JP2006300798A - Biological data processor, biological data measuring method, program and recording medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To sufficiently ensure a dynamic range of an estimable hybridized amount. <P>SOLUTION: In a case that a plurality of developed profile images acquired by scanning the spot 12 provided on a DNA chip 11 by exciting lights different in intensity are set to a processing target, in a preparation part 81, hybridized amounts of which the number is the same as that of the developed profile images are acquired from the fluorescence intensities of the developed profile images and a preliminarily prepared fluorescence intensity-hybridized amount conversion formula. One hybridized amount is calculated by applying weighting to the acquired hybridized amounts and the calculated hybridized amount is set as the estimate value of the hybridized amount produced in the spot 12. This biological data processor is adapted to an apparatus for measuring the fluorescence intensity of the DNA chip. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biological information processing apparatus and method, a program, and a recording medium, and in particular, a biological information processing apparatus and method, a program, and a recording medium that can sufficiently ensure a dynamic range of an estimation possible hybridization amount. About.

  In recent years, a DNA (deoxyribonucleic acid) chip or a DNA microarray (hereinafter simply referred to as a DNA chip when it is not necessary to distinguish between them) has been put into practical use. A DNA chip is a DNA chip in which various and many DNA oligo chains are integrated and immobilized on a substrate surface as nucleic acids for detection. Comprehensive analysis of gene expression in collected cells by detecting hybridization between probes immobilized on spots on the substrate surface and targets in samples collected from cells using a DNA chip Can do.

  With the improvement of hybridization detection technology in gene expression analysis using a DNA chip, it is becoming possible not only to detect the presence or absence of gene expression but also to quantitatively measure the gene expression level. For example, a technique for obtaining a quantitative numerical value indicating a gene expression level by quantitatively measuring fluorescence intensity at the time of hybridization detection has been partially put into practical use.

For example, Patent Documents 1 to 3 are prior art documents related to a method for analyzing and correcting a gene expression level obtained with a DNA chip or the like.
JP 2002-71688 A JP 2002-267668 Gazette JP 2003-28862 A

  By the way, conventionally, in the operation of estimating the amount of hybridization generated at a spot, only an expression profile image obtained with one kind of excitation light has been used. Here, the expression profile image is an image that is an observation target of the hybridization state obtained by irradiating the DNA chip with excitation light and scanning the generated fluorescence.

  Therefore, there is a problem that the dynamic range of the amount of hybridization that can be estimated cannot be sufficiently secured depending on the excitation light intensity of the target expression profile image.

  The present invention has been made in view of such circumstances, and is intended to ensure a sufficient dynamic range of the amount of hybridization that can be estimated.

  According to an aspect of the present invention, a state of hybridization between a first biological material fixed to a reaction region provided on a substrate and a second biological material that hybridizes to the first biological material is excited. A biological information processing apparatus for measuring using an expression profile image obtained by scanning fluorescence generated from a portion where a first biological material and a second biological material are hybridized when irradiated with light, Storage means for storing conversion information, which is information for which the amount of hybridization between the first biological material and the second biological material in the reaction region is uniquely determined for each intensity of excitation light, and excitation with different intensities When a plurality of expression profile images obtained by light are to be processed, a plurality of expression profiles are obtained based on a plurality of pieces of conversion information of the intensity of each excitation light used for acquiring the expression profile images. Respectively included in the file image is a biological information processing apparatus and a estimating means for estimating a hybridized amount of the corresponding reaction region.

  The estimation means calculates the first hybridization amount in the first reaction region from the fluorescence intensity of the first reaction region represented by the expression profile image obtained by the excitation light having the first intensity. From the fluorescence intensity of the second reaction region corresponding to the first reaction region represented by the expression profile image obtained by the excitation light of the second intensity, and using the conversion information of the excitation light, the second By calculating the second hybridization amount in the reaction region using the conversion information of the excitation light having the second intensity, and weighting the obtained first and second hybridization amounts, the corresponding reaction region is obtained. The amount of hybridization in can be estimated.

  In the present invention, conversion information, which is information in which the amount of hybridization between the first biological material and the second biological material in the reaction region is uniquely determined for each intensity of excitation light, is stored from the fluorescence intensity, and the different intensities are stored. When processing multiple expression profile images obtained by the excitation light of each of the plurality of expression profile images based on multiple conversion information of the intensity of each excitation light used to acquire the expression profile image The amount of hybridization included in the corresponding reaction region is estimated.

  According to the present invention, it is possible to sufficiently ensure the dynamic range of the amount of hybridization that can be estimated.

  Embodiments of the present invention will be described below. The correspondence relationship between the invention described in this specification and the embodiments of the invention is exemplified as follows. This description is intended to assure that embodiments supporting the claimed invention are described in this specification. Therefore, although there is an embodiment which is described in the embodiment of the invention but is not described here as corresponding to the invention, it means that the embodiment is not It does not mean that it does not correspond to the invention. Conversely, even if an embodiment is described herein as corresponding to an invention, that means that the embodiment does not correspond to an invention other than the invention. Absent.

  Further, this description does not mean all the inventions described in this specification. In other words, this description is for the invention described in the present specification and not claimed in this application, i.e., the existence of an invention that will be filed in the future or added by amendment. There is no denial.

