JPH0259953B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a signal processing method in autoradiography.

Autoradiography is already known as a method for obtaining positional information of radiolabeled substances that are distributed in at least one dimension to form a distribution array on a support medium.

For example, a radioactive label is added to a biologically derived polymeric substance such as a protein or a nucleic acid, and the radiolabeled polymeric substance, its derivative, or its decomposition product is subjected to a separation operation such as gel electrophoresis to form a gel-like support. The position of the radiolabeled substance on the gel-like support medium obtained from the exposed position by exposing the film to light by overlapping the gel-like support medium and a high-sensitivity X-ray film for a certain period of time. Based on this information, methods for separating and identifying the polymeric substances, or evaluating the molecular weight and characteristics of the polymeric substances have also been developed and are in actual use.

The above-mentioned autoradiography has the great advantage of being able to visually observe the positional information of radiolabeled substances on a cellular or molecular basis by using conventional radiography. ing. However, in other words, in autoradiography using conventional radiography, in order to obtain positional information of a radiolabeled substance, it is necessary to visualize an autoradiograph containing this positional information on a radiographic film. It has become a mandatory requirement.

Therefore, by judging the visualized autoradiograph with their own eyes, researchers can measure the distribution of radiolabeled substances in the sample and obtain location information about specific radiolabeled substances. Gaining knowledge.

Furthermore, by performing various analyzes based on the position information of the radiolabeled substance obtained as described above, it is possible to separate and identify the radiolabeled substance, or evaluate the molecular weight and characteristics of a specific substance. is also being carried out. For example, the above-mentioned autoradiography has been effectively used to determine the base sequence of nucleic acids such as DNA, especially in recent years, and is therefore an extremely useful means for determining the structure of polymeric substances derived from living organisms. However, the determination of the structure of such materials is also done through human visual judgment.

In other words, autoradiography is a useful method for elucidating the structure, function, etc. of living body tissues and/or living body-derived substances, but the analysis of the obtained autoradiographs usually involves This is done through human vision, which requires a great deal of time and effort.

Furthermore, since it relies on the human eye, there are limits to the accuracy of the information obtained, such as the positional information obtained by analyzing the autoradiograph differing depending on the researcher. In particular, the amount of sample is small, the radiation energy emitted from the radiolabeled substance is weak,
If the autoradiograph visualized on radiographic film does not have good image quality (sharpness, contrast), for example because suitable exposure conditions cannot be obtained, it is difficult to obtain satisfactory information. , there is also the problem that the accuracy tends to decrease.

Conventionally, in order to improve the accuracy of positional information, for example, the visualized autoradiograph has been measured using a measuring instrument such as a scanning densitometer. However, this increases the time required to analyze the autoradiograph and complicates the operation.

The present inventor has developed a system that uses a radiation image conversion method that uses a stimulable phosphor sheet instead of the radiographic method that uses a radiation film used in conventional autoradiography to provide information on the position of a radiolabeled substance. By obtaining the positional information of the autoradiograph as a digital signal without converting it into an image, and by performing suitable signal processing on the obtained digital signal, one-dimensional positional information of the radiolabeled substance can be expressed as a symbol and/or The present invention has been achieved by realizing displaying numerical values.

That is, the present invention allows the stimulable phosphor sheet to absorb radiation energy emitted from a radiolabeled substance distributed in at least one dimension on the support medium. After accumulating and recording an autoradiograph having positional information of the radiolabeled substance, the stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is photoelectrically transmitted. signal processing for a digital signal corresponding to the autoradiograph obtained by reading out the autoradiograph, including: ) determining a one-dimensional scanning direction for signal processing; ) detecting sampling points on the scanning direction. This method comprises a signal processing method in autoradiography, characterized in that one-dimensional positional information of a radiolabeled substance on the autoradiograph is obtained in the form of symbols and/or numerical values.

Here, the radiation image conversion method that replaces the conventional radiography method is, for example,
As described in the above publication, a stimulable phosphor sheet containing a photostimulable phosphor is used, and the radiation energy transmitted through or emitted from the subject is absorbed by the stimulable phosphor sheet. The stimulable phosphor is absorbed by the stimulable phosphor, and then exposed to electromagnetic waves (excitation light) such as visible light and infrared rays.
By scanning with a stimulable phosphor, the radiation energy stored in the stimulable phosphor is released as fluorescence (stimulated luminescence), this fluorescence is read photoelectrically to obtain an electrical signal, and this electrical signal is converted into a visible image. It can be reproduced or A/D converted to obtain a digital signal.

Further, the stimulable phosphor sheet contains, for example, a stimulable phosphor such as a divalent europium-activated alkaline earth metal fluorohalide phosphor. This stimulable phosphor can be used for X-rays, α-rays, β-rays,
After being irradiated with radiation such as rays, gamma rays, and ultraviolet rays and accumulating a portion of the radiation energy, when irradiated with electromagnetic waves (excitation light) such as visible light and infrared rays, photostimulated luminescence occurs depending on the accumulated energy. It has the property of indicating.

