JPS6118865A - Signal processing for autoradiography - Google Patents

Abstract

PURPOSE:To determine the base sequence of DNA at a high accuracy, by making a storage phosphor sheet absorb radiation energies from radioactive label substances in respective separate development trains of at lesst two groups of base-specified DNA synthetic matter mixtures to process. CONSTITUTION:Radioactive energies released from radioactive label substances in respective separate development trains made up of at least two groups of base-specified DNA synthetic matters or mixtures containing at least one kind of base-specified DNA synthetic matter and mixture of guanine-specified DNA synthetic matter, adenine-specified DNA synthetic matter, thymine-specified DNA synthetic matter and cytosine-specified DNA synthetic matter are absorbed by a storage phosphor sheet 1 and scanned in an electromagnetic wave 15 with a light deflector 18 to be photoelectrically read out with a photo detector 23 whereby the separate development train of the DNA synthetic matter mixture is established as reference train. Sampling points of adjacent separate development trains are identified based on the reference sampling points of the reference train.

Description

[Detailed description of the invention]

FIELD OF THE INVENTION The present invention relates to signal processing power methods in autoradiography. More specifically, the present invention relates to a method for comparatively identifying the sub-deployment position of a radiolabeled substance in digital signal processing for base sequencing of DNA or DNA fragments in autoradiography. BACKGROUND OF THE INVENTION Autoradiography is already known as a method for obtaining positional information of radiolabeled substances that are distributed in at least one dimension in a support medium to form a distribution array. For example, after attaching a radioactive label to a macromolecular substance derived from a biological body such as a protein or nuclear acid, the radiolabeled macromolecular substance, its derivative, or its decomposition product (hereinafter also referred to as radiolabeled substance) is used. By subjecting the gel-like support medium to a separation operation such as electrophoresis to separate and develop the radiolabeled substance, a separated and developed column (however invisible) of the radiolabeled substance is formed on the support medium. minute 1
An autoradiograph of the 11i development row is acquired as a visible image on a radiographic film, and positional information of the radiolabeled substance is obtained from the visible image. Furthermore, methods for separating and identifying the polymeric substance, or evaluating the molecular weight and characteristics of the polymeric substance based on the obtained positional information of the radiolabeled substance have already been developed and are currently in use. . Particularly in recent years, autoradiography has been
It is effectively used to determine the base sequence of A (or DNA fragment, hereinafter the same). The Sanger-Coulson method is known as one of the representative methods for determining the base sequence of DNA using autoradiography. In this method, DNA has a double helical structure consisting of two chain molecules, and each of the two chain molecules contains four types of bases, namely adenine (A), guanine (G), and cytosine. (C) and thymine (T), and the two chain molecules are cross-linked by hydrogen bonds between these four types of bases, and each Focusing on the characteristic structure of DNA, in which hydrogen bonds between structural units are realized only in two types of combinations: G-C and A, -T, DNA synthase (which synthesizes DNA fragments, This is a method of determining the base sequence of DNA by skillfully utilizing the means of Kell electrophoresis and autoradiography. There are several methods to synthesize NA fragments that are complementary to DNA).Basically, single-stranded sample DNA is used as a template (template 1. By allowing DNA polymerase to act in the presence of nucleoside triphosphates, D of various lengths complementary to the sample DNA are synthesized.