  The biological information processing apparatus according to claim 1, wherein a second biological material (for example, a probe) fixed to a reaction region provided on a substrate and a second biological material that hybridizes to the first biological material. The fluorescence generated from the portion where the first biological material and the second biological material are hybridized when irradiated with excitation light is scanned for the state of hybridization with the biological material (for example, target). A biological information processing apparatus for measuring using the obtained expression profile image, wherein the amount of hybridization between the first biological material and the second biological material in the reaction region is determined for each intensity of excitation light based on fluorescence intensity. Storage means (for example, fluorescence intensity-hybridization amount conversion formula storage unit in FIG. 1) that stores conversion information (e.g., fluorescence intensity-hybridization amount conversion formula) that is uniquely determined by 0) and a plurality of the expression profile images obtained with excitation light having different intensities, based on the plurality of conversion information of the intensity of each excitation light used for acquiring the expression profile image. And an estimation means (for example, the creating unit 81 in FIG. 1) for estimating the amount of hybridization in the corresponding reaction region included in each of the plurality of expression profile images.

  The biological information processing method according to claim 3, wherein a second biological material (for example, a probe) fixed to a reaction region provided on a substrate and a second biological material that hybridizes to the first biological material. The fluorescence generated from the portion where the first biological material and the second biological material are hybridized when irradiated with excitation light is scanned for the state of hybridization with the biological material (for example, target). A biological information processing method for measuring using the obtained expression profile image, wherein the amount of hybridization between the first biological material and the second biological material in the reaction region is determined for each intensity of excitation light based on fluorescence intensity. The acquisition step (for example, step S41 in FIG. 8) for acquiring conversion information (for example, fluorescence intensity-hybridization amount conversion formula) that is uniquely obtained from When processing a plurality of expression profile images obtained by light, a plurality of the expression profile images based on a plurality of the conversion information of the intensity of each excitation light used to acquire the expression profile image And an estimation step (for example, step S45 in FIG. 8) for estimating the amount of hybridization in the corresponding reaction region.

  Also in the program according to claim 4 and the program recorded in the recording medium according to claim 5, the embodiment (however, an example) corresponding to each step is the biological information processing method according to claim 3. It is the same.

  Here, the meanings of terms used in this specification will be described.

  A probe refers to a biological substance fixed on a bioassay substrate such as a DNA chip, which reacts biologically with a target.

  The target refers to a biological material that bioreacts with a biological material fixed on a bioassay substrate such as a DNA chip.

  Biological substances include genes generated in vivo such as proteins, nucleic acids, and sugars, as well as genes having mutually complementary base sequences or substances derived therefrom.

  Biological reaction means that two or more biological substances react biochemically. A typical example is hybridization.

  Hybridization refers to a complementary strand (double strand) forming reaction between nucleic acids having a complementary base sequence structure.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 illustrates a configuration example of a biological information processing apparatus according to an embodiment of the present invention. The biological information processing apparatus 1 includes a DNA chip 11, a pickup unit 21, a fluorescence intensity acquisition unit 22, an excitation light intensity calculation unit 23, a hybridization amount estimation unit 24, an expression amount calculation unit 25, a standardization unit 26, an output unit 27, It comprises an expression profile data storage unit 28, a user interface (UI) unit 29 having a display unit 29A, a fluorescence intensity-hybridization amount conversion type storage unit 30, and a mechanical learning unit 31.

  The DNA chip 11 has a spot 12 and a guide 13. FIG. 2 shows a more detailed configuration example of the DNA chip 11.

  The DNA chip 11 has an expression analysis reaction vessel 101 and a cell number counting reaction vessel 102 on the substrate 11A. A linear start position guide 13A is provided at the lower end of the substrate 11A in the figure, and an end position guide 13B is provided at the upper end in the figure. Specifically, the guide 13 shown in FIG. 1 includes a start position guide 13A and an end position guide 13B.

  The expression analysis reaction tank 101 and the cell number counting reaction tank 102 are arranged between the start position guide 13A and the end position guide 13B.

  In the expression analysis reaction tank 101, a plurality of spots 12 as reaction regions are formed, and in each spot 12, a hybridization verification probe 111 as a biological material (first biological material), an expression analysis A probe 112 and an expression standardization control probe 113 are fixed. When a sample is dropped into the expression analysis reaction tank 101, a target 111A as a biological material (second biological material) having a base having a complementary structure to the base is hybridized to the hybridization verification probe 111. To do. Similarly, the target 112A as a biological material (second biological material) having a base having a complementary structure to the base is hybridized to the expression analysis probe 112. In addition, the expression standardization control probe 113 is hybridized with a target 113A as a biological material (second biological material) having a base complementary to the base.

  In the cell number counting reaction tank 102, a hybridization verification probe 114 as a biological material (first biological material) and a cell number counting control probe 115 are respectively attached to a spot 12 as a reaction region. When a sample is dropped into the cell number counting reaction tank 102, a target 114A as a biological material (second biological material) having a base complementary to the base is hybridized to the hybridization verification probe 114. The target 115A as a biological material (second biological material) having a base complementary to the base hybridizes with the control probe 115 for counting the number of cells.

  An intercalator 116 is bonded to a probe and a target as a hybridized (bioreacted) biological material. The intercalator 116 generates fluorescence when irradiated with excitation light.

  FIG. 2 shows a state in which the target is hybridized to each probe as described above. In FIG. 2, for convenience, only one probe is shown for one spot 12, but actually, a plurality of probes of the same type are fixed to one spot 12. Further, in each reaction tank, an arbitrary number of spots to which the same type of probe is fixed are arranged at predetermined positions.

  The pickup unit 21 shown in FIG. 1 includes a fluorescence intensity acquisition pickup 41, a guide signal acquisition pickup 42, a control unit 43, an objective coordinate calculation unit 44, and a convolution expansion unit 45.

  The fluorescence intensity acquisition pickup 41 is a pickup that acquires images of the expression analysis reaction tank 101 and the cell number counting reaction tank 102 of the DNA chip 11 of FIG. On the other hand, the guide signal acquisition pickup 42 is a pickup for reading the start position guide 13A and the end position guide 13B.