That is, the present invention utilizes the radiation image conversion method described above in autoradiography to obtain positional information of a radiolabeled substance distributed in a one-dimensional direction on a support medium, particularly through imaging. It is obtained directly as a digital signal.

The radiographic image conversion method has the very practical advantage that radiographic images can be recorded over an extremely wide radiation exposure latitude compared to conventional radiographic methods. In other words, in the stimulable phosphor used in this method, the amount of stimulable light emitted by irradiation with excitation light after accumulating the radiation energy is proportional to the amount of radiation exposure over an extremely wide range. It is permitted to do so. Therefore, according to the above method, a highly accurate digital signal corresponding to the autoradiograph of the subject can be directly obtained.

In addition, by setting the reading gain to an appropriate value at the stage of photoelectrically reading the photostimulated light that contains the autoradiograph of the test object and obtaining it as an electrical signal, it is possible to control changes in the exposure amount depending on the conditions of the test object, Even if the level of radiation energy accumulated in the stimulable phosphor sheet varies due to factors such as variations in the sensitivity of the phosphor and the sensitivity of the photodetector, a digital signal that is not affected by variations in these factors can be obtained. be able to. Furthermore, it becomes possible to reduce the amount of radiation emitted from a subject compared to the conventional method, thereby making it possible to reduce the amount of radiolabeled substances in samples that are harmful to the health of researchers.

Furthermore, by passing the digital signal having the positional information of the radiolabeled substance obtained by the radiation image conversion method using the above-mentioned stimulable phosphor sheet through an appropriate signal processing circuit having a signal processing function, the radiolabeled substance can be detected. position information can be automatically displayed as desired symbols and/or numerical values without relying on human vision. Then, in the signal processing circuit, the positional information displayed as symbols and/or numerical values is further subjected to appropriate arithmetic processing and other related information, thereby obtaining desired information, such as material information, without human intervention. It is possible to obtain information regarding the structure, functions, etc. of

Therefore, the present invention automates the analysis of autoradiographs by digital signal processing of digital signals corresponding to autoradiographs having positional information of radiolabeled substances, thereby saving time and labor required in conventional methods. This makes it possible to significantly shorten the time. It also makes it possible to obtain highly accurate position information.

In addition, in the radiation image conversion method described above, based on the amount of stimulated light emitted from the stimulable phosphor sheet, information on the one-dimensional position of the radiolabeled substance and the relative position of the radiolabeled substance at each position are obtained. Information on quantity can also be obtained.
That is, it becomes possible to obtain quantitative information such as the concentration distribution of a radiolabeled substance without using a separate measurement means for the visualized autoradiograph as in conventional autoradiography.

Therefore, in the present invention, "position information" refers to various information centered on the position of a radioactively labeled substance or an aggregate thereof in a sample, for example, the position of an aggregate of radioactive substances present in a support medium. Refers to various types of information obtained as one or any combination of information such as shape, concentration of radioactive substances at that location, distribution, etc.

Examples of samples used in the present invention include:
Examples include a support medium in which a radiolabeled substance is separated and developed in one dimension to form a separation and development row. Examples of radioactively labeled substances include biopolymer substances to which radioactive labels have been added, derivatives thereof, and decomposition products thereof.

For example, in the case where the biopolymer substances to which a radioactive label has been given are macromolecular substances such as proteins, nucleic acids, derivatives thereof, and decomposition products thereof, the present invention provides for separation of these biopolymer substances. , useful for identification, etc. Furthermore, the present invention can be effectively used to analyze the entire or partial molecular weight, molecular structure, or basic unit configuration of these biopolymer substances.

In addition, methods for separating and developing radiolabeled substances using support media include, for example, gel-like support media (any shape such as layered or columnar), polymer moldings such as acetate, or various types of supports such as filter paper. Representative methods include electrophoresis using a medium and thin layer chromatography using a support medium such as silica gel, but the separation and development method is not limited to these methods.

However, the sample that can be used in the present invention is not limited to the above sample, but is a support medium containing a radiolabeled substance distributed in at least one dimension, and a stimulable phosphor sheet. Any device may be used as long as it can store and record an autoradiograph having positional information of the radiolabeled substance.

The stimulable phosphor sheet used in the present invention has a base structure including a support, a phosphor layer, and a transparent protective film. The phosphor layer is made of a binder containing and supporting the stimulable phosphor in a dispersed state,
For example, it can be obtained by dispersing divalent europium-activated barium fluoride bromide (BaFBr: Eu ²⁺ ) phosphor particles in a mixture of nitrocellulose and linear polyester. A stimulable phosphor sheet is, for example, one in which a sheet of polyethylene terephthalate or the like is used as a support, the above-mentioned phosphor layer is provided on this sheet, and a polyethylene terephthalate sheet or the like is further provided as a protective film on the phosphor layer. .