Synthesize the NA fragment. At this time, some of the mononucleoside triphosphates are radioactively labeled, and the synthesis conditions are modified to include a 1' base, making it specific for any one of the four types of bases. By doing so, the base-specific synthesized N
A fragment (DNA synthesis product) is obtained. Next, the mixture consisting of a large number of DNA compounds obtained by this operation is separated and developed on a support medium by gel electrophoresis (however, it cannot be seen visually). Conventionally, the separation and development array on the support medium is visualized on an X-ray film to obtain an autoradiograph, and based on the obtained autoradiograph, the base sequence of the chain molecule is determined sequentially from the end. In this way, the sequence of all bases in the sample DNA is determined. Note that the characteristics and operation of the Sanger-Coursin method summarized above are described, for example, in the following literature. ■゛Reading genetic information in its original language: Surprising DNA sequence analysis method J Yoshiro Miura, Gendai Ilaku, September 1977 issue, pages 46-54 (■Tokyo Kagaku Doujin Publishing) Sanger,
F., N1cklen, S., & Coalson
, A, R,. Proc, Natl, Acad, Sci, US
A. 74. pp, 54e3 - As mentioned above, in the use of conventional radiography, in order to obtain positional information of a radioactively labeled substance, an autoradiograph with this positional information is visualized on a radiographic film. It has become essential. 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 substances that have been radiolabeled. Gaining knowledge. That is, the DNA base sequence is determined by visually determining the separation position for each of the radioactively labeled base-specific DNA compounds or mixtures thereof, and then determining the separation and development sequence of these base-specific DNA=A compounds. are determined by comparing them with each other. Therefore, analysis of the obtained autoradiograph is usually performed 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, if the autoradiograph visualized on radiographic film does not have good image quality (sharpness, contrast), it is difficult to obtain satisfactory information and its accuracy tends to decrease. There's a problem. Conventionally, in order to improve the accuracy of the location information obtained, 1. For example, a method is also used in which 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, during an exposure operation in which a radiation film is brought into close contact with a support medium on which the separation development rows are formed, a shift may occur in the overlapping of the two, and in this case, a visible image may be obtained on the radiation film. Separation and development columns (e.g., electrophoresis columns) are not parallel to the length of the film, but are shifted, making it easy for errors to occur when visually determining the positional information of radioactively labeled substances, resulting in poor accuracy. tends to decline. Furthermore, depending on the support medium and the separation and development position, the resulting separation and development rows are often not parallel to the length direction of the support medium or are distorted. Furthermore, when using gel as a support medium, this gel does not have self-supporting properties, so it is usually separated and developed by holding both sides of glass, but the thickness of the gel may be uneven due to deformation of the coating. The radiolabeled substance may not necessarily be separated and developed uniformly on the support body. Similar non-uniform separation and development also occurs when the gel contains air bubbles or when the composition of the gel is non-uniform. For this reason, a so-called smiling effect often appears, in which, for example, the moving distance of the separation and development rows at both ends is relatively short compared to the movement distance of the separation and development rows near the center of the support medium. Alternatively, when performing separation and development by electrophoresis, the voltage may not be applied uniformly to the support medium, and in such cases, the separation and development position (position) differs locally depending on the support medium, so the resulting separation and development Distortion tends to occur in the column. There is no suitable method to correct such distortion in the separation/development column other than artificially correcting it. Therefore, in the above cases, it is necessary to Analysis becomes particularly difficult.1 Even when using the above-mentioned measuring instruments, it is difficult to obtain positional information of the separated and developed radiolabeled substance, that is, the base sequence of DNA or DNA fragments, with sufficient accuracy. [Summary of the Invention] The present invention utilizes a radiographic image conversion method using a stimulable phosphor sheet instead of the radiographic method using a radiographic film used in conventional autoradiography. After obtaining the position information as a digital signal from an autoradiograph that captures the position information of the substance without specifically converting it into an image, the distribution position of the radiolabeled substance is detected by applying signal processing to this digital signal. DNA or D
The fifth invention has been achieved by realizing the simple and highly accurate determination of the base sequence of NA fragments. That is, the present invention provides a signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, which has a base sequence complementary to the DNA or DNA fragment, and has a radioactive label. 1) At least one type of base-specific DN
A base-specific DNA compound containing the A compound, 2) guanine-specific DNA compound, adenine-specific DNA
A compound, a mixture of a thymine-specific DNA compound and a cytosine-specific DNA compound; By absorbing the emitted radiation energy into the stimulable phosphor sheet, the radiolabeled substance group force 1 of the separated and expanded array formed by the dimensional expansion of the stimulable phosphor sheet After accumulating and recording an autoradiograph having positional information of the group of radiolabeled substances, the single stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is emitted. Regarding the digital signal corresponding to the autoradiograph of each separated development column obtained by photoelectrically reading out,

[) a step of detecting a reference sampling point for the separation and development column of the DNA compound mixture as a reference column, 11) a step of detecting a sampling point for a separation and development column other than the reference column; Provided is a signal processing method in autoradiography that includes the step of identifying the sampling points of adjacent separation expansion columns by comparing and collating the sampling points of adjacent separation expansion columns using the reference sampling point of the reference column as a reference. It is something to do. The present invention involves superimposing a sample and a stimulable phosphor sheet so that the radiation energy emitted from the sample is absorbed by the stimulable phosphor sheet, and then absorbing electromagnetic waves such as visible light and infrared rays into the stimulable phosphor sheet. ([1il
The radiation energy stored in the stimulable phosphor sheet is emitted as fluorescence (primary luminescence) by scanning with 1 luminescence), and this fluorescence is read photoelectrically to obtain an electrical signal. This method utilizes a radiation image conversion method that involves A/D conversion to obtain a digital signal. Regarding the radiation image conversion method described above, for example, Japanese Patent Application Laid-open No.
