproof facilities (normal environment)".

After a predetermined period of time, the amount of liquid in each tube was measured with a pipetman, and sterilized water was added to make the initial volume (50 μL). The absorbance of this solution was measured with an absorptiometer. That is, the absorbance of Cy5 solution was measured at OD650 nm, and Cy3 solution was measured at OD550 nm.
3. result
The measurement results of the absorbance of Cy3 solution at OD550nm are as shown in Table 1 (clean room) and Table 2 (normal environment). The Cy3 solution showed almost no decrease in absorbance after 16 hours at any concentration tested in any environment.

On the other hand, the absorbance at OD 650 nm of the Cy5 solution is as shown in Table 3 (clean room) and Table 4 (normal environment). For example, when a Cy5 solution with a concentration of 1 μmol / L is left in a normal environment for 4 hours, the decrease rate of the OD650 value is about 20%, and when left for 16 hours, the decrease rate is about 60%. When the concentrated Cy5 solution was left in a clean room, a decrease of 50% was observed after 4 hours and as much as 98% after 16 hours.

From the above results, it was confirmed that the inspected clean room was an environment that deteriorates Cy5.

  Hereinafter, as a reference example, a method for preventing Cy5 degradation is illustrated.

  The same inspection as in Example 1 was performed in a clean room at a temperature of 65 ° C. The results are as shown in Table 5, and no decrease in the OD650 value of the Cy5 solution was observed. From this result, it was confirmed that the deterioration of Cy5 was suppressed at a high temperature of about 65 ° C.

  Installed a hood with activated carbon in the front part of the hepa filter at the top of the clean booth (As One, Model 600, outer dimensions 600 x 480 x 720 mm), and Wako Pure Chemicals activated carbon 660 g (activated carbon layer: 200 x 200 x height) 66 mm) was filled into the hood. A mesh for holding the activated carbon was attached to the lower part of the activated carbon hood, and the activated carbon was held in the hood so that the outside air permeated only through the activated carbon layer. This clean booth was operated at a flow rate of 250 L / min in the clean room determined as a Cy5 deteriorated environment in Example 1, and after 15 minutes, a 3.3 μmol / L Cy5 solution tube was placed in the clean booth.

As a result, the OD650 value of the Cy5 solution decreased 80% after 16 hours outside the clean booth, but the OD650 value decreased 5-10% inside the clean booth. From this result, it was confirmed that Cy5 deterioration was suppressed in the clean booth with activated carbon hood.

  In the clean room determined as the Cy5 deteriorated environment in Example 1, it was confirmed that Cy5 deterioration was suppressed as in Examples 2 and 3 even when air was allowed to pass through in the washing environment.

As described above in detail, the method of this application makes it possible to detect in advance the environment that degrades Cy5. In DNA microarray inspection using Cy5 labeling, this pre-detection provides accurate test results and eliminates the risk of retesting due to Cy5 degradation.

Claims (6)

Following formula:
Wherein X is the same or different and represents CR ³ R ⁴ , Y is the same or different and is selected from a functional group having a hydrogen atom, a hydrocarbon group, or a hetero atom, and R ¹ or R ² is Any one represents an active ester group, the other represents a hydrocarbon group which may have a functional group, R ³ and R ⁴ are a hydrogen atom or a hydrocarbon group, at least one is a hydrocarbon group, and n Is 2 or 3)
A test solution containing the above-mentioned cyanine organic dye is exposed to the test environment, and the absorbance of the test solution is determined. And the test environment is judged to be a cyanine-based organic dye-degrading environment. In the formula 1, R ¹ or R ² is dUTP (5-Amino-propargyl-2'-deoxyuridine5'-triphosphate) or dCTP (5-Amino-propargyl-2'-deoxycytidine5'-triphosphate). Method.   The method of claim 1, wherein the exposure time is from 4 to 24 hours.   The method according to claim 1, wherein the test environment is determined to be a cyanine-based organic dye-degraded environment when the rate of decrease in absorbance of the test solution is 25% or more.   A method for converting a cyanine-based organic dye-deteriorated environment detected by the method according to any one of claims 1 to 4 into a cyanine-based organic dye-deteriorated environment, wherein the air in the environment is filtered by an adsorbent or water washing. A method characterized by processing. The method of claim 4, wherein the adsorbent is activated carbon.