In the present invention, the operation (exposure operation) of transferring and accumulating radiation energy emitted from a support medium containing a radiolabeled substance onto a stimulable phosphor sheet is performed as follows:
By overlapping the support medium and the stimulable phosphor sheet for a certain period of time, at least a portion of the radiation emitted from the radiolabeled substance on the support medium is absorbed by the stimulable phosphor sheet. This exposure operation can be carried out by placing the support medium and the stimulable phosphor sheet in close proximity to each other, for example, by keeping them in this state for at least several seconds at room temperature or low temperature.

For details of the stimulable phosphor sheet and the exposure operation, please refer to the patent application No. 57-193418 filed by the present applicant.
No. 59-83057).

Next, in the present invention, a method for reading out one-dimensional positional information of a radiolabeled substance on a support medium transferred and accumulated on a stimulable phosphor sheet and converting it into a digital signal will be described in FIG. 1 of the attached drawings. This will be briefly described with reference to an example of the reading device (or reading device) shown in FIG.

Figure 1 shows a pre-reading system for temporarily reading out one-dimensional positional information of a radiolabeled substance accumulated and recorded on a stimulable phosphor sheet (hereinafter sometimes abbreviated as phosphor sheet) 1. Part 2
1 shows a schematic diagram of an example of a reading device comprising a main reading reading section 3 having a function of reading out an autoradiograph stored and recorded on a phosphor sheet 1 in order to output positional information of a radiolabeled substance. There is.

In the prefetch reading unit 2, the following prefetch operation is performed.

The laser light 5 generated from the laser light source 4 passes through the filter 6, whereby a portion of the wavelength range corresponding to the wavelength range of stimulated luminescence generated from the phosphor sheet 1 in response to excitation by the laser light 5 is cut out. Ru. Next, the laser beam is deflected by an optical deflector 7 such as a galvano mirror, reflected by a flat reflecting mirror 8, and then reflected by a phosphor sheet 1.
It is deflected one-dimensionally and incident on the top. The laser light source 4 used here is selected so that the wavelength range of its laser light 5 does not overlap with the main wavelength range of stimulated luminescence emitted from the phosphor sheet 1.

The phosphor sheet 1 is transported in the direction of the arrow 9 under irradiation with the above-mentioned polarized laser light. Therefore, the entire surface of the phosphor sheet 1 is irradiated with the polarized laser light. Note that regarding the output of the laser light source 4, the beam diameter of the laser light 5, the scanning speed of the laser light 5, and the transport speed of the phosphor sheet 1, the energy of the laser light 5 for the pre-reading operation is smaller than the energy used for the main reading operation. It will be adjusted so that

When the phosphor sheet 1 is irradiated with the laser light as described above, it exhibits stimulated luminescence with an amount of light proportional to the accumulated and recorded radiation energy, and this light enters the light guide sheet 10 for prereading. The light guide sheet 10 has a linear incident surface and is placed close to the scanning line on the phosphor sheet 1 so as to face it, and its exit surface forms an annular ring to allow light such as a photoprint to pass through. It communicates with the light receiving surface of the detector 11. The light guide sheet 10 is made by processing a transparent thermoplastic resin sheet such as acrylic synthetic resin, and allows light incident from the incident surface to be transmitted to the exit surface while being totally reflected inside. It is configured to The stimulated luminescence from the phosphor sheet 1 is guided through the light guide sheet 10 and reaches the exit surface, is emitted from the exit surface, and is received by the photodetector 11.

A filter is attached to the light-receiving surface of the photodetector 11, which transmits only light in the stimulated emission wavelength range and cuts out light in the excitation light (laser light) wavelength range.
It is designed to detect only stimulated luminescence. Stimulated luminescence detected by the photodetector 11 is converted into an electrical signal, which is further amplified by the amplifier 12 and output. The accumulated recording information output from the amplifier 12 is input to the control circuit 13 of the main reading reading section 3. The control circuit 13 outputs an amplification factor setting value a and a recording scale factor b so that a signal of an appropriate level can be obtained according to the obtained accumulated recording information.

The phosphor sheet 1 whose pre-reading operation has been completed as described above is transferred to the main reading reading section 3.

In the main reading reading section 3, the following main reading operation is performed.

The laser beam 15 emitted from the main reading laser light source 14 passes through a filter 16 having the same function as the filter 6 described above, and then the beam
The beam diameter is precisely adjusted by the expander 17. Next, the laser beam is deflected by a light deflector 18 such as a galvano mirror, reflected by a plane reflecting mirror 19, and then one-dimensionally deflected and incident on the phosphor sheet 1. Note that an fθ lens 2 is provided between the optical deflector 18 and the plane reflecting mirror 19.
0, etc., so that when the polarized laser beam scans the phosphor sheet 1, a uniform beam speed is always maintained.

The phosphor sheet 1 is transported in the direction of the arrow 21 under irradiation with the above-mentioned polarized laser light. Therefore, as in the pre-reading operation, the entire surface of the phosphor sheet 1 is irradiated with the polarized laser light.

