JPH0259956B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a signal processing method in autoradiography. More specifically, the present invention relates to a method for removing noise on digital image data in digital signal processing for obtaining position information of a radiolabeled substance as symbols and/or numerical values 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, after attaching a radioactive label to a biologically derived polymeric substance such as a protein or a nucleic acid, the radioactively labeled polymeric substance, its derivative, etc.
Alternatively, the decomposition products (hereinafter also referred to as radiolabeled substances) can be separated and developed by performing separation operations such as electrophoresis on a gel-like support medium.
A separated and developed column (but invisible) of the radiolabeled substance is formed on the support medium, an autoradiograph of this separated and developed column is acquired as a visible image on a radiation film, and the radiolabeled substance is detected from the visible image. Obtaining location information. In addition, based on the obtained positional information of the radiolabeled substance, it is possible to separate and identify the polymeric substance, or to determine the molecular weight of the polymeric substance.
Methods for evaluating characteristics have already been developed and are in actual use. In recent years, autoradiography as described above has become increasingly popular, especially in recent years.
It is effectively used to determine the base sequence of nucleic acids such as

As mentioned above, in autoradiography that uses conventional radiography, it is essential to visualize an autoradiograph containing this positional information on radiographic film in order to obtain positional information about radiolabeled substances. It's summery.

Therefore, researchers determine the distribution of radiolabeled substances on the support medium by visually observing the visualized autoradiograph.
Furthermore, the characteristics, functions, etc. of radiolabeled substances are evaluated by performing various analyzes based on visually obtained positional information of the radiolabeled substances.

However, in conventional autoradiography, the analysis work relies on the human eye as mentioned above, so the position information of the radiolabeled substance obtained by analyzing the autoradiograph, which is a visible image, is There are problems such as differences between researchers, and there are limits to the accuracy of the information that can be obtained. In particular, if the autoradiograph visualized on the radiographic film does not have good image quality (sharpness, contrast), it is difficult to obtain satisfactory information, and its accuracy tends to decrease. Conventionally, in order to improve the accuracy of the positional information to be obtained, a method has been used in which, for example, the visualized autoradiograph is measured using a measuring instrument such as a scanning densitometer. However, there is a limit to the improvement of accuracy in methods that simply use such measuring instruments.

For example, if the sample contains radioactively labeled impurities, if the support medium is radioactively contaminated by natural radioactivity, or if the separation development conditions are insufficient, automatic Noise is more likely to appear on the radiograph,
It becomes difficult to analyze the location information of radioactively labeled substances,
Therefore, the accuracy of the information obtained will be reduced.

In the above cases, it becomes particularly difficult to analyze the positional information of the radiolabeled substance, and even if the above-mentioned measuring instruments are used, it is difficult to obtain the positional information of the separated and expanded radiolabeled substance with sufficient accuracy. It is difficult.

The inventors of the present invention obtained positional information of radiolabeled substances by using a radiation conversion method using a stimulable phosphor sheet instead of the radiographic method using a radiation film used in conventional autoradiography. By obtaining the positional information of the autoradiograph as a digital signal without converting it into an image, and by performing appropriate signal processing on the obtained digital signal, one-dimensional positional information of the radiolabeled substance can be converted into a symbol and/or The present invention was achieved by realizing numerical display.

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 accumulatively recording an autoradiograph having positional information of the radiolabeled substance,
Regarding the digital signal corresponding to the autoradiograph obtained by scanning the stimulable phosphor sheet with electromagnetic waves to emit the autoradiograph as photostimulated light, and photoelectrically reading out the photostimulated light, ( i) obtaining a graph in which the horizontal axis represents the position in the scanning direction and the vertical axis represents the signal level; (ii) detecting sampling points by smoothing and/or thresholding the graph; Provided is 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 performing signal processing including the following. It is something.

In the present invention, by overlapping a sample and a stimulable phosphor sheet, the stimulable phosphor sheet absorbs radiation energy emitted from the sample, and then the stimulable phosphor sheet is used to absorb visible light and infrared rays. By scanning with electromagnetic waves (excitation light), the radiation energy stored in the stimulable phosphor sheet is released as fluorescence (stimulated luminescence),
A radiation image conversion method is utilized in which this fluorescence is read photoelectrically to obtain an electrical signal, and this electrical signal is A/D converted to obtain a digital signal.