  The fluorescence intensity acquisition pickup 41 includes an objective lens 51, a prism 52, a semiconductor laser 53, and a photodiode 54. Laser light (excitation light) emitted from the semiconductor laser 53 is incident on the objective lens 51 via the prism 52, and the objective lens 51 irradiates the incident laser light on the substrate 11A (spot 12). The objective lens 51 also makes the light from the spot 12 incident on the photodiode 54 via the prism 52. A plurality of probes are fixed to each spot 12, and when the probe and the target are hybridized, an intercalator 116 is further coupled to both. That is, when the probe and the target are not hybridized, there is no intercalator 116 between them, and only when they are hybridized, there is an intercalator 116 between them. The intercalator 116 generates fluorescence when irradiated with excitation light. The fluorescence condensed by the objective lens 51 is separated from the excitation light by the prism 52 and is incident on the photodiode 54.

  The greater the amount of hybridization, the greater the amount of intercalator 116, and hence the greater the amount of fluorescence generated therefrom. Therefore, it is possible to measure the state of hybridization (obtain information on hybridization) based on the intensity of fluorescence.

  The control unit 43 controls the current of the semiconductor laser 53 and adjusts the intensity of the excitation light. Further, the control unit 43 reads the output (current amount change) of the photodiode 54.

  The convolution developing unit 45 receives a signal based on the change in the amount of current output from the photodiode 54 from the control unit 43, and generates image data in units of pixels.

  The guide signal acquisition pickup 42 includes an objective lens 61, a prism 62, a semiconductor laser 63, and a photodiode 64. The semiconductor laser 63 generates laser light based on control from the control unit 43 (this laser light functions as guide detection light). The prism 62 makes the laser light from the semiconductor laser 63 incident on the objective lens 61, and the objective lens 61 irradiates the substrate 11A with this laser light. The objective lens 61 receives the reflected light from the substrate 11A, and the prism 62 separates the reflected light from the irradiation light and emits it to the photodiode 64. The photodiode 64 photoelectrically converts the reflected light incident from the prism 62 and outputs it as a guide signal to the control unit 43. The control unit 43 outputs the guide signal input from the photodiode 64 to the objective coordinate calculation unit 44. The guides 13 (the start position guide 13A and the end position guide 13B) are formed so that the reflectance is higher (or lower) than other areas of the substrate 11A. The objective coordinate calculation unit 44 determines the positions of the start position guide 13A and the end position guide 13B and the start position guide 13A based on the level of the guide signal supplied from the guide signal acquisition pickup 42 via the control unit 43. The position (coordinates) of the guide signal acquisition pickup 42 moved at a constant speed toward the end position guide 13B is calculated.

  The control unit 43 controls the position of the fluorescence intensity acquisition pickup 41 (objective lens 51) based on the position of the guide signal acquisition pickup 42 calculated by the objective coordinate calculation unit 44. The guide signal acquisition pickup 42 and the fluorescence intensity acquisition pickup 41 are fixed to each other in a predetermined positional relationship, and the fluorescence intensity acquisition pickup 41 is arranged between the start position guide 13A and the end position guide 13B in FIG. The guide signal acquisition pickup 42 is disposed at a predetermined position between the start position guide 13A and the end position guide 13B.

The fluorescence intensity acquisition unit 22 receives the input of the fluorescence intensity (pf _{x, y} ) from each spot 12 (its coordinates (x, y)) output from the photodiode 54 of the fluorescence intensity acquisition pickup 41, and receives this fluorescence intensity. Is output to the creation unit 81 of the hybridization amount estimation unit 24. The fluorescence intensity acquisition unit 22 also receives control signals for controlling the objective coordinates (x, y), the objective area radius (r), and the excitation light intensity of the objective lens 51 of the fluorescence intensity acquisition pickup 41 on the substrate 11A. Output to 43. The control unit 43 controls the objective lens 51 based on this control signal. Thereby, the objective lens 51 is arranged at predetermined coordinates (x, y) on the substrate 11A, and the radius (objective area radius) (r) of the irradiation range of the laser light emitted from the objective lens 51 becomes a predetermined value. The laser light intensity (excitation light intensity) is adjusted to a predetermined value.

  The fluorescence intensity acquisition unit 22 outputs the fluorescence intensity supplied from the control unit 43 to the excitation light intensity calculation unit 23. The excitation light intensity calculation unit 23 calculates the optimum excitation light intensity based on the fluorescence intensity-hybridization amount conversion equation stored in the fluorescence intensity-hybridization amount conversion equation storage unit 30, and obtains the calculation result. The obtained excitation light intensity is output to the fluorescence intensity acquisition unit 22. The fluorescence intensity acquisition unit 22 controls the current of the semiconductor laser 53 based on the excitation light intensity from the excitation light intensity calculation unit 23 and causes the semiconductor laser 53 to emit excitation light having a predetermined intensity.

  The hybridization amount estimation unit 24 includes a creation unit 81, an image processing unit 82, a verification unit 83, and a hybridization amount calculation unit 84.

  The creation unit 81 creates an expression hybridize (pf) based on the data from the fluorescence intensity acquisition unit 22, and uses the hybridization amount obtained from the created expression hybridize (pf) as an estimated value of the hybridization amount.

  As will be described later, when a plurality of expression profile images obtained using different excitation light intensities have been supplied as images to be processed, the creation unit 81 has the fluorescence intensity-hybridization amount of each excitation light intensity. A conversion equation (an equation representing the relationship between the fluorescence intensity and the hybridization amount at each excitation light intensity) is read from the fluorescence intensity-hybridization amount conversion equation storage unit 30, and the read fluorescence intensity-hybridization amount conversion equation is read out. Based on, the expression hybridize (pf) is created. The amount of hybridization determined from the created expression hybridize (pf) is output to the image processing unit 82 together with the image data supplied from the fluorescence intensity acquisition unit 22.