When the phosphor sheet 1 is irradiated with laser light as described above, it emits stimulated luminescence with an amount of light proportional to the accumulated and recorded radiation energy, as in the pre-reading operation, and this light is used as a guide for main reading. The light enters the optical sheet 22. This light-guiding sheet 22 for main reading has the same material and structure as the light-guiding sheet 10 for pre-reading, and the stimulated luminescence guided through the interior of the light-guiding sheet 22 for main reading through repeated total reflections. The light is emitted from the exit surface and is received by the photodetector 23. Note that a filter that selectively transmits only the wavelength region of stimulated luminescence is attached to the light receiving surface of the photodetector 23, so that the photodetector 23 detects only stimulated luminescence.

The stimulated luminescence detected by the photodetector 23 is converted into an electrical signal, which is amplified to an electrical signal at an appropriate level in the amplifier 24 whose sensitivity is set according to the amplification factor setting value a, and then A/D converted. The signal is input to the device 25. The A/D converter 25 converts the signal into a digital signal using a scale factor suitable for the signal fluctuation width according to the recorded scale factor setting value b.

Regarding the method for reading the positional information of the radiolabeled substance on the support medium that has been transferred and accumulated on the stimulable phosphor sheet in the present invention, the readout operation consisting of the pre-reading operation and the main reading operation has been described above. The read operations that can be utilized in the present invention are not limited to the above examples. For example, if the amount of radiolabeled substance on the support medium and the exposure time of the stimulable phosphor sheet for the support medium are known in advance, the look-ahead operation can be omitted in the above example.

Furthermore, the method for reading the positional information of the radiolabeled substance on the support medium transferred and accumulated on the stimulable phosphor sheet in the present invention is not limited to the method exemplified above.

The digital signal corresponding to the autoradiograph of the radiolabeled substance thus obtained is then input to the signal processing circuit 26 shown in FIG. The signal processing circuit 26 determines the scanning direction for signal processing, and then detects sampling points.

The digital signal processing of the present invention will be described below, taking as an example an autoradiograph of a separated and developed column obtained by separating and developing a mixture of radiolabeled substances on a support medium by electrophoresis or the like.

FIG. 2 shows an example of an autoradiograph of a sample in which multiple types of radiolabeled substances are linearly separated and developed in the length direction of the support medium. By applying the radiation image conversion method to this sample as described above, the digital signal input to the signal processing circuit 26 is converted to an address (x, y) and the signal level (z) at that address, and the signal level corresponds to the amount of photostimulated light. That is, the digital signal corresponds to the autoradiograph shown in FIG. Therefore, digital image data having positional information of the radiolabeled substance is input to the signal processing circuit 26. In the present invention, digital image data refers to a collection of digital signals corresponding to an autoradiograph of a radiolabeled substance.

First, a scanning direction for digital signal processing is determined for the digital signal. here,
In FIG. 2, if the vertical direction (separation and development direction) is the y-axis direction and the horizontal direction is the x-axis direction, the scanning direction can be determined, for example, as follows. That is, the obtained digital image data is numerically scanned in the x-axis direction to detect the x-coordinate (x _a ) where the signal level is maximum. This x
Axial scanning can be performed at any position in the y-axis direction (for example, at a position where y=y _a ). However, it is necessary to scan with a width centered on a scanning position that covers at least one separation and deployment site of a radiolabeled substance.

In the signal processing method of the present invention, the digital signal obtained by reading out the stimulable phosphor sheet is temporarily stored in a memory in the signal processing circuit 26 (i.e., a buffer memory or a non-volatile memory such as a magnetic disk). stored in memory). In signal processing, scanning digital image data means selectively retrieving only the digital signal at this scanning location from memory.

Therefore, the x-coordinate (x _a ) is, for example, the level of the signal obtained when the digital signals within the above scanning width are repeatedly extracted in the y-axis direction and the signal levels are added for each x-coordinate. The maximum x coordinate (x _a ) can be used. Alternatively, the digital signal within the above scanning width is repeatedly extracted in the x-axis direction, the x-coordinate where the signal level is maximum is found for each y-coordinate, and then the average coordinate of each x-coordinate is calculated. It can be the x coordinate (x _a ). Note that in order to avoid peaks of noise mixed in during such scanning, the signal level may be binarized using a preset threshold.

A straight line passing through the x-coordinate (x _a ) detected in this way and parallel to the y-axis direction is the scanning direction for the following signal processing.

Next, sampling points in this scanning direction are detected. Sampling points for detecting the separated development site of the radiolabeled substance can be all points where the signal level is maximum when only the digital signal in the scanning direction is extracted.
Also in the above scanning, it is desirable to scan with a constant width. Therefore, the maximum point of the signal level is, for example, the position (y) in the scanning direction on the horizontal axis.
When expressed in a graph in which the vertical axis is the average value (z) of the signal level within the scanning width, it means all the peak points that appear in this graph.
Hereinafter, the average value of the signal level at each position in the scanning direction will be simply referred to as the signal level at that position.