The radiation image conversion method described above is described in, for example, Japanese Patent Laid-Open No. 12145/1983.

The stimulable phosphor sheet used in the present invention contains, for example, a stimulable phosphor such as a divalent europium-activated alkaline earth metal fluorohalide phosphor. This stimulable phosphor is
After being irradiated with radiation such as rays, alpha rays, beta rays, gamma rays, and ultraviolet rays and accumulating a portion of that radiation energy, when irradiated with electromagnetic waves (excitation light) such as visible light and infrared rays, the accumulated energy is It has the property of exhibiting stimulated luminescence depending on the

According to the present invention, the positional information of a radiolabeled substance is directly obtained as a digital signal without going through any imaging process, using the radiation image conversion method using the above-mentioned stimulable phosphor sheet.

In the present invention, "position information" refers to various types of information centered on the position of a radiolabeled substance or an aggregate thereof in a sample, such as the location and shape 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 the concentration and distribution of radioactive substances at that location.

According to the present invention, by performing specific processing on digital image data for an autoradiograph containing noise, only noise can be easily removed and true image data can be obtained. In other words, it is possible to obtain positional information with high precision without being affected by, for example, noise generated by radioactively labeled impurities contained in a sample or noise generated by poor separation development conditions. It is something that can be done. Furthermore, by providing a reference (internal standard) column in the separation and development operation, it is possible to easily detect a detection target substance to which a radioactive label has been attached with high precision.

In addition, in the present invention, the reference string (internal standard string) refers to, for example, when the purpose is signal processing for determining the base sequence of DNA or DNA partial decomposition products, On the other hand, it refers to a separation and development array formed by separating and developing a mixture of cleavage products (base-specific cleavage products) obtained by specifically cleaving each base. In other words, this reference column is a column used as a reference when performing signal processing to obtain position information of radiolabeled substances in other separation and development columns. However, the reference column does not necessarily have to be a single separated and expanded column, and may be obtained by virtually combining a plurality of separated and expanded columns in the course of a signal processing operation.

Examples of samples used in the present invention include:
A support medium in which a radiolabeled substance is separated and developed in a one-dimensional direction can be mentioned. 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 basic structure of the stimulable phosphor sheet used in the present invention is 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 radioactive 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. These exposure operations 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 incident on the top with a one-dimensional deflection. 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 is transported in the direction of 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 readout section 3 . The control circuit 13 outputs an amplification factor setting value a and a recording scale factor b so that a signal at 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. Note that an fθ lens 20 is provided between the optical deflector 18 and the plane reflecting mirror 19.
etc. are arranged 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 appropriate level electrical signal 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 recording 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 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 separated and developed in the length direction on a 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 of FIG. Therefore, digital image data having positional information of the radiolabeled substance is inputted to the signal processing circuit 26.
As used herein, digital image data refers to a collection of digital signals corresponding to an autoradiograph of a radiolabeled substance.

First, in the autoradiograph shown in Figure 2,
The scanning direction along the one-dimensional distribution direction of the radiolabeled substance is defined as the y-axis direction, and the direction perpendicular thereto is defined as the x-axis direction.

This scanning direction can be determined, for example, by scanning the digital signal twice at different positions on the digital image data across the one-dimensional distribution direction of the radiolabeled substance on each scan. It is determined by detecting the distribution points of the radiolabeled substance, connecting the two distribution points to obtain a straight line, and using the obtained straight line as the scanning direction for sampling point detection.

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 the signal processing of the present invention, scanning digital image data means selectively extracting only the digital signal at this scanning location from the memory.

Next, sampling points for detecting the separation and development site of the radiolabeled substance are determined, for example, as follows.

By scanning the digital image data along the scanning direction, the horizontal axis represents the position (w) in the scanning direction.
and obtain a graph with the signal level (z) on the vertical axis. This scanning is performed with a constant width along the scanning direction. That is, the graph shows the signal level obtained by adding the signal levels of the digital signals within the above scanning width for each W coordinate. Due to this,
For example, a graph as shown in FIG. 3 is obtained.