  The image processing unit 82 processes the image data input from the creation unit 81 and outputs the processed image data to the verification unit 83 and the user interface unit 29. The user interface unit 29 displays the image input from the image processing unit 82 on the display unit 29A. The image processing unit 82 removes components of debris (substance that is an obstacle to observation) from the image of the DNA chip 11 based on an input instructed by the user via the user interface unit 29, and the spot 12 A process of disassembling each image is performed.

  The verification unit 83 verifies that the hybridization is correctly performed based on the amount of hybridization in the spot 12 of the hybridization verification probes 111 and 114 in the image data input from the image processing unit 82.

  The hybridizing amount calculation unit 84 estimates the hybridizing amount in spot units and obtains the reliability by a predetermined method. The amount of hybridization and the reliability are output to the expression level calculator 25.

  The expression level calculation unit 25 estimates the expression level corresponding to the fluorescence intensity by obtaining the binding strength of the target to the probe based on the output from the hybridization level calculation unit 84. The standardization unit 26 performs a standardization process using the expression standardization control probe 113 and the cell number counting control probe 115. The output unit 27 supplies the standardized data to the expression profile data storage unit 28. The expression profile data storage unit 28 stores the data supplied from the output unit 27 as expression profile data. The data stored in the expression profile data storage unit 28 is supplied to the user interface unit 29 as necessary and displayed on the display unit 29A. Data output from the expression level calculation unit 25 is also displayed on the display unit 29A as necessary.

  The fluorescence intensity-hybridization amount conversion formula storage unit 30 is a conversion formula as conversion information for uniquely determining the relationship between the fluorescence intensity and the corresponding hybridization amount (although it is not always necessary to construct a formula, (May be data).

Figure 3 is a fluorescence intensity - is a diagram showing an example of a formula hybridize _e (pf) which defines the hybridized amount conversion equation fluorescence intensity stored in the storage unit 30 and the hybridized amount relational.

  As shown in the figure, when the fluorescence intensity is given, the corresponding hybridization amount is uniquely determined based on the function (curves 121 to 124). However, as shown in the figure, the curve 121 shown on the uppermost side in the figure represents the curve when the level of the excitation light intensity is the weakest. Hereinafter, the lower curve 122, the curve 123, and the excitation are sequentially shown. The light intensity level becomes strong, and the lowermost curve 124 represents the curve when the excitation light intensity level is strongest. That is, the fluorescence intensity-hybridization amount conversion formula storage unit 30 is provided with a fluorescence intensity-hybridization amount conversion formula that can uniquely determine the hybridization amount from the fluorescence intensity for each excitation light intensity.

  Each of the curves 121 to 124 shows a slight change in the fluorescence intensity in the portion 121A to 124A on the left end portion in the drawing and the portion 121B to 124B on the right end portion in the drawing. Thus, since the amount of hybridization has changed significantly, it is difficult to uniquely determine the amount of hybridization corresponding to the fluorescence intensity in these regions.

  Therefore, since the hybridization amount obtained by inputting the fluorescence intensity from these portions 121A to 124A and the portions 121B to 124B can be said to be low in reliability, for example, in the center except for such a low reliability section. Only the solid line section is used for the calculation of the amount of hybridization corresponding to the fluorescence intensity.

  Returning to the description of FIG. 1, the mechanical learning unit 31 includes an SVM (Support Vector Machine) 91 and a spot removal pattern database 92 as means for mechanical learning. In the learning mode, the SVM 91 performs learning based on data from the user interface unit 29 and the expression profile data storage unit 28, and stores the learning result in the spot removal pattern database 92. In the determination mode, the SVM 91 also determines the data from the expression profile data storage unit 28 based on the pattern stored in the spot removal pattern database 92 and outputs the determination result to the hybridization amount calculation unit 84.

  The quantitative measurement of the gene expression level is performed by the experimental process processor 131 shown in FIG. The biological information processing apparatus 1 in FIG. 1 constitutes a part of the experimental process processing apparatus 131 in FIG.

  That is, the experimental process processing device 131 includes an adjustment unit 141, a hybridization unit 142, an acquisition unit 143, an expression level estimation unit 144, a standardization unit 145, an output unit 146, and a storage unit 147. Among these, the acquisition unit 143, the expression level estimation unit 144, the standardization unit 145, the output unit 146, and the storage unit 147 are configured by the biological information processing apparatus 1. Specifically, the acquisition unit 143 includes a pickup unit 21, a fluorescence intensity acquisition unit 22, an excitation light intensity calculation unit 23, and a fluorescence intensity-hybridization amount conversion expression storage unit 30, and the expression level estimation unit 144 includes: The hybridizing amount estimating unit 24, the expression amount calculating unit 25, and the mechanical learning unit 31 are configured, the standardizing unit 145 is configured by the standardizing unit 26, the output unit 146 is configured by the output unit 27, and the storage unit 147 is expressed. The profile data storage unit 28 is used.

  The adjustment unit 141 adjusts the target. The hybridizing unit 142 performs hybridization between the probe and the target. The acquisition unit 143 acquires the fluorescence intensity. The expression level estimation unit 144 performs expression level estimation processing. The standardization unit 145 standardizes data. The output unit 146 outputs expression profile data. The storage unit 147 stores expression profile data.

  Next, the process of the experimental process processor 131 of FIG. 4 will be described with reference to the flowchart of FIG.