In Figure 3, as described above, the horizontal axis represents the position (y) in the scanning direction, and the vertical axis represents the signal level (z).
It shows a graph with .

In this way, a sampling point S _o having a coordinate point and a signal level (x _a , _yo , zo ₎ at this coordinate point is determined. Here, n is a positive integer and represents the number of sampling points.

By applying signal processing to the digital signal as described above, it is possible to express one-dimensional positional information of the radiolabeled substance in terms of the position in one-dimensional direction and the signal level (y _o , z _o ) at that position. can. Here, the signal level (z _o ) at each position can be considered to represent the relative amount (concentration) of the radiolabeled substance.

Alternatively, for example, by recording the starting position for separating and developing a radioactively labeled substance on a stimulable phosphor sheet with a marker containing a radioactively labeled substance, this starting position (y _p ) can be detected in the same manner as above. Furthermore, the starting position (y _p ) can be set in the exposure operation by processing the stimulable phosphor sheet itself using physical means such as punching holes. Therefore, detection is possible. Then, by subtracting {y _o −y _p =y _o ′} using this y _p , the above position information can be calculated as the moving distance from the separation deployment start position and the signal level at that position (y _o ′, z _o )
It can be expressed as

Furthermore, regarding the evaluation of the relative amount of the radiolabeled substance, in addition to the signal level at the above-mentioned sampling points, various calculations such as the integral value near each maximum point are possible.

An autoradiograph having one-dimensional positional information of a radiolabeled substance can be output from the signal processing circuit 26 as numerical values as described above. Note that the one-dimensional position information of the radiolabeled substance obtained as the coordinate point of the sampling point S _o and the signal level (x _a , y _o , z _o ) at this coordinate point is limited to the above display format. Instead, any display format is possible. In this way, one-dimensional positional information of the radiolabeled substance can be obtained as symbols and/or numerical values.

The obtained symbols and/or numerical values are then transmitted directly or, if necessary, via storage means such as magnetic tape to a recording device (not shown).

Examples of recording devices include those that optically record by scanning a photosensitive material with a laser beam or the like;
Recording devices based on various principles, such as those that display electronically on CRTs, etc., those that record symbols and numbers displayed on CRTs, etc. on video printers, etc., and those that record on heat-sensitive recording materials using heat rays. can be used.

The present invention also provides a signal processing method in autoradiography of a sample in which radiolabeled substances are distributed one-dimensionally in multiple rows.

That is, by causing the stimulable phosphor sheet to absorb radiation energy emitted from a plurality of rows of radiolabeled substances, each of which is distributed in at least one dimension on the support medium,
After accumulating and recording an autoradiograph having positional information of the group of radiolabeled substances on this stimulable phosphor sheet, scanning the stimulable phosphor sheet with electromagnetic waves to emit the autoradiograph as photostimulated light; With regard to the digital signal corresponding to the autoradiograph obtained by photoelectrically reading out the stimulated light, the following steps are performed: ) determining a one-dimensional scanning direction for signal processing for each of the plurality of columns; Detecting sampling points on the autoradiograph for each of the plurality of columns by performing signal processing including the following: () performing comparative identification of the sampling points detected in the plurality of columns. The present invention also provides a signal processing method in autoradiography, which is characterized in that one-dimensional positional information of a group of radiolabeled substances in a column is obtained as a signal and/or a numerical value.

The sample used in the above method generally consists of a support medium in which a plurality of rows of radiolabeled substances are distributed in parallel to each other in a one-dimensional direction. Here, the parallel relationship does not necessarily mean that the plurality of rows are in completely parallel positions with each other, but it means that they are in a positional relationship that can be considered to be locally or generally parallel.

The above signal processing method in autoradiography is particularly useful for analyzing the molecular weight, molecular structure, or basic unit structure of polymeric substances such as proteins, nucleic acids, derivatives thereof, and decomposition products thereof. This is an effective method.

Therefore, the present invention further provides DNA or
A signal processing method in autoradiography for determining the base sequence of a partially degraded product, the method comprising:
1) A base-specific cleavage product containing at least a guanine-specific cleavage product, 2) A base-specific cleavage product containing at least an adenine-specific cleavage product obtained by base-specific cleavage degradation of radioactively labeled DNA or DNA partial decomposition product. 3) a base-specific cleavage product comprising at least a thymine-specific cleavage product; 4) a base-specific cleavage product including at least a cytosine-specific cleavage product; Each of the specific cleavage and decomposition products is one-dimensionally separated and developed on a support medium in a parallel relationship, and the radiation energy emitted from the group of radiolabeled substances in the separation and development row is applied to the stimulable phosphor sheet. By absorbing the stimulable phosphor sheet, an autoradiograph containing positional information of the radiolabeled substance group is stored and recorded, and then the stimulable phosphor sheet is scanned with electromagnetic waves to record the autoradiograph. Regarding the digital signal corresponding to the autoradiograph of each separated development column obtained by emitting photostimulated light and photoelectrically reading out this photostimulated light, determining a scanning direction;) detecting a sampling point in the scanning direction of the separation expansion column for each of the separation expansion columns; ) detecting a corresponding sampling point in the scanning direction for each of the four groups of separation expansion columns; DNA characterized by performing signal processing including the step of obtaining positional information of each of guanine, adenine, thymine, and cytosine by comparing sampling points between positions.
Alternatively, the present invention also provides a signal processing method in autoradiography for obtaining the base sequence of a partial DNA decomposition product.

