KR101510351B1 - Microarray and method of analyzing signal from microarray analysis - Google Patents

Abstract

One or more embodiments provide a microarray comprising a combination of a dark reference point marker and a bright reference point marker and a method for analyzing the microarray signal using the same.

Microarray, dark reference point marker, bright reference point marker

Description

[0001] The present invention relates to a microarray and a method for analyzing microarray signals,

One or more embodiments relate to a microarray having a fiducial marker and a method for analyzing the microarray analysis signal.

A microarray generally refers to a probe substance that binds to a target substance on a substrate and is fixed to a plurality of divided regions. Microarrays are used for the analysis of many target substances. The analysis of the target substance is carried out by contacting a sample containing the target substance labeled with the fluorescent substance to the probe substance on the microarray and measuring the light obtained therefrom.

Since the region of the microarray where the probe material is immobilized (hereinafter also referred to as a spot) is generally arranged at a high density, the number of irradiated and detected spots used in one experiment may be several thousands to several tens of thousands. Thus, the operator analyzing the image data obtained from the microarray analysis results is able to determine the brightness of each hybridized spot and the local background prior to quantifying the image signal obtained from the microarray, ) Or a pattern. Microarray gridding is a template used by detection software to more efficiently locate the true position of each spot within the pattern. Therefore, there is a need to efficiently specify the position of each spot from the optical data obtained from the microarray having many spots.

As a method of determining the position of a conventional spot, there is a method of manually specifying a spot on an optical image based on known spot information and a method using a robotic spot placement equipment.

However, there is still a need for an improved method for easily locating and analyzing the position of each spot from the optical data obtained from the microarray.

One example provides a microarray having a structure that is efficient for distinguishing signals obtained from microarray analysis.

Another embodiment provides a method for analyzing a microarray analysis signal using the microarray.

A microarray comprising a first divided region, a second divided region and a third divided region on a surface of a substrate, wherein the probe nucleic acid is immobilized on the third divided region, and the probe nucleic acid And the binding strength between the first divided region and the target nucleic acid labeled with the detectable label or the binding property with the target substance labeled with the detectable label is determined by the ratio between the second divided region and the target nucleic acid labeled with the detectable label, The binding force between the second divided region and the target substance labeled with the detectable label is lower than the binding force between the probe nucleic acid of the third divided region and the detectable label, Wherein the microarray is the same or larger than the binding force with the target substance.

Wherein the first divided region and the second divided region are obtained by reacting a detection signal obtained after reacting with a target nucleic acid labeled with a detectable label or a target substance labeled with a detectable label in comparison with a signal obtained from the first divided region 2 The signal obtained from the segmented region may be larger. From the difference of these signals, the first divided region and the second divided region can act as a fiducial marker for discriminating the signal obtained from the microarray analysis. Hereinafter, the first divided region and the second divided region are referred to as a dark fiducial marker (DF) and a bright fiducial marker (BF), respectively. Also, the third divided area is also referred to as a data spot.

The signal is a fluorescence signal and the fluorescence signal obtained from the second segmented region may be brighter than the fluorescence signal obtained from the first segmented region.

The detectable label may be an optical label, a radioactive label, and an enzyme capable of converting the substrate into a coloring material. The optical label includes a fluorescent substance. Such enzymes include alkaline phosphatase and horseradish peroxidase.

The first, second and third divided regions may have a dimension of 0.1 mu m to 10 mu m. The dimension means a diameter in the case of a circle, and a length of a line passing through the center of gravity of the region and not meeting the contour of the region when the region is not circular.

The first divided region and the second divided region are obtained by reacting a detection signal obtained after reacting with a target nucleic acid labeled with a detectable label or a target substance labeled with a detectable label, So that the signals are separated from each other.

The combination may have a low probability that the signal obtained from the third segmented region that was reacted identically would have the same arrangement by chance. Here, the term "arrangement with a low probability of having the same arrangement by chance" means that the target nucleic acid labeled with the detectable label or the target nucleic acid labeled with the detectable label is reacted with the probe nucleic acid immobilized in the third separated region, Means that the batch is not statistically significant. The low-probability arrangement may be, for example, a linear arrangement or an L-arrangement. The combination may have a letter or symbol shape. The first segmented area may be a combination of surrounding the second segmented area or having an opposite shape.