  First, in step S11, the adjustment unit 141 adjusts the target. Specifically, a sample containing cells is taken out, the protein is denatured and removed from the sample, RNA (ribonucleic acid) extraction, fragmentation, DNA (deoxyribonucleic acid) extraction, fragment The target (target 112A for the expression analysis probe 112) is generated.

  In step S12, the hybridizing unit 142 executes a hybridizing process. Specifically, in the solution containing the target generated in step S11, the targets 111A and 114A for the hybridization verification probes 111 and 114, the target 113A for the expression standardization control probe 113, and the cell count counter A target 115A for the control probe 115 is added, and this solution is dropped into the expression analysis reaction tank 101 and the cell number counting reaction tank 102, whereby the target and the probe are hybridized. Then, the intercalator 116 is introduced and combined with the hybridized target and probe to obtain the DNA chip 11 as shown in FIG. As shown in the figure, in the spot 12 of the expression analysis reaction tank 101, the target 112A is hybridized to the expression analysis probe 112, and the target 113A is hybridized to the expression standardization control probe 113. The target 111A is hybridized with the probe 111 for hybridization verification. And the intercalator 116 has couple | bonded between the probe and target which couple | bonded those double strands.

  Similarly, also in the spot 12 of the cell number counting reaction tank 102, the target 114A is hybridized to the hybridization verification probe 114, and the target 115A is hybridized to the cell number counting control probe 115. ing. An intercalator 116 is also coupled between these hybridized probe and target.

  In step S13, the acquisition unit 143 acquires the fluorescence intensity. In this process, the fluorescence intensity acquisition unit 22 constituting the acquisition unit 143 drives the fluorescence intensity acquisition pickup 41 via the control unit 43 and causes the semiconductor laser 53 to emit laser light as excitation light. The excitation light is incident on the objective lens 51 via the prism 52, and the objective lens 51 irradiates the expression analysis reaction tank 101 on the substrate 11A.

  The intercalator 116 generates fluorescence when irradiated with excitation light. This fluorescence is collected by the objective lens 51 and is incident on the photodiode 54 via the prism 52. The photodiode 54 outputs a current corresponding to the fluorescence. The control unit 43 causes the convolutional expansion unit 45 to convert a signal corresponding to the current into an image signal, and outputs a signal corresponding to the fluorescence intensity generated by the conversion to the fluorescence intensity acquisition unit 22.

  The control unit 43 moves the position of the objective lens 51 from the start position guide 13A toward the end position guide 13B. At this time, laser light as guide detection light emitted from the semiconductor laser 63 of the guide signal acquisition pickup 42 is incident on the objective lens 61 via the prism 62, and the objective lens 61 irradiates the substrate 11A with this guide detection light. To do. The intensity of the reflected light of the guide detection light increases when it is applied to the start position guide 13A and the end position guide 13B. This reflected light is incident on the prism 62 via the objective lens 61, and is incident on the photodiode 64 from the prism 62. The objective coordinate calculation unit 44 acquires a guide signal from the photodiode 64 via the control unit 43, and on the basis of this signal, the guide signal acquisition pickup 42 (therefore, the fluorescence intensity acquisition pickup 41 integrated therewith). Is located between the start position guide 13A and the end position guide 13B of the substrate 11A. Based on the coordinates, the control unit 43 moves (scans) the guide signal acquisition pickup 42 (fluorescence intensity acquisition pickup 41) from the start position guide 13A to the end position guide 13B at a constant speed.

  In this way, the fluorescence intensity acquisition pickup 41 is moved from the start position guide 13A to the end position guide 13B in FIG. 2, and the scanning position is further changed to the start position guide 13A (end position guide 13B). ) In the direction parallel to () in the figure (x-coordinate direction) by one pitch, and similarly, from the start position guide 13A to the end position guide 13B at the new movement position. In this manner, the entire expression analysis reaction tank 101 and cell number counting reaction tank 102 are scanned, and image signals at respective coordinates are output from the fluorescence intensity acquisition pickup 41.

  FIG. 6 is a diagram illustrating an example of a format of image data obtained by the fluorescence intensity acquisition unit 22.

  The image data consists of the excitation light intensity at the time of scanning, the index that specifies the probe gene sequence, the number of pixels in the vertical and horizontal directions of the image of the entire spot, and the fluorescence image (image taken by irradiating excitation light) for each spot. Is done. Image data having such a format is output from the fluorescence intensity acquisition unit 22 to the hybridization amount estimation unit 24, and various processes are performed based thereon. For example, a plurality of pieces of image data having the format of FIG. 6 acquired by scanning the same DNA chip 11 using excitation light having different intensities are output to the hybridization amount estimation unit 24.

A plurality of image data obtained by scanning the DNA chip 11 (DNA chip 11 in which the same amount of probes of the same type is fixed to each spot 12 by the same amount) using different excitation lights. When comparing the data of the corresponding spots (when there are a plurality of image data as shown in FIG. 6, for example, when the data of the spot S ₁ which is the same spot is compared), the excitation light intensity and Only data representing the fluorescence image is different data. As described above, the difference in the fluorescence intensity appearing in the image data is caused by the difference in the excitation light intensity, and the hybridization actually occurring in the corresponding spots 12 between the DNA chips 11 obtained in the same process. The amount of is the same.

  In step S14, the expression level estimation unit 144 executes an expression level estimation process. The expression level estimation process will be described later with reference to the flowchart of FIG. 7, and the hybridization level and reliability are calculated by this process, and the expression level is calculated.

  In step S15, the standardization unit 145 (standardization unit 26) performs processing for standardizing data. As the standardization, standardization by the expression standardization control probe 113 and standardization by the cell number counting control probe 115 are performed. Standardization by the expression standardization control probe 113 is performed as follows.