Next, the embodiment of signal processing in autoradiography using the signal processing method of the present invention is as follows.
The following describes the DNA base sequencing method as an example.

DNA has a double helical structure consisting of two chain molecules, and each of the two chain molecules contains four types of bases: adenine (A), guanine (G),
It is composed of structural units containing the bases cytosine (C) and thymine (T). These two chain molecules are cross-linked by hydrogen bonds between these four types of bases, and the hydrogen bonds between each constituent unit are formed by two types of combinations: G-C and A-T. Since this method has been realized only in this method, once the base sequence of one chain molecule is determined, the base sequence of the other chain molecule can also be automatically determined.

A typical example of DNA base sequencing using autoradiography is Maxam.
The Gilbert (Maxam-Gilbert) method is known. This method involves bonding a group containing a radioactive isotope of phosphorus (P) to one end of a chain molecule of DNA or DNA decomposition product whose base sequence is to be determined. After being made into a radiolabeled substance, the bonds between each constituent unit of the chain molecule are cleaved base-specifically using chemical means. Next, the DNA obtained by this operation or a mixture of a large number of base-specific cleavage products of the DNA degradation product is separated and developed by gel electrophoresis, and a large number of cleavage products are separated and developed to form a separated product. You get an expanded column (but you can't see it visually).

Conventionally, this separation and development array is visualized on an X-ray film to obtain an autoradiograph, and from the obtained autoradiograph and each base-specific cleavage means, a chain molecule to which a radioactive isotope is bound is determined. The bases in a certain positional relationship can be sequentially determined from the end of the target, and in this way, the sequence of all the bases of the object can be determined.

Using the above Maxam-Gilbert method
Taking DNA base sequencing as an example, a case will be described in which the following four types of cleavage products are used as typical combinations of base-specific cleavage products for base sequencing.

1) Guanine (G)-specific cleavage product, 2) Guanine (G)-specific cleavage product + adenine (A)-specific cleavage product, 3) Thymine (T)-specific cleavage product + cytosine (C) Specific cleavage products, 4) Cytosine (C)-specific cleavage products, First, the sample was prepared by gel-supporting a mixture of base-specific cleavage products of the above four groups, which were radiolabeled with ³² P, using a conventional method. It can be obtained by separation and development on the body by electrophoresis. Next, this sample (gel) and a stimulable phosphor sheet are overlapped for several minutes at room temperature to perform an exposure operation, and the autoradiograph of the sample is transferred and accumulated on the stimulable phosphor sheet. Details of the above exposure operation are described in the above-mentioned Japanese Patent Application No. 193418/1983.

FIG. 4 shows an autoradiograph of a separation and development column (electrophoresis column) formed by separation and development of the above-mentioned four types of base-specific cleavage products to which radioactive labels have been added.

That is, the first to fourth columns in FIG. (1)−(G) Specific cleavage product (2)−(G) Specific cleavage product +(A) Specific cleavage product (3)-(T) Specific cleavage product +(C)Specific cleavage product (4)−(C) Specific cleavage product Each electrophoresis row is shown.

The autoradiograph transferred and stored on the stimulable phosphor sheet is loaded into the reading device shown in FIG. 1 and read out, thereby obtaining a digital signal corresponding to the autoradiograph.

The obtained digital signal is subjected to digital signal processing in the signal processing circuit 26 as described above.
First, for each of the four columns shown in the autoradiograph of FIG. 4, the scanning direction for signal processing is determined in the same manner as described above. The scanning direction of each column can be, for example, a straight line passing through the x-coordinate (x _a ) and parallel to the y-axis direction. However, a
is a positive integer and represents the number of each column.

Next, in each scanning direction, all points where the signal level is maximum are determined as sampling points to be detected in the same manner as in the above-described method. In this way, a sampling point S _ao having a coordinate point and a signal level (x _a , y _ao , z _ao ) at this coordinate point is determined for each column. However, n is a positive integer and represents the number of sampling points.