The combination may be arranged for each panel of the microarray, and a plurality of combinations may be arranged for each panel. For example, the combination may be arranged at four corners of each panel if each panel of the microarray is square. The term "panel " refers to a unit region in which a detector reads a signal from a microarray reacted with a target nucleic acid. For example, when a detector is a camera for measuring fluorescence, it refers to a unit area in which a fluorescent signal is read from a microarray reacted with a target nucleic acid. The panel may be part of the microarray, in which case the signal obtained from the microarray can be analyzed by combining the signals obtained from the panel.

The first divided region is composed of a hydrophobic substance, and the target nucleic acid labeled with the detectable label and the target substance labeled with the detectable label may be composed of a hydrophilic substance.

In addition, the second divided region may have a fixed substance capable of binding to a target substance labeled with a detectable label, or may have such a surface property. For example, the second segmented region may be one in which biotin is immobilized, and the target substance labeled with a detectable label is streptavidin labeled with a detectable label. The binding force between biotin and streptavidin is generally stronger than the binding force between the probe nucleic acid and the target nucleic acid so that the signal obtained therefrom can be stronger than the signal from the probe nucleic acid and the target nucleic acid. In addition, the second divided region may have a probe having a longer length than the probe nucleic acid, and the target substance labeled with a detectable label may be a nucleic acid complementary to the long probe.

According to the microarray according to one embodiment, the area used as the reference point marker can be minimized, and the time can be reduced in distinguishing signals obtained from the microarray. It can alleviate pollution by panel.

Another embodiment includes the steps of reacting a sample comprising a target nucleic acid labeled with a detectable label and a target substance labeled with a detectable label on the microarray and obtaining a signal from the reaction product; And

And separating the signal from the third segmented region based on the signal from the first and second segmented regions.

The method comprises reacting a sample comprising a target nucleic acid labeled with a detectable label and a target labeled with a detectable label on the microarray and obtaining a signal from the reaction product. The reaction may be a hybridization reaction, and conditions of the hybridization reaction are known in the art. For example, the reaction may be overnight at 4 ° C in a hybridization buffer. The microarray is as described above. The step of obtaining a signal from the reaction product can be appropriately selected by those skilled in the art depending on the labeling substance selected. For example, when a fluorescent label is selected, excitation light may be irradiated and the emitted light emitted therefrom may be measured. Such measurements can be made by the camera.

The microarray according to one embodiment can be used for efficiently analyzing signals obtained from a microarray.

According to the method according to another embodiment, the microarray signal can be analyzed efficiently.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

1 is a diagram schematically showing an example of an image obtained as a result of microarray analysis. The microarray analysis may be a fluorescence image obtained by hybridizing a microarray with a target nucleic acid labeled with a fluorescent substance, and irradiating the excitation light to the fluorescent material with the result of hybridization and measuring the emitted light emitted therefrom. 1, a dark reference point marker is a dark rectangular shape composed of divided regions at a second position from the edge of the image, and a bright reference point marker is an L-shaped marker having a divided region at the third position from the left and lower edges of the image, Shape, and a L-shaped bright reference mark marker consisting of the dark reference marker portion consisting of the divided regions of the second position from the left and bottom edges of the image and the divided regions of the third position are adjacent to each other. Therefore, even if the signal from the data spot is bright or equal to the bright reference point marker, the position of the reference point marker can be confirmed. That is, by combining the dark reference point marker and the bright reference point marker, the position of the reference point marker can be more accurately found in the microarray analysis image, and the number of spots used can be reduced. The number of spots used in the reference point marker decreases, so that the influence of the reaction between the reference point marker and the substance in the sample on the reaction between the data spot and the target substance in the sample may be reduced, thereby increasing the strength or accuracy of the data spot. . The image shown in Figure 1 represents an image of a panel on a microarray.