  That is, in FIG. 2, the expression standardization control probe 113 is shown only in one place, but actually, this expression standardization control probe 113 is a predetermined predetermined in the expression analysis reaction tank 101. It is distributed and arranged at a plurality of positions (for example, four corners of the expression analysis reaction tank 101 and approximately five at the center). Then, based on the fluorescence value of the expression standardization control probe 113 arranged at each position, a correction curved surface is calculated based on, for example, a B-spline curved surface, and each pixel is calculated based on the fluorescence value obtained by the correction curved surface. Normalization is performed by dividing the fluorescence value. By this normalization, variation in hybridization due to the position of the spot 12 in the reaction tank 101 for expression analysis is corrected.

  Further, the standardization by the cell number counting control probe 115 is performed based on the value of the amount of hybridization to the cell number counting control probe 115 (fluorescence value based on the cell number counting control 115). This is done by dividing the fluorescence value of the pixel on the spot 12. As the control probe 115 for calculating the number of cells, a repetitive sequence (for example, Alu sequence for humans) in the genome of the living body from which the expression analysis probe 112 is extracted is used. By this processing, the expression level of the acquired gene can be converted into a value per certain number of cells.

  Furthermore, in step S16, the output unit 146 (output unit 27) outputs the expression profile data. Specifically, the image data obtained as described above is supplied to the storage unit 147 (expression profile data storage unit 28) and recorded.

  Next, the expression level estimation process performed in step S14 of FIG. 5 will be described with reference to the flowchart of FIG.

  In step S31, the creation unit 81 inputs image information (image data). Specifically, image information is input from the fluorescence intensity acquisition unit 22.

  In step S <b> 32, the creation unit 81 determines whether the input image information is image information of an image captured with a plurality of excitation light intensities. If it is image information of an image photographed with a plurality of excitation light intensities, the process proceeds to step S33, where the creating unit 81 creates an expression hybridize (pf) based on the images photographed with a plurality of excitation light intensities and hybridizes. The amount of soybean is estimated, and the obtained estimated value is output to the image processing unit 82 together with the image information. The process performed in step S33 will be described later with reference to the flowchart of FIG.

  If it is determined in step S32 that the input image data is not image data of an image captured with a plurality of excitation light intensities, the process of step S33 cannot be executed and is skipped.

  In step S34, the image processing unit 83 performs image processing. By this process, a debris region straddling the spot boundary is removed from the image of the DNA chip 11, and the image is decomposed into an image for each spot.

  In step S35, the verification unit 83 executes a process for verifying hybridization. Specifically, as shown in FIG. 2, a hybridization verification probe 111 is provided in the expression analysis reaction tank 101, and a hybridization verification probe 114 is provided in the cell number counting reaction tank 102, respectively. It is fixed to. As the hybridization verification probes 111 and 114, gene sequences that do not exist in the biological species to be experimented are used. For example, when the experiment target is an animal (when the expression analysis probe 112 is an animal gene), a plant chlorophyll gene is used as the hybridization verification probes 111 and 114, and the targets 111A and 114A are as follows: Its complementary sequence is used.

  That is, as the hybridization verification probes 111 and 114 and the targets 111A and 114A, those that reliably cause hybridization regardless of the hybridization of the expression analysis probe 112 and the target 112A are used. In addition, since a completely different species from that of the experiment target is used, when the hybridization verification probes 111 and 114 are sufficiently hybridized, in this experiment (in the measurement), the hybridization surely occurred. Can be verified. On the contrary, when the hybridization verification probes 111 and 114 are not sufficiently hybridized, there is a possibility that this measurement is an environment in which hybridization is difficult to occur for some reason. Therefore, by measuring the fluorescence value of the hybridization verification probes 111 and 114, if the fluorescence value is equal to or higher than a preset reference value, it is verified that correct hybridization processing is being performed. be able to.

  In step S <b> 36, the hybridization amount calculation unit 84 calculates the hybridization amount and reliability, and outputs the obtained hybridization amount and reliability to the expression amount calculation unit 25. In addition, for example, when the hybridization amount is estimated in step S33, the hybridization amount calculation unit 84 calculates the reliability of the estimated value, and uses the obtained reliability as the estimated value of the hybridization amount. At the same time, it is output to the expression level calculator 25.

  In step S <b> 37, the expression level calculation unit 25 executes a process of calculating the expression level based on the hybridization level and the reliability supplied from the hybridization level calculation unit 84. Based on this processing, the expression level corresponding to the calculated (acquired) fluorescence value is calculated.

  Next, the process of creating the expression hybridize (pf) performed in step S33 of FIG. 7 will be described with reference to the flowchart of FIG.

  When a plurality of image data (data of a plurality of expression profile images) obtained with different excitation light intensities have been supplied, in step S41, the creating unit 81 converts the fluorescence intensity-hybridization amount of these excitation light intensities. The equation is read from the fluorescence intensity-hybridization amount conversion equation storage unit 30.

In this example, it is assumed that image data obtained with two different excitation light intensities is supplied to the creation unit 81 and is a processing target. Also, as in the curve indicated by a solid line in FIG. 9, the formula hybridize _s (pf) the relationship of the fluorescence intensity of hybridizing amount is represented by the curve 124, the relationship between fluorescence intensity and hybridizing amount is represented by curve 122 It is assumed that hybridize _w (pf) is read from the fluorescence intensity-hybridization amount conversion expression storage unit 30.

As shown in FIG. 9, by using the formula hybrid _s (pf), the hybridize amount hybrid _s (pf _s ) is uniquely determined from the fluorescence intensity pf _s , and the formula hybrid _w (pf) is used. Thus, the hybridizing amount hybrid _w (pf _w ) is uniquely determined from the fluorescence intensity pf _w . The fluorescence intensities pf _s and pf _w are the fluorescence intensities of a corresponding spot 12 represented by the image data to be processed.