Next, the above four columns are reorganized. That is,
Sampling point S _1o expressed as (x ₁ , y _1o , z _1o )
and a second column with a sampling point S _2o represented by (x ₂ , y _2o , z _2o ),
Set of y coordinates of sampling points in the first example {y _1o }
is included in the set of sampling points {y _2o } in the second column, so we create a new set {y _5o } expressed by the operation { _1o }∩{y _2o }={y _5o },
Along with the corresponding signal level (y _5o ,
We obtain a fifth column with sampling point S _5o denoted by z _5o ). The obtained fifth column has position information only for adenine (A). Similar arithmetic processing is performed at the sampling point represented by (x ₃ , y _3o , z _3o )
By doing this also between the third column with S _3o and the fourth column with sampling point S _4o denoted by (x ₄ , y _4o , z _4o ), we get the result expressed by (y _6o , z _6o ) Obtain the 6th column with sampling point S _6o .
This sixth column has position information only for thymine (T). In this way, one-dimensional positional information (y _ao , z _ao ) consisting of the following four columns is newly obtained.

(1) - (G) Specific cleavage and decomposition product (5) - (A) Specific cleavage and decomposition product (6) - (T) Specific cleavage and decomposition product (4) - (C) Specific cleavage and decomposition product Part 5 The figure diagrammatically shows the position information of the four columns obtained by reorganization through the above calculation process.

The positions (y _ao ) of these four columns are compared with each other and arranged in descending order of y _ao . For example, we can obtain the following diagram:

S ₁₁ , S ₄₁ , S ₁₂ , S ₄₂ , S ₅₁ , S ₅₂ , S ₆₁ , S ₁₃ , S ₄₃ ,
... In the above diagram, S _1o = G, S _4o = C, S _5o
By replacing =A and S _6o =T, the following diagram is obtained.

G-C-G-C-A-A-T-G-C-... In this way, the base sequence of one chain molecule of DNA can be determined. In addition,
Information about the obtained DNA base sequence is not limited to the above display format, and any display format is possible. For example, if desired, it is also possible to simultaneously display the signal level (z _ao ) at the sampling point in the signal processing described above as the relative amount of each base-specific cleavage product separated and expanded. Alternatively, a diagram as shown in FIG. 5 is also possible.

Alternatively, it is also possible to display the base sequences of both two stranded molecules of DNA. That is, in the diagram represented by the above symbols, the combinations corresponding to each base are A→T, G→
By providing information such as C, C→G, and T→A, the DNA base sequence represented by the following diagram can be obtained.

G-C-G-C-A-A-T-G-C-... C-G-C-G-T-T-A-C-G-... In addition, the above (G, G+A, T+C , C) The DNA base sequencing method using the combination of
The signal processing method of the present invention, which is an example of a DNA base sequencing method, is not limited to the above combinations, and various combinations are possible. It is possible to determine the base sequence in a similar manner by a method similar to the above method. For example, the base sequence of DNA can also be determined using a combination of (G, A, T, C). Alternatively, it is also possible to determine the sequence of a specific base from a combination of at least one group of base-specific cleavage products and an appropriate reference substance (for example, a mixture of each base-specific cleavage product).

Furthermore, in the above example, the explanation was given using four rows of radiolabeled substance groups that are separated and developed in a one-dimensional direction on the support medium, but the separation and development rows are not limited to four rows. There may be four or more rows, or fewer than four rows. Alternatively, two or more types of DNA can be simultaneously produced using one support medium.
Alternatively, it is also possible to determine the base sequence of a partial DNA decomposition product.

determined by the signal processing method described above.
After the information about the DNA base sequence is output from the signal processing circuit 26, it can be recorded using, for example, the recording device described above.

In addition to this, the information obtained as described above may also be used in other ways, such as other information that has already been recorded.
It is also possible to perform genetic linguistic information processing such as matching with DNA base sequences.

[Brief explanation of drawings]

FIG. 1 shows an example of a reading device (or reading device) for reading positional information of a radiolabeled substance in a sample that has been transferred and accumulated on a stimulable phosphor sheet in the present invention. 1: stimulable phosphor sheet, 2: readout section for pre-reading, 3: readout section for main reading, 4: laser light source,
5: Laser light, 6: Filter, 7: Optical deflector, 8: Planar reflector, 9: Transfer direction, 10: Light guide sheet for prereading, 11: Photodetector, 12: Amplifier, 13: Control circuit, 14: Laser light source, 1
5: laser beam, 16: filter, 17: beam expander, 18: optical deflector, 19: plane reflecting mirror, 20: fθ lens, 21: transport direction, 2
2: Light guide sheet for main reading, 23: Photodetector, 2
4: Amplifier, 25: A/D converter, 26: Signal processing circuit. FIG. 2 is a diagram showing an example of an autoradiograph of a sample in which a radiolabeled substance is separated and developed in a one-dimensional direction on a support medium. FIG. 3 is a graph showing an example of the relationship between the position in the scanning direction and the level of the digital signal for signal processing. Figure 4 shows
FIG. 2 is a diagram showing an example of an autoradiograph of a sample in which a base-specific cleavage product of DNA is separated and developed on a gel support. FIG. 5 is a diagram schematically showing an example of a DNA base sequence determined by the signal processing method of the present invention.