Next, smoothing processing is performed on this graph. Smoothing can be performed, for example, by performing convolution on the graph to be processed using an appropriate filter function. An example of a filter function used for this convolution is a function g(w) as shown in FIG. Here, when the graph shown in Figure 3 is expressed by a function f(w), by convolution with the above filter function, h=(w)=g(w)*f(w) (where * is A smoothed function h(w), which is a convolution operator, is obtained. That is, a graph as shown in FIG. 5 can be obtained.

Separately, regarding the digital signal on the above scanning,
A graph, ie, a histogram, is obtained in which the horizontal axis represents the signal level and the vertical axis represents the frequency of occurrence of the signal level. It is preferable to perform smoothing processing using convolution similar to the above method on this histogram as well.

FIG. 6 is a histogram corresponding to the graph shown in FIG. 3, which has been subjected to smoothing processing. The peak point (α) in the histogram of FIG. 6 represents the background level of the digital signal. Then, a value (α _p ) obtained by adding a certain value to the level (α) of this signal is set as a threshold value.

Based on the threshold value (α _p ) thus obtained, threshold processing is then performed on the graph of FIG. 5. That is, in the graph of FIG. 5, the signal level is set to 1 when the signal level is above the threshold value, and 0 when it is below the threshold value. Obtain a graph represented by 1 or 0. In this graph, each midpoint of the region where the signal level is 1 can be used as a sampling point to be detected.

Furthermore, in the signal processing method of the present invention, the sampling points may be all the maximum points appearing in the smoothed graph of FIG. 5.

In this way, a sampling point with a position (w _n ) in the separation development direction of the radiolabeled substance
S _n is determined. Here, m is a positive integer and represents the number of sampling points.

By subjecting the digital signal to signal processing as described above, one-dimensional positional information of the radiolabeled substance can be expressed as a position (w _n ) in one-dimensional direction.

For example, by recording the starting position for separating and developing a radiolabeled substance on a stimulable phosphor sheet with a marker containing a radiolabeled substance, this starting position (w _p ) can be recorded on the digital image data. can be detected in the same manner as above. Alternatively, the starting position can be detected by processing the stimulable phosphor sheet itself using physical means such as punching holes, and then setting the starting position during the exposure operation. It is. Then, using this W _p, {w _n −w _p
=w _n ′}, the desired position information can be expressed as a moving distance (w _n ′) from the separation development start position.

Furthermore, for example, one-dimensional positional information of the radiolabeled substance can be obtained by regarding the signal level value at each maximum point on the graph of FIG. 5 as the relative amount (concentration) of the radiolabeled substance at each separation and development site. It can also be displayed in terms of travel distance and relative amounts (w _n ′, z _n ).
This relative amount can be calculated in various ways, such as by taking the integral value near each maximum point in the graph of FIG.

An autoradiograph having one-dimensional positional information of the 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 _n and the signal level (x _n , y _n , z _n ) 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, the stimulable phosphor sheet absorbs the radioactive energy emitted from a plurality of rows of radiolabeled substances, each including a reference row separated and expanded in at least one dimension on a support medium. After accumulating and recording an autoradiograph having positional information of the group of radiolabeled substances on a stimulable phosphor sheet, scanning the stimulable phosphor sheet with electromagnetic waves to emit the autoradiograph as photostimulated light; Regarding the digital signal corresponding to the autoradiograph obtained by photoelectrically reading out this photostimulated light, (i) regarding the reference column, the horizontal axis represents the position in the scanning direction, and the vertical axis represents the signal level; (ii) detecting candidate points for sampling by performing smoothing and/or threshold processing on the graph; (iii) performing statistical processing on the candidate points for sampling; (iv) detecting sampling points in each scanning direction of the remaining separated expansion rows from the basic sampling point; 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 symbols and/or numerical values. Note that the reference column in the above signal processing method of the present invention does not necessarily have to be actually arranged on the support medium, and may be virtually synthesized from a plurality of separated and expanded columns as described above.