It is described in Publication No. 7355-12145 and the like. The stimulable phosphor sheet used in the present invention is, for example,
It contains a stimulable phosphor such as a divalent europium-activated alkaline earth metal fluorohalide phosphor. This stimulable phosphor is irradiated with radiation such as X-rays, alpha-rays, beta-rays, gamma-rays, and ultraviolet rays and accumulates a portion of the radiation energy. ) has the property of exhibiting stimulated luminescence depending on the accumulated energy. 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, by the radiation image conversion method using the stimulable phosphor sheet described in -1-. 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 turbidity and distribution of radioactive substances at that location. In addition, in the present invention, the reference sequence refers to a separation containing all of the base-specific DNA compounds of the four types of DNA bases, namely, guanine, adenine, thymine, and cytosine.
In determining the base sequence of sample DNA, it serves as an internal standard for a separation and development column in which a radioactively labeled base-specific DNA compound is separated and developed on a support medium. . The labeled DNA used to obtain the reference column was sample D.
It may be made using NA or another DNA, but the former is more preferable. According to the present invention, the positional distortion during separation and development of the radiolabeled substance on the support medium as described above, or the autoradiograph of the radiolabeled substance distributed in the -dimensional direction to form a distribution row. High accuracy (DNA
Alternatively, the base sequence of the DNA fragment can be determined. In particular, it is possible to identify the liI and expansion site of each column without correcting the distortion in the direction of separation and expansion. Therefore, it is possible to identify the base with high precision and rationally. This can be set as Array 4. In addition, in order to conduct experiments efficiently, -□Generally, as many separation and development columns as possible are provided on the support medium. As a result, distortions in the separation and development sequence, such as the above-mentioned smiling effect, are likely to occur, but the present invention uses four types of base-specific DNA as internal standards.
By providing a reference row that includes all of the composites, it is possible to correct the distortions of all of the separated spread rows on the support medium. [Structure 1 of the Invention An example of a sample used in the present invention is a deoxynucleoside triphosphinic acid labeled with a radioactive label using DNA or a DNA fragment as a template]
Each base-specific DNA compound synthesized using (dNTP) and a DNA synthase and a mixture thereof are -
Examples include support media that are separated and expanded in a dimensional direction to form separated and expanded rows. In the present invention, the radioactive element used in the radioactive label may be any nuclide as long as it emits radiation (α rays, β rays, γ rays, neutron rays, X rays, etc.). As a specific example, 32F! 4C, 353,3H,
Examples include usl. In addition, the above radiolabeled substance was added in 1i1 minutes using a support medium.
11 Development methods include, for example, electrophoresis using gel-like support media (any shape such as layered or columnar), polymer moldings such as acetate, or various support media such as filter paper, and supports such as silica gel. A typical method is thin layer chromatography using a medium. Among these, electrophoresis using a gel-like support medium is a typical separation and development method and is preferred. 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 a stimulable phosphor in a dispersed state. For example, divalent europium-activated barium fluoride bromide (BaFBr:Eu2+) phosphor particles are bonded to nitrocellulose, a linear polyvarnish, and a chilled phosphor layer. It is obtained by dispersing and containing it in a mixture with. A stimulable phosphor sheet is, for example, one in which a sheet such as polyethylene terephthalate is used as a support, the above-mentioned phosphor layer is provided on this sheet, and a polyethylene terephrate sheet or the like is further provided as a protective film on the phosphor layer. It is. For details of the support medium and stimulable phosphor sheet used in the present invention, please refer to the patent application filed by the applicant in
It is described in the specification of No. 193419. In the present invention, the operation (exposure operation) of accumulating and recording radiation energy emitted from a support medium containing a radiolabeled substance onto a stimulable phosphor sheet is performed by overlapping the support medium and the stimulable phosphor sheet for a certain period of time. Accordingly, 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 sample preparation method, stimulable phosphor sheet, and exposure operation in the first traradiography using the Sanger e-Coulson method, please refer to the patent application filed in 1973 by the applicant.