Claims (1)

[Scope of Claims] 1. The stimulable phosphor sheet is produced by absorbing radiation energy emitted from a radiolabeled substance distributed in at least one dimension on the support medium into the stimulable phosphor sheet. After accumulating and recording an autoradiograph having positional information of the radiolabeled substance, the stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is converted into photoelectrons. a digital signal corresponding to the autoradiograph obtained by reading the autoradiograph automatically, the signal comprising: ) determining a one-dimensional scanning direction for signal processing; ) detecting sampling points on the scanning direction. 1. A signal processing method in autoradiography, characterized in that one-dimensional positional information of a radiolabeled substance on the autoradiograph is obtained as symbols and/or numerical values by processing. 2. A signal processing method in autoradiography according to claim 1, characterized in that all points at which the signal level reaches a maximum in the scanning direction are taken as sampling points in the scanning direction. 3. It is confirmed that the radiolabeled substance distributed on the support medium is a biopolymer substance, a derivative thereof, or a decomposition product thereof, to which a radiolabel is attached and which is separated and developed in one-dimensional direction on the support medium. A signal processing method in autoradiography according to claim 1 or 2, characterized by: 4. Claim 3, characterized in that the biopolymer substance is a nucleic acid, a derivative thereof, or a decomposition product thereof, and the symbol and/or numerical value obtained by signal processing represents the base sequence thereof. Signal processing method in autoradiography described. 5. By causing the stimulable phosphor sheet to absorb radiation energy emitted from a plurality of rows of radiolabeled substances, each of which is distributed in at least one dimension on the support medium, the stimulable phosphor sheet is After accumulating and recording an autoradiograph having positional information of the group of radiolabeled substances, the stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is emitted. With respect to the digital signal corresponding to the autoradiograph obtained by photoelectrically reading out, the steps of: ) determining a one-dimensional scanning direction for signal processing for each of the plurality of columns; ) determining sampling points on the scanning direction; A step of detecting each of the plurality of columns; and) a step of performing comparative identification of the sampling points detected in the plurality of columns. A signal processing method in autoradiography characterized by obtaining one-dimensional position information as symbols and/or numerical values. 6. The signal processing method in autoradiography according to claim 5, characterized in that all points at which the signal level reaches a maximum in the scanning direction are taken as sampling points in the scanning direction. 7. The autoradiograph according to claim 5 or 6, wherein the comparative identification of the sampling points is performed by numerical calculation of the sampling points between corresponding positions in the scanning direction of each of the plurality of columns. Signal processing method in E. 8. The group of radiolabeled substances distributed on the support medium is a group of radioactively labeled biopolymer substances, derivatives thereof, or decomposition products thereof, which are separated and developed in one-dimensional direction on the support medium. Claims 5 to 7 are characterized in that:
A signal processing method in autoradiography according to any one of paragraphs. 9. Claim No. 9, wherein the biopolymer substances are nucleic acids, derivatives thereof, or decomposition products thereof, and the symbols and/or numerical values obtained by signal processing represent their base sequences. The signal processing method in autoradiography according to item 8. 10 A signal processing method in autoradiography for determining the base sequence of DNA or a DNA partial decomposition product, which is obtained by base-specific cleavage of radioactively labeled DNA or a DNA partial decomposition product, 1) a base-specific cleavage product that includes at least a guanine-specific cleavage product; 2) a base-specific cleavage product that includes at least an adenine-specific cleavage product; 3) a base-specific cleavage product that includes at least a thymine-specific cleavage product. 4) base-specific cleavage products including at least a cytosine-specific cleavage product; each of at least four groups of base-specific cleavage products, including: By causing the stimulable phosphor sheet to absorb the radiation energy emitted from the radiolabeled substance group in the separated and expanded array formed by separating and developing the stimulable phosphor sheet, the position of the radiolabeled substance group is determined by the stimulable phosphor sheet. After storing and recording an autoradiograph containing information, the stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is read out photoelectrically. For the digital signal corresponding to the autoradiograph of each separated development column, a step of: ) determining a scanning direction for signal processing for each of the separation development column; ) determining a sampling point in the scanning direction of the separation development column; a step of detecting each of the separation and development columns;) for each of the four groups of separation and development columns, by comparing sampling points between corresponding positions in the scanning direction, each of guanine, adenine, thymine, and cytosine is detected; DNA characterized by performing signal processing including the step of obtaining location information of
Or a signal processing method in autoradiography to obtain the base sequence of partial DNA degradation products. 11 The base-specific cleavage products of DNA or DNA partial decomposition products are: 1) guanine-specific cleavage product, 2) guanine-specific cleavage product + adenine-specific cleavage product, 3) thymine-specific cleavage product + A signal in autoradiography according to claim 10, characterized in that the signal comprises at least four groups of base-specific cleavage products consisting of: 4) cytosine-specific cleavage products; Processing method.