Since the amount of hybridization occurring in the corresponding spot 12 is the same, the hybridizing amount hybridize _s (pf _s ) and the hybridizing amount hybridize _w (pf _w ) are supposed to coincide with each other. 9 may be measured as different amounts as shown in FIG. 9. In this case, in the subsequent processing, the two hybridizing amounts hybridize _s (pf _s ) and hybridize _w (pf _w ) are combined (weighted). The amount of hybridization that is considered to have actually occurred is estimated.

Returning to the description of FIG. 8, in step S42, the creation unit 81 calculates the fluorescence intensities upper _s and lower _s which are the fluorescence intensities at the boundary point with the saturation region set in the expression hybrid _s (pf) (curve 124). Set. In addition, the creation unit 81 sets fluorescence intensity upper _w and lower _w , which are the fluorescence intensity at the boundary point with the saturated region set in the expression hybrid _w (pf) (curve 122).

As described above, regions (saturation regions) where the hybridization amount cannot be correctly determined from the fluorescence intensity are set at both ends of the curve representing the fluorescence intensity-hybridization amount conversion formula. FIG. 10 shows the fluorescence intensity upper _s and lower _s and the fluorescence intensity upper _w and lower _w set here.

In step S43, the creation unit 81 selects the weight W _s from the positional relationship between the fluorescence intensities upper _s and lower _s and the fluorescence intensity pf _s set in step S42. In step S44, the creation unit 81 selects the weight W _w from the positional relationship between the fluorescence intensity upper _w and lower _w and the fluorescence intensity pf _w set in step S42.

For example, the weights W _s and W _w are obtained by the following expressions (1) and (2), respectively.

That is, as shown in equation (1), when the fluorescence intensity pf _s is less than the fluorescence intensity lower _s, the weight W _s is 1, the fluorescence intensity pf _s fluorescence intensity lower _s or more, less than the fluorescence intensity upper _s , The weight W _s is (upper _s −pf _s ) / (upper _s −lower _s ). Further, when the fluorescence intensity pf _s is equal to or greater than the fluorescence intensity upper _s , the weight W _s is set to zero.

Similarly, as shown in Equation (2), when the fluorescence intensity pf _w is less than the fluorescence intensity lower _w , the weight W _w is set to 0, the fluorescence intensity pf _w is greater than or equal to the fluorescence intensity lower _w , and the fluorescence intensity upper _w If it is less, the weight W _w is (pf _w −lower _w ) / (upper _w −lower _w ). Further, when the fluorescence intensity pf _w is greater than or equal to the fluorescence intensity upper _w , the weight W _w is set to 1.

In step S45, the creation unit 81 creates an expression hybridize (pf) by substituting the weights W _s and W _w selected in steps S43 and S44 into the following expression (3), respectively. Further, the creating unit 81 uses the value (hybridize (pf)) obtained by the substitution as an estimated value of the hybridization amount obtained from the two image data.

That is, as shown in FIG. 11, the weight W _s acts on the hybridizing amount hybridize _s (pf _s ) and the weight W _w acts on the hybridizing amount hybrid _w (pf _w ), respectively, and the hybridizing amount hybridize (pf) becomes Desired. By obtaining the weights W _s and W _w from the equations (1) and (2), the hybridized amount estimated by the action of the weights W _s and W _w is the hybridized amount hybrid _s (pf _s ). And hybridize _w (pf _w ). After the estimated value of the hybridization amount is obtained, the process returns to step S33 in FIG. 7 and the subsequent processes are performed.

  By estimating the hybridizing amount of each spot 12 from the image data photographed with a plurality of excitation light intensities as described above, a sufficient dynamic range of the hybridizing amount that can be estimated is ensured. It becomes possible.

That is, by using only the hybridize _s (pf) equation in FIG. 10, the range in which the hybridizing amount and the reliable hybridizing amount can be measured is from the hybridizing amount hybridize _s (lower _s ) to hybridize _s (upper _s ). Similarly, the range in which a reliable hybridizing amount can be measured by using only the formula hybrid _w (pf) is the hybridizing amount hybridize _w (lower _w ) to hybridize _w (upper _In the range up to _w ), by synthesizing the hybridizing amount obtained from these two equations, the range from hybridizing amount hybrids _s (lower _s ) to hybridize _w (upper _w ) can be trusted. It can be ensured as a range in which the soybean amount can be estimated.

Further, the weights W _s and W _w are selected according to the expressions (1) and (2) and are applied to prevent the estimated value of the amount of hybridization to be a value in the saturation region. And the reliability of the estimated value can be improved.

  In the above description, the case where two hybridized amounts are synthesized has been described. Naturally, a predetermined number of hybridized amounts of two or more are synthesized to obtain an estimated value of one hybridized amount. May be.

  As described above, the embodiment in the case of measuring the hybridization of the DNA chip has been described. However, the present invention is not limited to the DNA chip. In the case of measuring whether various biological materials are biologically bound to other predetermined biological materials. It is possible to apply to.

  The series of processes described above can be executed by hardware or can be executed by software. In this case, for example, the biological information processing apparatus 1 is configured by a personal computer as shown in FIG.

  In FIG. 12, a CPU (Central Processing Unit) 201 executes various processes according to a program stored in a ROM (Read Only Memory) 202 or a program loaded from a storage unit 208 to a RAM (Random Access Memory) 203. To do. The RAM 203 also appropriately stores data necessary for the CPU 201 to execute various processes.

  The CPU 201, the ROM 202, and the RAM 203 are connected to each other via the bus 204. An input / output interface 205 is also connected to the bus 204.