8-201231. Next, in the present invention, a method for reading out one-dimensional positional information of a radiolabeled substance on a support medium stored and recorded 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 is for pre-reading to temporarily read out the one-dimensional positional information of the radiolabeled substance accumulated and recorded on the stimulable phosphor sheet (hereinafter sometimes abbreviated as phosphor sheet). The device is composed of a readout section 2 and a main reading readout section 3 having a function of reading out the autoradiograph stored and recorded on the phosphor sheet 1 in order to output positional information of the radiolabeled substance. 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, so that 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 removed. be cut. Next, the laser beam is deflected by a light deflector 7 such as a galvanometer mirror, reflected by a plane reflecting mirror 8, and then one-dimensionally deflected and incident on the phosphor sheet l. In the laser light source 4 used here, the wavelength range of the laser light 5 is
It is selected so as not to overlap with the main wavelength region of stimulated luminescence emitted from the phosphor C. 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 beam. In addition, the output of the laser light source 4, the beam diameter of the laser light 5, and the laser. The scanning speed of the light 5 and the transport speed of the phosphor sheet 1 are adjusted so that the energy of the laser beam 5 for the pre-reading operation is smaller than the energy used for the main reading operation. 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 pre-reading light guiding sheet IO. The light guide sheet 10 has a linear incident surface and is disposed close to the scanning line on the phosphor sheet 1-1, and its exit surface forms an annular ring.・It is connected to the light receiving surface of the photodetector 1l, such as a circle. 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 guiding 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 1f, which transmits only light in the wavelength region of stimulated luminescence and cuts light in the wavelength region of excitation light (laser light), and detects only stimulated luminescence. It's getting wet. Stimulated luminescence detected by photodetector l1 is converted into an electrical signal, which is further amplified and output by amplifier l2. 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 l3 sets the amplification factor setting value a in accordance with the obtained accumulated recording information so that a signal at an appropriate level is obtained.
and output the recording scale factor b. Phosphor sheet 1 whose pre-reading operation has been completed as described above
is transferred to the reading section 3 for main reading. In the main reading reading section 3, the following main reading operation is performed. Laser light emitted from the main reading laser light source 14]
After passing through a filter 5 having the same function as the filter 6 described above, the beam diameter is adjusted to scatter by a beam expander 17. Next, the laser beam is deflected by a light deflector 18 such as a galvanometer mirror, reflected by a plane reflecting mirror 19, and then one-dimensionally deflected and incident on the phosphor sheet 1. Note that there is an fθ lens 20 between the optical deflector 18 and the plane reflecting mirror 19!