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 effective for analyzing the molecular weight, molecular structure, or basic unit composition of polymeric substances such as proteins, nucleic acids, their derivatives, and their decomposition products. This is a great 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:
Obtained by base-specific cleavage of radioactively labeled DNA or DNA partial decomposition products, 1. Guanine-specific cleavage product 2. Guanine-specific cleavage product + adenine-specific cleavage product, 3. Thymine-specific cleavage product At least four groups of base-specific cleavage products, including 4 cytosine-specific cleavage products, are separated and developed one-dimensionally in a parallel relationship on a support medium. By making the stimulable phosphor sheet absorb the radiation energy emitted from the group of radiolabeled substances in the separated and expanded array formed by the stimulable phosphor sheet, an autograph having positional information of the group of radiolabeled substances is formed in the stimulable phosphor sheet. After accumulating and recording a radiograph, the stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and each separation development obtained by photoelectrically reading out this photostimulated light. Regarding the digital signal corresponding to the autoradiograph of a column, (i) a reference column (internal standard column) is synthesized from the plurality of separated and expanded columns, the horizontal axis is the position in the scanning direction, and the vertical axis is the position of the reference column in the scanning direction. (ii) detecting sampling candidate points by performing smoothing and/or threshold processing on the graph; (iii) performing statistical processing on the sampling candidate points; (iv) detecting sampling points in each scanning direction of the remaining separation expansion columns from the basic sampling points; (v) separation expansion of at least the four groups; For each column, by comparing sampling points between corresponding positions in the scanning direction, signal processing is performed including the step of obtaining position information for each of guanine, adenine, thymine, and cytosine. DNA
The present invention also provides a signal processing method in autoradiography for obtaining the base sequence of.

Note that in the above signal processing method of the present invention,
DNA or DNA partial decomposition product was obtained by specifically cleaving each of the four types of bases that are its constituent units during separation and development without using a method of synthesizing a reference string. It is also possible to actually provide a reference row on the support medium by simultaneously separating and developing the cutting mixture on the same support medium.

Next, embodiments of signal processing in autoradiography using the signal processing method of the present invention will be described using an operation for determining a DNA base sequence 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 many base-specific cleavage products of the DNA decomposition product is separated and developed by gel electrophoresis, and each of the many base-specific cleavage products forms a band. You get a separated separation expansion 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 specific cutting means, it is determined that a certain number of molecules are detected from the end of the chain molecule to which the radioactive isotope is bound. The bases in the positional relationship can be sequentially determined, and in this way, the sequence of all the bases of the object can be determined.

Using the Maxim Gilbert method mentioned above
Taking DNA base sequencing as an example, a case will be described in which the following four types of base-specific 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 a medium by electrophoresis. Next, an exposure operation is performed by overlapping this sample (supporting medium) and a stimulable phosphor sheet at room temperature for several minutes.
An autoradiograph of the sample is stored on a stimulable phosphor sheet. For more information on the above exposure operations,
It is described in the specification of Japanese Patent Application No. 57-193418.

FIG. 7 shows an autoradiograph of separation and development arrays (electrophoretic arrays) of the above-mentioned four types of cleavage products formed by the separation and development 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. 7, the scanning direction for signal processing is determined in the same manner as described above.

Next, by scanning the digital image data along each scanning direction, for each column, the horizontal axis represents the position (w) in the scanning direction, and the vertical axis represents the signal level (z). Obtain an ivy graph. Here, the position in the scanning direction is preferably expressed by the migration distance from the migration start position by detecting the migration start position ( _wkp) of each column with a marker. However, k is a positive integer, Represents the number of each column.

For the obtained graphs in the second column and the graph in the third column, extract and synthesize the level values of the signals in the columns where the signal level (z) shows the maximum value at the same position (w) in each scanning direction. By this, four types of base-specific cleavage decomposition products: (G) specific cleavage decomposition products, (A) specific cleavage decomposition products, (T) specific cleavage decomposition products, and (C) specific cleavage decomposition products. You will get a graph that includes everything. The obtained graph is a graph regarding a column that should also be called an internal standard (reference) column.