 FIG. 2 is a diagram showing an example of gridding the reference point markers and the data spots correctly based on the reference point markers in the image shown in FIG. Once the location or shape of the reference point marker is identified, it is known in the art to identify the location of other data spots relative to it. The confirmation of the reference point marker may be performed by referring to a reference file storing information on visual confirmation of an image or reference point marker. The reference file refers to a file that stores information such as the shape of a reference point marker disposed at the time of manufacturing a microarray, and a relative position of a reference point marker and a data spot. In FIG. 2, it can be seen that a certain grid is allocated so as to correspond exactly to each spot in the dark reference mark marker of the rectangular shape, the bright reference mark marker of L shape, and the data spot area. In the analysis of the target substance using the microarray, information on the target substance can be analyzed with reference to only the signal of the lattice content, for example, a fluorescence signal.

  FIG. 3 is a diagram showing an example in which the reference point marker and the data spot are incorrectly gridded on the basis of the reference point marker in the image shown in FIG. 3, the lower portion of the dark reference point marker is composed of spots in the third row shifted from the lower outline of the image by one spot above the actual position, as compared with the reference point marker actually formed in the microarray, The lower part consists of the spots in the fourth row moved above one spot above the actual position from the bottom outline of the image. As a result, the positions of the spots separated by the lattice are not coincident with the actual spot positions on the microarray, and even if the signals read from the lattices are analyzed, the target substances in the sample can not be analyzed accurately.

Fig. 4 is a diagram showing an image of one panel in a microarray in which a plurality of combinations of dark reference point markers and bright reference point markers are included, and the plurality of combinations have different shapes. According to FIG. 4, the combination of the dark reference point marker and the bright reference point marker are respectively located at the four corners of the image of the panel, and their shapes are different from each other. In FIG. 4, B represents a bright reference point marker spot, D represents a dark reference point marker spot, and M and P represent a data spot, respectively a mismatch spot and a perfect match spot. The microarray of FIG. 4 is a diagram of one panel, and includes a total of 144 reference mark spot (BF: DF = 60: 84). The bright reference point marker spot may be a region where the oxide film of the substrate having the oxide film is removed. The area may be the surface of the substrate itself from which the oxide film is removed, or the surface may be coated with a hydrophobic material. The hydrophobic material may be derived from a dry etch material such as a fluorocarbon comprising tetrafluoromethane. The dry etching may be a reaction of a material with plasma, and these are known.

The substrate may have a probe immobilized compound immobilized on its surface. The surface may be the entire surface excluding the surface of the reference point mark or the surface on which the probe is to be immobilized. Wherein the immobilized compound is selected from the group consisting of compounds having biotin, avidin, streptavidin, poly L-lysine, imino group, aldehyde group, thiol group, carbonyl group, succinimide group, maleimide group, epoxide group, isothiocyanate group Or more. Examples of the compound having an amino group include 3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA), trimethoxysilylpropyldiethylenetriamine (DETA) , 3- (2-aminoethylaminopropyl) trimethoxysilane, 3-aminopropyltriethoxysilane, and the compound having an aldehyde group includes glutaraldehyde. An example of a compound having a thiol group includes 4-mercaptopropyltrimethoxysilane (MPTS). Examples of the compound having an epoxide group include 3-glycidoxypropyltrimethoxysilane, and examples of the compound having an isothiocyanate group include 4-phenylenediisothiocyanate (PDITC), succinimide, Examples of compounds having a mid group include disuccinimidyl carbonate (DSC) or succinimidyl 4- (maleimide phenyl) butyrate (SMPB). In Figure 4, the dark reference point marker spot may be located in an area adjacent to the first reference point mark.

The second reference mark may be defined by patterning the surface of the substrate. The patterning can be performed by a known method. For example, the patterning may be performed by photolithography. As a result of the patterning, the second reference mark mark may have a columnar structure formed by etching and removing the substrate surface around the reference spot spot. The shape viewed from the top of the column, that is, the shape of the plane, for example, may be a rectangle including a circle, a rectangle and a normal square, but is not limited thereto.