:f is 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 during irradiation with the above-mentioned polarized laser light. Therefore, the entire surface of the phosphor sheet 1 is irradiated with the polarized laser beam, as in the pre-reading operation. 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 range 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 sent to the A/D converter 25. is input. A/D converter 2
5 is converted into a digital signal with a scale factor suitable for the signal fluctuation range according to the recording scale factor setting value. Regarding the method for reading out the positional information of the radiolabeled substance on the support medium stored and recorded on the stimulable phosphor sheet in the present invention, Section 2 describes the readout operation consisting of the pre-reading operation and the main reading operation. However, 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 material in the support medium and the exposure time of the stimulable phosphor sheet for that support medium are known in advance, the look-ahead operation can be omitted in the above example. Furthermore, as a method for reading out the positional information of the radiolabeled substance in the support medium stored and recorded on the stimulable phosphor sheet in the present invention, it is of course possible to use any method other than those exemplified above. be. The stimulable phosphor sheet used in the present invention can be reused by irradiating the sheet with appropriate light or heating it to eliminate residual radiation energy after the above readout operation is completed. can. 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. Signal processing circuit 26
Then, the base sequence of the target DNA is determined by converting the dimensional positional information of the radiolabeled substance into symbols and/or numerical values. Hereinafter, the embodiments of signal processing in autoradiography for DNA IiA group sequencing using the signal processing method of the present invention will be explained using the following five types of signal processing using the above-mentioned Sanger or Courland method as an example. base specific dna
A case will be described in which an electrophoretic column (separation and development column) formed by a combination of a synthetic compound and a DN'A compound mixture is used. 1) Guacone-specific DNA compound, 2) Azocone-specific DNA compound, 3) Thymine-specific DNA compound, 4) Cytosine-specific DNA compound, 5.) Guanine-specific DNA compound + Adenine-specific DNA compound + Thymine-specific DNA compound + Cytosine-specific DNA compound First, using the sample DNA as a template, a radioactive label ("
By base-specific synthesis using a mononucleoside triphosphate to which P) has been added and a DNA polymerase, base-specific DNA compounds of the five groups 1) to 5) are synthesized by a conventional method using a mononucleoside triphosphate and a DNA polymerase. A DNA synthesis mixture is obtained. Next, the above five groups of base-specific DNA compounds and the DNA compound mixture were separated by electrophoresis on a gel support medium.
Expand to obtain each separated expansion sequence. Next, an exposure operation is performed by overlapping this sample (gel-like support medium with separation and development columns formed) and a stimulable phosphor sheet at room temperature for several minutes, and the autoradiograph of the sample is transferred to the stimulable phosphor sheet. Accumulate and record. For details of the above exposure operation, please refer to the above-mentioned patent application No. 58-2012.
No. 3'1, described in the specification. Figure 2 shows that each base-specific DNA compound and DNA compound mixture of radiolabeled DNA has a molecular weight of 48%.
An example of an autoradiograph of a separation development column (electrophoresis column) consisting of the five types described above is shown below. That is, the first to fifth columns in FIG. 2 are (1) -
(G) Specific DNA compound (2) - (A) Specific DNA compound (3) - (G) Specific DNA compound + (A) Specific DNA compound + (T) Specific DNA compound + (C) #Different DNA composite (4) - (T) Specific DNA composite (5) - (C) Each separation development column of specific DNA composite is shown. The third column is (G, A, T, C
) - Contains all base-specific DNA compounds and is an internal standard column (reference column) for base sequencing. By loading the autoradiograph stored and recorded on the stimulable phosphor sheet into the reading device shown in FIG. 1 and reading it out, the digital signal input to the signal processing circuit 26 is
The stimulable phosphor sheet has an address (x, y) expressed by a grid system fixed on the stimulable phosphor sheet and a signal level (Z) at that address, and the signal level is determined by the amount of photostimulant light. It corresponds to That is, the digital signal corresponds to the autoradiograph of FIG. Therefore, the signal processing circuit 26
Digital image data having the positional information of the radiolabeled substance is inputted to the field. In this specification,
Digital image data means a collection of digital signals corresponding to an autoradiograph of a radiolabeled substance. First, using digital image data, the direction of separation and development of the radiolabeled substance is detected for each of the five separation and development rows, and these are used as sampling points. The sampling point can be detected, for example, as follows. ``By scanning different positions on the digital image data twice across the one-dimensional distribution direction (separation development direction) of the radiolabeled substance, each column on each scanning area is The scan to detect the distribution point of the radiolabeled substance is called a preliminary scan), connect the two distribution points for each separation development column to obtain five straight lines, and connect the obtained straight lines to each This is the scanning direction for sampling point detection in each column. In the signal processing method of the present invention, the digital signal obtained by reading out the stimulable phosphor sheet is stored in the -H memory in the signal processing circuit 26 (i.e.,
(stored in non-volatile memory such as hard disk, hard memory, or magnetic disk). In signal processing, scanning digital image data means selectively retrieving only the digital signals at the scanning location from memory. Next, by scanning the digital image data ``2'' along the scanning direction, a function f (w) representing the level of the signal in the scanning area ``2'' [w represents the position of the scanning direction ``2]]