Note that the internal standard column is not synthesized from a separation and development column of a mixture of base-specific cleavage products as described above, but by actually preparing an electrophoresis column containing the base-specific cleavage products of the four types of DNA mentioned above in advance. , this may be used as an internal standard column.

Smoothing and/or threshold processing is performed on the internal standard column in the same manner as described above to obtain sampling candidate points S _po expressed by migration distance (w _po ). However, o represents an internal standard sequence, and n is a positive integer and represents the number of the sampling point corresponding to the candidate point.

Next, basic sampling points are determined by performing statistical processing on the obtained sampling candidate points S _po . The number of constituent units containing one of the four types of bases increases by one in the radiolabeled substances present at sampling candidate points on the internal standard column in the order of decreasing migration distance, that is, in the order of increasing sampling point number. Since it has been experimentally determined that there is a linear relationship between the migration distance of these radiolabeled substances and the logarithm of the molecular weight of the radiolabeled substances, sampling is not recommended. Statistical processing can be performed by approximating candidate points using the following relationship.

w _po = a−b log(A+M _o ) (1) (However, a and b are numerical values determined experimentally according to electrophoresis conditions, and A and M are values for DNA
This is a numerical value related to the molecular weight of the base-specific cleavage product of . ) By substituting the migration distance w _po of each sampling candidate point S _po and the corresponding sampling point number n into equation (1) and performing statistical processing, the most probable value is calculated.
Calculate a _p and b _p , and convert this a _p and b _p into (1)
By substituting again into the equation, it is possible to determine the basic sampling point S _po ', which is represented by the most probable migration distance (w _po ').

Next, based on this basic sampling point S _po ′, among the digital signals existing within a certain width centered on the basic sampling point for each basic sampling point in each scanning direction for each of the four columns, The number of digital signals exhibiting a signal level equal to or higher than the threshold determined in the threshold processing described above is calculated. Then, by repeatedly performing more suitable threshold processing as necessary while considering the number of each obtained digital signal, sampling points to be detected are detected for each column.

Through the above processing, each column is represented by a set {S _po ′} _k of basic sampling points S _po ′ having the most probable migration distance (w _po ′). Then, the basic sampling points detected in each column in this manner are taken as sampling points to be detected.

Figure 8 shows, in order: (0)-(G) Specific cleavage product +(A) Specific cleavage product +(T)Specific cleavage product +(C)Specific cleavage product (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 The sampling points in each column are shown.

Next, the first to fourth columns are compared. That is, using a virtual first column with a set of sampling points {S _po ′} ₁ and a second column with a set of sampling points {S _po ′} ₂ , we have { _po ′} ₁ ∩{S _po ′} ₂ = {S _po ′} ₅ A virtual fifth column having a new set of sampling points {S _po ′} ₅ is obtained. The obtained fifth column has position information only for adenine (A). By performing the same subtraction process between the third column, which has a set of sampling points {S _po ′} ₃ , and the fourth column, which has another set of sampling points {S _po ′} ₄ , another A virtual sixth column having a set of sampling points {S _po ′} ₆ is obtained. The sixth column obtained as described above has position information of only thymine (T).

Through the processing described above, new one-dimensional position information consisting of the next four columns is obtained.

(1) - (G) Specific cleavage product (5) - (A) Specific cleavage product (6) - (T) Specific cleavage product (4) - (C) Specific cleavage product For the basic sampling point S _po ′, (i) {S _po ′} the sampling point belonging to ₁ is G (ii) the sampling point belonging to {S _po ′} ₄ is C (iii) the sampling point belonging to {S _po ′} ₅ After replacing the sampling points belonging to A (iv) {S _po ′} ₆ with T and arranging them in order of sampling numbers, we obtain the following diagram.

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 display the relative amount of each separated and developed cut and decomposed product by arbitrarily processing the signal level in the scanning direction in each column.

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-... By the signal processing method of the present invention, In addition to the DNA base sequencing method using the above-mentioned combinations of (G, G+A, T+C, C), at least one group of base-specific cleavage products and an appropriate reference substance (for example, the above-mentioned It is also possible to determine the sequence of a specific base from the combination with 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 more than four rows,
Also, there may be fewer than four rows. Alternatively, it is also possible to simultaneously determine the base sequences of two or more types of DNA using one support medium.