  The input / output interface 205 includes an input unit 206 including a keyboard and a mouse, a display including a CRT (Cathode Ray Tube) and an LCD (Liquid Crystal display), an output unit 207 including a speaker, and a hard disk. A communication unit 209 including a storage unit 208 and a modem is connected. The communication unit 209 performs communication processing via a network including the Internet.

  A drive 210 is connected to the input / output interface 205 as necessary, and a removable medium 211 such as a magnetic disk, an optical disk, a magneto-optical disk, or a semiconductor memory is appropriately attached, and a computer program read from them is It is installed in the storage unit 208 as necessary.

  When a series of processing is executed by software, a program constituting the software executes various functions by installing a computer incorporated in dedicated hardware or various programs. For example, a general-purpose personal computer is installed from a network or a recording medium.

  As shown in FIG. 12, the recording medium is distributed to provide a program to the user separately from the apparatus main body, and includes a magnetic disk (including a floppy disk) on which the program is recorded, an optical disk (CD- Not only is it composed of removable media 211 consisting of ROM (compact disk-read only memory), DVD (digital versatile disk), magneto-optical disk (including MD (mini-disk)), or semiconductor memory. The program is configured by a ROM 202 in which a program is recorded and a hard disk included in the storage unit 208 provided to the user in a state of being incorporated in the apparatus main body in advance.

  In the present specification, the step of describing the program recorded on the recording medium is not limited to the processing performed in chronological order according to the described order, but is not necessarily performed in chronological order. It also includes processes that are executed individually.

It is a block diagram showing the example of a structure of the biometric information processing apparatus as embodiment of this invention. It is a perspective view showing the structural example of a DNA chip. It is a figure showing the relationship between fluorescence intensity and the amount of hybridization. It is a block diagram showing the structural example of an experimental process processing apparatus. It is a flowchart explaining the process of an experiment process. It is a figure which shows the example of the format of image data. It is a flowchart explaining an expression level estimation process. It is a flowchart explaining the creation process of expression hybridize (pf). It is a figure which shows the example of a fluorescence intensity-hybridization amount conversion type | formula. It is a figure which shows the example of fluorescence intensity upper _s and lower _s , and fluorescence intensity upper _w and lower _w . It is a figure which shows the example of weighting. And FIG. 11 is a block diagram illustrating a configuration example of a personal computer.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 Biological information processing apparatus, 11 DNA chip, 21 Pickup part, 22 Fluorescence intensity acquisition part, 23 Excitation light intensity calculation part, 24 Hybridization amount estimation part, 25 Expression amount calculation part, 28 Expression profile data storage part, 29 User interface , 30 fluorescence intensity-hybridization amount conversion type storage unit, 81 creation unit, 82 image processing unit, 83 verification unit, 84 hybridization amount calculation unit

Claims (5)

When the excitation light is applied to the hybridized state between the first biological material fixed in the reaction region provided on the substrate and the second biological material that hybridizes to the first biological material. In a biological information processing apparatus for measuring using an expression profile image obtained by scanning fluorescence generated from a portion where the first biological material and the second biological material are hybridized to each other,
Storage means for storing conversion information, which is information that is uniquely determined for each intensity of excitation light, from the fluorescence intensity, the amount of hybridization between the first biological material and the second biological material in the reaction region;
When processing a plurality of the expression profile images obtained by excitation light having different intensities, based on a plurality of the conversion information of the respective excitation light intensities used for acquiring the expression profile image, A biological information processing apparatus comprising: estimation means for estimating the amount of hybridization in a corresponding reaction region included in each of the expression profile images. The estimation means calculates the first hybridization amount in the first reaction region from the fluorescence intensity of the first reaction region represented by the expression profile image obtained by the excitation light having the first intensity. A second reaction region corresponding to the first reaction region represented by the expression profile image obtained by the excitation light having the second intensity and obtained using the conversion information of the excitation light having the first intensity. From the fluorescence intensity, the second hybridization amount in the second reaction region is determined using the conversion information of the excitation light having the second intensity, and the obtained first and second hybridization amounts are calculated. The biological information processing apparatus according to claim 1, wherein the amount of hybridization in the corresponding reaction region is estimated by weighting the information. When the excitation light is applied to the hybridized state between the first biological material fixed in the reaction region provided on the substrate and the second biological material that hybridizes to the first biological material. In a biological information processing method for measuring using an expression profile image obtained by scanning fluorescence generated from a portion where the first biological material and the second biological material are hybridized to each other,
An acquisition step of acquiring conversion information, which is information that is uniquely determined for each intensity of excitation light, from the fluorescence intensity, the amount of hybridization between the first biological material and the second biological material in the reaction region;
When processing a plurality of the expression profile images obtained by excitation light having different intensities, based on a plurality of the conversion information of the respective excitation light intensities used for acquiring the expression profile image, A biological information processing method, comprising: an estimation step of estimating the amount of hybridization in the corresponding reaction region included in each of the expression profile images. When the excitation light is applied to the hybridized state between the first biological material fixed in the reaction region provided on the substrate and the second biological material that hybridizes to the first biological material. In a program for causing a computer to execute a process of measuring using an expression profile image obtained by scanning fluorescence generated from a portion where the first biological material and the second biological material are hybridized,
An acquisition step of acquiring conversion information, which is information that is uniquely determined for each intensity of excitation light, from the fluorescence intensity, the amount of hybridization between the first biological material and the second biological material in the reaction region;
When processing a plurality of the expression profile images obtained by excitation light having different intensities, based on a plurality of the conversion information of the respective excitation light intensities used for acquiring the expression profile image, An estimation step of estimating the amount of hybridization in the corresponding reaction region, each included in the expression profile image.   A recording medium on which the program according to claim 4 is recorded.

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