can be obtained. Then, compile this function f (w) using, for example, an appropriate filter function. , - John to perform smoothing processing and obtain the function g(W). Next, threshold processing is performed on this function g(w). That is, for the threshold value (α0), when g (w)≧α0, g (w) −1g (w)
When <α0, the function g (w) is converted into a continuous function of 1 or O by performing processing to set g(w)=0. The sampling points are detected by setting each midpoint of the area of g(w)=1. Note that the threshold value (α0) in the above threshold processing is determined based on, for example, the relationship between the signal rail and its frequency for digital signals on the scanning area,
That is, it can be determined from the histogram. In this way, sampling points 5icn can be detected for each column. Here, k is a positive integer and represents the number of each column, and n is a positive integer and represents the number of the sampling point. Note that the method for detecting sampling points is not limited to the above method. Next, the comparison identification between each column specifically includes the task of searching for the same separation expansion between the reference column and the remaining separation expansion columns [for example, (G) + (A) + - ( T) + (C
) and CG), use (G,)+
Find the separated expansion that is an element of (G) from the column (A) + (T) + (C)]. However, when this comparative identification is distorted, the positions of the equivalent separation and expansion columns of each column are not necessarily equal on the X coordinate as shown in FIG. Conventionally, correction of such distortions has been left to human visual judgment. However, according to the method of the present invention, by using a reference column and a reference sampling point, distortion can be automatically corrected without relying on humans, and accurate comparison and identification between columns can be performed. This will be explained based on FIG. FIG. 3 is a diagram showing a portion of the reference column (third column) and the fourth column. Here, the black squares represent sampling points in each column corresponding to the separation and deployment site of the radiolabeled substance, and the hollow squares represent interpolated reference sampling points. Using the sampling point 33n of the third column, which is the reference column, as the reference sampling point, the sampling point s of the adjacent fourth column
By performing comparison identification with an, interpolation of reference sampling points is performed for the fourth column. For example, regarding the sampling point S41 in the fourth column,
The position (X41) of sampling point S41 is compared with the position (X32) of reference sampling point 332 and the position (X33) of reference sampling point 333 in the third column. For example, if lX32 X411=b lX33 In this way, all the sampling points in the fourth column are assigned to one of the reference sampling points in order. Then, based on the assigned sampling point san of the fourth column, each of the remaining reference sampling points of the third column is interpolated into the fourth column, thereby creating a virtual set of reference sampling points in the fourth column. Create (54m). However, m is a positive integer and matches the number n of the sampling point in the third column. In this way, a virtual reference column can be obtained by moving the reference column obtained in the third column to the position of the fourth column. Next, based on the created reference sampling point sam of the fourth column, the reference sampling point s is connected to the adjacent sampling point S5n of the fifth column via the reference sampling point 54m.
3n and further interpolates the reference sampling point S4m in the fourth column to the fifth column, thereby creating a virtual set of reference sampling points (35m) in the fifth column. In this way, based on the reference sampling point s3n of the reference column, while creating a virtual reference sampling point set (Skm) in each column in turn, all the sampling points Skn are set to the reference sampling point S, 3n. Attribute to either. That is, the above method comprises: 1) at least three groups of base-specific DNA compounds each containing at least one type of base-specific DNA compound that is different from each other;
NA compounds, and 2) guanine-specific DNA? fm
substance, adenine-specific DNA compound, thymine-specific DNA
At least a positive group of base-specific DNA compounds and a mixture of cytosine-specific DNA compounds, each of which includes a compound and a mixture of cytosine-specific DNA compounds, are separated and developed one-dimensionally in parallel 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 column formed by
After this stimulable phosphor sheet accumulates and records 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 each fraction g! obtained by photoelectrically reading out this stimulated light! :
Regarding the digital signal corresponding to the autoradiograph of the development column, l) using the separation development column of the DNA compound mixture as a reference column, detecting a reference sampling point for the reference column; 11) each separation development other than the reference column; iii) comparing and checking the sampling points of the separation expansion column adjacent to the reference column using the reference sampling point of the reference column as a reference; a step of identifying a sampling point and establishing a new reference sampling point based on the identified sampling point, iv
) The reference sampling point newly determined in step iii) above is compared with the sampling point of the separate expansion column adjacent to the one-separation expansion column in which the reference sampling point has been determined, and the adjacent The method can be summarized as a signal processing method including the step of identifying sampling points of a separation expansion sequence. Next, regarding the first column, second column, fourth column, and fifth column,
The virtual reference sampling point (Sk The sampling point S 3'n of column ) is replaced with the matching sampling point 5krl. Then, if we follow the reference column in order of decreasing n, we get
For example, you can obtain the following diagram. 5111551, 5411SI2. S21°S22+,
S12+Sf3+"52+...In the above diagram, 3.:=G, 52n=A, 54n=T, 55n=
By substituting C, we obtain the following diagram.