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
It is a graph showing an example of a filter function used for smoothing processing. FIG. 5 is a graph obtained by smoothing the graph of FIG. 3. FIG. 6 shows a histogram. Figure 7 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. 8 is a chart schematically showing sampling points on a DNA separation and development array detected 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. (i) obtaining a graph in which the horizontal axis represents the position in the scanning direction and the vertical axis represents the signal level; (ii) the digital signal corresponding to the autoradiograph obtained by reading the By performing signal processing including the step of detecting sampling points by performing smoothing and/or threshold processing on the graph, one-dimensional positional information of the radiolabeled substance on the autoradiograph can be expressed as a symbol and/or A signal processing method in autoradiography characterized by obtaining numerical values. 2. The automatic system according to claim 1, wherein smoothing of a graph in which the horizontal axis represents the position in the scanning direction and the vertical axis represents the signal level is performed by convolution with a filter function. Signal processing methods in radiography. 3. The signal processing method in autoradiography according to claim 1 or 2, characterized in that all maximum points on the graph obtained by smoothing are used as sampling points. 4. The autoradio according to claim 1 or 2, characterized in that in the graph obtained by smoothing and threshold processing, each midpoint of a region where the signal level is positive is taken as a sampling point. Signal processing method in Graphee. 5. The signal processing method in autoradiography according to claim 4, wherein the threshold value in the threshold value processing is determined by a histogram. 6. Claim No. 6, characterized in that the radiolabeled substance separated and developed in one-dimensional direction on the support medium is a radioactively labeled biopolymer substance, a derivative thereof, or a decomposition product thereof. A signal processing method in autoradiography according to any one of items 1 to 5. 7. By causing a stimulable phosphor sheet to absorb radiation energy emitted from a plurality of rows of radiolabeled substances, each including a reference row separated and expanded in at least one dimension on a support medium, After accumulating and recording an autoradiograph having positional information of the group of radiolabeled substances on a stimulable phosphor sheet, scanning the stimulable phosphor sheet with electromagnetic waves to emit the autoradiograph as photostimulated light; Regarding the digital signal corresponding to the autoradiograph obtained by photoelectrically reading out this photostimulated light, (i) regarding the reference column, the horizontal axis represents the position in the scanning direction, and the vertical axis represents the signal level; (ii) detecting candidate points for sampling by performing smoothing and/or threshold processing on the graph; (iii) performing statistical processing on the candidate points for sampling; (iv) detecting sampling points in each scanning direction of the remaining separated expansion rows from the basic sampling point; 1. A signal processing method in autoradiography, characterized in that one-dimensional positional information of a group of radiolabeled substances in a column is obtained as symbols and/or numerical values. 8. Claim No. 8, characterized in that the radiolabeled substance separated and developed in one dimension on the support medium is a radioactively labeled biopolymer substance, a derivative thereof, or a decomposition product thereof. The signal processing method in autoradiography according to item 7. 9. Claim 8, 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. 10 This stimulable phosphor sheet is produced by absorbing radiation energy emitted from a plurality of rows of radiolabeled substances, each of which is separately developed in at least one dimension on a support medium, into the stimulable phosphor sheet. After accumulating and recording an autoradiograph having positional information of the group of radiolabeled substances, scanning the stimulable phosphor sheet with electromagnetic waves to emit the autoradiograph as photostimulated light;
Then, regarding the digital signal corresponding to the autoradiograph obtained by photoelectrically reading out this stimulated light, (i) a reference column is synthesized from a plurality of separated and expanded columns, and the reference column is plotted along the horizontal axis in the scanning direction. take the position of
(ii) detecting sampling candidate points by performing smoothing and/or threshold processing on the graph; (iii) performing statistical analysis on the sampling candidate points; (iv) detecting sampling points in each scanning direction of the remaining separation expansion columns from the basic sampling point; 1. A signal processing method in autoradiography, characterized in that one-dimensional positional information of radiolabeled substance groups in a plurality of rows on the autoradiograph is obtained as symbols and/or numerical values. 11. Claim No. 1, characterized in that the radiolabeled substance separated and developed in one-dimensional direction on the support medium is a radioactively labeled biopolymer substance, a derivative thereof, or a decomposition product thereof. The signal processing method in autoradiography according to item 10. 12. Claim 11, wherein 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. 13 A signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, comprising: 1 Radioactively labeled DNA or DNA.