When using a plurality of combinations different from each other as shown in Fig. 4, for example, in the case of using a combination which is unlikely to occur accidentally in a data spot, the probability of erroneous misgridding is reduced. In the case of the experiment of FIG. 4, no false lattice distinction occurred in 216 panels (72 panels / microarray x 3 microarrays). When a plurality of different combinations are used, since a minimum area per panel is used as a reference point marker spot, an area that can be used as a data spot increases. In addition, since the number of reference spot marker spots decreases, the time required for the grid segment can be reduced accordingly. When a plurality of different combinations are used, it is possible to reduce confusion between the adjacent panels and the panel.

1 is a diagram schematically showing an example of an image obtained as a result of microarray analysis.

FIG. 2 is a diagram showing an example of gridding the reference point markers and the data spots correctly based on the reference point markers in the image shown in FIG.

FIG. 3 is a diagram showing an example in which the reference point marker and the data spot are incorrectly gridded on the basis of the reference point marker in the image shown in FIG.

Fig. 4 is a diagram showing an image of one panel in a microarray in which a plurality of combinations of dark reference point markers and bright reference point markers are included, and the plurality of combinations have different shapes. In Fig. 4, the rectangle of the four corners is an enlarged portion of the four corners of the microarray.

Claims (13)

1. A microarray comprising a first divided region, a second divided region and a third divided region on a surface of a substrate, wherein a probe nucleic acid is immobilized in the third divided region, and the probe nucleic acid is complementary to a target nucleic acid Wherein binding force between the first segmented region and the target nucleic acid labeled with the detectable label or binding force with the target substance labeled with the detectable label is determined by labeling the second divided region with a detectable label And the binding force between the second divided region and the target substance labeled with the detectable label is smaller than the binding force between the probe nucleic acid of the third divided region and the target substance labeled with the detectable label The microarray being the same or larger than the binding force. The method according to claim 1, wherein the detection signal obtained after the first divided region and the second divided region are reacted with a target nucleic acid labeled with a detectable label or a target substance labeled with a detectable label, Wherein the signal obtained from the second segmented region is larger than the signal obtained from the region. 3. The microarray according to claim 2, wherein the signal is a fluorescence signal, and the fluorescence signal obtained from the second segmented region is brighter than the fluorescence signal obtained from the first segmented region. The method according to claim 1, wherein the first divided region and the second divided region are obtained by reacting a detection signal obtained after reacting with a target nucleic acid labeled with a detectable label or a target substance labeled with a detectable label, And the second signal is separated from the signal obtained from the third divided region. 5. The microarray according to claim 4, wherein the combination has a low probability that the signal obtained from the third divided region reacted identically will have the same arrangement by chance. 5. The microarray according to claim 4, wherein the combination has a letter or symbol shape. 5. The microarray according to claim 4, wherein the combination is arranged for each panel of the microarray, and a plurality of the panels are arranged for each panel. 5. The microarray of claim 4, wherein the combination is disposed at four corners of each panel when each panel of the microarray is square. The microarray according to claim 1, wherein the first segmented region is composed of a hydrophobic substance, and the target nucleic acid labeled with the detectable label and the target substance labeled with the detectable label are composed of hydrophilic substances. The microarray according to claim 1, wherein the second segmented region is immobilized or has surface properties capable of binding to a target substance labeled with a detectable label. 2. The microarray according to claim 1, wherein the second segmented region is biotin-immobilized, and the target substance labeled with a detectable label is streptavidin labeled with a detectable label. The microarray according to claim 1, wherein the second divided region is a probe having a length longer than that of the probe nucleic acid, and the target substance labeled with the detectable label is a complementary nucleic acid to the long probe. Reacting a sample comprising a target nucleic acid labeled with a detectable label and a target substance labeled with a detectable label in a microarray according to any one of claims 1 to 12 and obtaining a signal from the reaction product; And And separating the signal from the third segmented region based on signals from the first and second segmented regions.

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