. G-C-T-G-A-A-T-G-C-...In this way, the base sequence of one chain molecule of DNA'A can be determined. In addition, the obtained DNA
The information about the 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 performing arithmetic processing on the signal level in the scanning direction of each column. Furthermore, it is also possible to display the base sequences of both two stranded molecules of DNA. In other words, by providing the information A-T, G-C1C-G, and T-+A as combinations corresponding to each base in the diagram represented by the two symbols, the following diagram can be obtained. D.N.
Obtain the base sequence of A. G-C-T-G-A-A-T-(,-C-...
C-G-A-C-T-T-A-C-G-...The signal processing method of the present invention allows the above (G+A+T
The DNA base sequencing method using the combination of (10C, G, A, T, C) is an example of the NA base sequencing method, and the signal processing method of the present invention uses the above combination. The combination is not limited to this, and various combinations are possible, and the base sequence can be determined in the same manner as the above-mentioned method using the combination. However, in any combination, as an internal standard,
It is necessary to provide a row (reference row) of a mixture of all base-specific DNA compounds of G, A, T, and C. The separation and development position of this reference column does not necessarily need to be placed in the center of the support medium as described above, but in order to determine the DNA base sequence with higher accuracy, it is necessary to place it in the center of the support medium. is preferable. Furthermore, the reference column is not applied only to the combination of base-specific DNA compounds of the DNA set, but also to each of the radiolabeled substance groups consisting of other DNA compounds separated and developed on the support medium. It can be applied to In the present invention, for example, a sequence for a specific base is determined from a combination of at least one group of base-specific DNA compounds and an appropriate reference substance (for example, a mixture of each base-specific DNA compound). It is also possible to do so. In addition, in the example described in -■2, the explanation was made using five rows of radiolabeled substance groups that are spread in one-dimensional direction on the support medium, but the number of separated and developed rows is limited to five rows. The signal processing method of the present invention is particularly effective when there are more than five columns. That is, the present invention is a method of performing identification while sequentially correcting deviations in the direction of minute glI expansion with the reference column as the center, so the present invention can be used more effectively as the number of separated expansion columns increases. It's Chino. Alternatively, it may be one column or less. Alternatively, it is also possible to simultaneously determine the base sequences of two or more types of DNA using one support medium. Information about the DNA base sequence determined by the signal processing method described above is output from the signal processing circuit 26, and then stored in a recording device directly or, if necessary, via a storage means such as a magnetic tape. (not shown). Recording devices include, for example, those that optically record by scanning a photosensitive material with a laser beam, etc., those that electronically display on a CRT, etc., and those that record numerical values in symbols displayed on a CRT, etc. with a video fist. Recording devices based on various principles can be used, such as those that record on a printer or the like, and those that record on a heat-sensitive recording material using heat rays. In addition, the information obtained as described in Section 2 can also be subjected to genetic linguistic information processing, such as comparing it with other DNA base sequences that have already been recorded. It is.