A mixture of a guanine-specific cleavage product, an adenine-specific cleavage product, a thymine-specific cleavage product, and a cytosine-specific cleavage product obtained by base-specific cleavage degradation of a partial decomposition product, 2. Guanine-specific cleavage product , 3 guanine-specific cleavage products + adenine-specific cleavage products, 4 thymine-specific cleavage products + cytosine-specific cleavage products, 5 cytosine-specific cleavage products, at least five groups of base-specific cleavage products. A phosphor sheet capable of accumulating radioactive energy emitted from a group of radiolabeled substances of a separated product formed by one-dimensionally separating and developing each product or a mixture of cut and decomposed products in a parallel relationship on a support medium. An autoradiograph having positional information of the group of radiolabeled substances is stored and recorded on this stimulable phosphor sheet, 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 reading out this photostimulated light photoelectrically, (i) the separated development column of the cleavage and decomposition product mixture; (ii) by performing smoothing and/or threshold processing on the graph; (iii) determining basic sampling points by performing statistical processing on the sampling candidate points; (iv) determining each of the remaining separation expansion sequences from the basic sampling points; (v) For each separation expansion column, by comparing the sampling points between corresponding positions in the scanning direction, each position of guanine, adenine, thymine, and cytosine is detected. A DNA characterized by performing signal processing including the step of obtaining information.
Or a signal processing method in autoradiography to obtain the base sequence of partial DNA degradation products. 14 In the step of determining basic sampling points by performing statistical processing on sampling candidate points, the statistical processing calculates each sampling candidate point as follows: w _po = a - b log (A + Mn) (1) (where w _po represents the distance of the sampling candidate point from the reference point in the scanning direction; n represents the number of the sampling point corresponding to the candidate point; A and M are constants) The autoradiography system according to claim 13, characterized in that a and b are determined by approximating a and b, and then a basic sampling point is determined by equation (1) based on a and b. signal processing method. 15 A signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, which is obtained by base-specific cleavage degradation of radioactively labeled DNA or DNA partial decomposition products. 1 guanine-specific cleavage product, 2 guanine-specific cleavage product + adenine-specific cleavage product, 3 thymine-specific cleavage product + cytosine-specific cleavage product, 4 cytosine-specific cleavage product. Each base-specific cleavage product of the group is one-dimensionally separated and developed on a support medium in a parallel relationship, and the radiation energy emitted from the radiolabeled substance group of the separated product is converted into an accumulative fluorescent material. An autoradiograph containing positional information of the group of radiolabeled substances is stored and recorded on this stimulable phosphor sheet by being absorbed into the body sheet, 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 the radiograph as photostimulated light and reading out this photostimulated light photoelectrically, (i) from the plurality of separated development columns; (ii) performing smoothing and/or threshold processing on the graph; combining the reference columns and obtaining a graph in which the horizontal axis represents the position in the scanning direction and the vertical axis represents the signal level; (iii) determining a basic sampling point by performing statistical processing on the sampling candidate point; (iv) determining the remaining separation expansion sequence from the basic sampling point. (v) detecting sampling points in each scanning direction; (v) matching sampling points between corresponding positions in the scanning direction for each separation expansion column; DNA characterized by performing signal processing including the step of obtaining position information.
Or a signal processing method in autoradiography to obtain the base sequence of partial DNA degradation products. 16 The statistical processing in the process of determining basic sampling points by performing statistical processing on the sampling candidate points is such that each sampling candidate point is calculated as follows: w _po = a - b log (A + Mn) (1) (where w _po represents the distance of the sampling candidate point from the reference point in the scanning direction; n represents the number of the sampling point corresponding to the candidate point; A and M are constants) The autoradiography system according to claim 15, characterized in that a and b are determined by approximating a and b, and then a basic sampling point is determined by equation (1) based on a and b. signal processing method.