[Brief explanation of drawings]

FIG. 1 shows an example of a reading device (or reading device) 6' for reading the positional information of a radiolabeled substance in a sample stored and recorded on a stimulable phosphor sheet in the present invention. 1: Stimulative 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: plane reflecting mirror, 9: transport direction, 10: light guide sheet for pre-reading, 11: photodetector, 12: amplifier, 13: control circuit, 14: laser light source, 15 : Laser light, 16: Filter, 17: Beam expander - 118: Optical deflector, 19: Planar reflecting mirror, 20: fθ lens, 21: Transfer direction, 22 Light guiding sheet for dual reading, 23: Photo detection instrument, 24: 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 base-specific DNA compounds of sample DNA are separated and developed on a gel support medium. FIG. 3 is a partially enlarged view of the autoradiograph of FIG. 2. Patent applicant Fuji Photo Film Co., Ltd. Agent Patent attorney Yasuo Yanagawa Figure 1 Figure 2 Figure 3

Claims (1)

[Scope of Claims] 1. A signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, which has a base sequence complementary to the DNA or DNA fragment and is radioactive. Labeled: 1) a base-specific DNA compound containing at least one type of base-specific DNA compound; 2) a guanine-specific DNA compound; an adenine-specific DNA compound;
A compound, a mixture of a thymine-specific DNA compound and a cytosine-specific DNA compound, respectively, each of at least two groups of base-specific DNA compounds and a mixture of DNA compounds, comprising: A compound, a mixture of a thymine-specific DNA compound and a cytosine-specific DNA compound; By causing the stimulable phosphor sheet to absorb the radiation energy emitted from the group of radiolabeled substances in the separated and expanded array formed by one-dimensionally separating and expanding,
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; Then, regarding the digital signal corresponding to the autoradiograph of each separation and development column obtained by photoelectrically reading out this photostimulated light, i) With the separation and development column of the DNA composite mixture as a reference column, regarding the reference column; ii) detecting sampling points for a separation expansion column other than the reference column; and iii) comparing sampling points of adjacent separation expansion columns using the reference sampling point of the reference column as a reference. 1. Identifying sampling points of adjacent separation expansion sequences by performing a signal processing method in autoradiography. 2. Claim 1, characterized in that the sampling points are detected by performing smoothing and/or threshold processing on the digital image data in the scanning direction of each of the plurality of separation expansion columns. Signal processing method in autoradiography described. 3. A signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, which has a base sequence complementary to the DNA or DNA fragment and is given a radioactive label. , 1) at least two types of base-specific DNA compounds, each containing at least one type of base-specific DNA compound that is different from each other; 2) a guanine-specific DNA compound, an adenine-specific DNA compound;
A compound, a mixture of thymine-specific DNA compound and cytosine-specific DNA compound; 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 one-dimensional separation and development, the stimulable phosphor sheet absorbs the radiolabeled substance group. After accumulating and recording an autoradiograph having positional 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 separation and development column obtained, i) using the separation and development column of the DNA composite mixture as a reference column, detecting a reference sampling point for the reference column; ii) a reference column; iii) comparing sampling points of separation expansion columns adjacent to the reference column using the reference sampling points of the reference column as a reference; step of identifying the sampling point of and determining a new virtual standard sampling point based on the identified sampling point, iv) the standard sampling point newly determined in the step of iii) above and its standard sampling A signal processing method in autoradiography, comprising the step of: identifying a sampling point of an adjacent separation expansion sequence by comparing and collating a separation expansion sequence with defined points with a sampling point of an adjacent separation expansion sequence. 4. Separation and expansion columns other than the reference column are arranged on both sides of the reference column, and the step iii) is performed for both of the two adjacent separation and expansion columns. Signal processing methods in autoradiography. 5. Claim 3, characterized in that the sampling points are detected by performing smoothing and/or threshold processing on the digital image data in the scanning direction of each of the plurality of separation development columns. Or the fourth
Signal processing method in autoradiography as described in section. 6. Base-specific DNA synthesis of DNA or DNA fragments includes: 1) guanine-specific DNA synthesis, 2) adenine-specific DNA synthesis, 3) thymine-specific DNA synthesis, 4) cytosine-specific DNA synthesis. 5) at least five groups of base-specific DNA compounds and DNA compound mixtures consisting of guanine-specific DNA compounds + adenine-specific DNA compounds + thymine-specific DNA compounds + cytosine-specific DNA compounds; A signal processing method in autoradiography according to any one of claims 3 to 5, characterized in that: