JPH0570111B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an autoradiographic analysis method.

[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 to form a distribution array on a support medium.

For example, a radiolabel is added to a biologically derived polymeric substance such as a protein or a nucleic acid, and the radiolabeled polymeric substance, its derivative, its decomposed product, or its composite is separated using gel electrophoresis or other methods. By overlapping the gel-like support medium and a high-sensitivity X-ray film for a certain period of time, the film is exposed and the radioactivity on the gel-like support medium obtained from the exposed area Methods for separating, identifying, or evaluating the molecular weight and characteristics of a polymeric substance based on the positional information of a labeling substance have also been developed and are in actual use.

Particularly in recent years, autoradiography has been effectively used to determine the base sequence of nucleic acids such as DNA and RNA. There are also southern blotting, northern blotting, and western blotting.
It is also an indispensable tool in genetic screening using hybridization methods such as blotting.

The present applicant has already filed a patent application for a method of utilizing a radiation image conversion method using a stimulable phosphor sheet in place of the conventional radiography method using the above-mentioned radiation film in autoradiography (Japanese Patent Application Laid-Open No. No. 59-83057, JP-A-60-10174
No., Japanese Patent Application No. 173393/1982 (Japanese Patent Application No. 66998/1983)).
The stimulable phosphor sheet is made of stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, it is excited with electromagnetic waves (excitation light) in the visible to infrared region. This allows radiation energy to be emitted as fluorescence. According to this method, the exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur.
Furthermore, an autoradiograph of a radiolabeled substance is once stored as radiation energy in a phosphor sheet and then read out in chronological order as photostimulated light, so it can be displayed and recorded in any format such as symbols and numbers in addition to images. Is possible.

Traditionally, those who wish to analyze autoradiographs have been able to detect the distribution of radiolabeled substances on the support medium by visually judging the visualized autoradiographs and detecting the distribution of radiolabeled substances on the support medium. We have obtained knowledge of location information (and various information based on it, such as identification of polymeric substances, evaluation of their molecular weights and properties, etc.) for specific substances. For example, DNA base sequences can be determined by visually determining the separation and development position of each radioactively labeled base-specific DNA fragment or a mixture thereof, and then mutually aligning the separation and development positions of these base-specific DNA fragments. It is determined by comparing the Therefore, a great deal of time and effort is spent on analyzing autoradiographs.

Furthermore, since it relies on the human eye, there are limits to the accuracy of the information that can be obtained, such as the positional information obtained by analyzing an autoradiograph differing depending on the analyst.

Therefore, the present applicant obtained an autoradiograph of a radiolabeled substance distributed in at least one dimension on a support medium as a digital signal, and then subjected this digital signal to appropriate signal processing to obtain a radiolabeled substance. A patent application has already been filed for a method for automatically obtaining positional information of substances as desired symbols and/or numerical values (Japanese Patent Application Laid-Open No. 1983-1999).
No. 126527, JP-A-59-126278, etc.). When using a conventional radiation film, the digital signal corresponding to the autoradiograph can be obtained by first making the autoradiograph into a visible image on the film and then reading it photoelectrically using reflected or transmitted light. can get. When a stimulable phosphor sheet is used, an autoradiograph can be obtained by directly reading out the phosphor sheet on which the stimulable phosphor sheet has been stored.

[Summary of the Invention] The present inventor has proposed a method for analyzing an autoradiograph having two-dimensional positional information of a radiolabeled substance separated and developed on a support medium. Rather than completely automating the process, we aim to obtain the desired position information with high precision by inputting the necessary information for analysis as appropriate and displaying images at each stage of signal processing based on this input information. It was realized.

That is, the present invention performs signal processing on a digital signal corresponding to an autoradiograph having two-dimensional positional information of a radiolabeled substance separated and developed on a support medium, thereby converting the positional information of a radiolabeled substance into a symbol. and/or the method for analyzing an autoradiograph, which comprises: (1) electrically displaying an image of the autoradiograph based on a digital signal; (2) a radioactive label determined from the displayed image; A step of extracting a signal in a certain range along the separation and expansion sequence based on input information about the separation and expansion sequence of the substance, and (3) performing one-dimensional signal processing on the extracted signal to determine the separation and expansion site. The present invention provides an autoradiographic analysis method characterized by including the step of detecting.

The present invention also provides a method for analyzing an autoradiograph, which comprises obtaining positional information of a radiolabeled substance as a symbol and/or numerical value by performing signal processing on a digital signal corresponding to the autoradiograph. (1) electrically displaying the autoradiograph as an image based on digital signals; (2) displaying the autoradiograph along the separation and development line based on input information regarding the separation and development line of the radiolabeled substance determined from the displayed image; a step of extracting a signal in a certain range; (3) a step of detecting a separation development region by performing one-dimensional signal processing on the extracted signal and then displaying it in a superimposed manner; and (4) a step of superimposing and displaying a separation development region. An autoradiographic analysis method characterized by comprising the steps of: correcting the separation deployment distance for the signal based on input information regarding the position of one separation deployment site determined from the displayed image, and (1) digital (2) electrically displaying an image of the autoradiograph based on the signal; (2) displaying a range of signals along the separation line based on input information about the separation line of the radiolabeled substance determined from the displayed image; (3) performing one-dimensional signal processing on the extracted signal to detect and display the separated development site in a superimposed manner; and (4) displaying the signal corresponding to the separation development site. The present invention also provides an autoradiographic analysis method comprising: correcting the starting position of separation development based on input information regarding the position of one separation development site determined from the image. .

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 position of an aggregate of radioactive substances on a support medium,
It means various types of information obtained as one or any combination of information such as concentration, distribution, identification of radioactive substances, identification of samples based on these, etc.

[Effects of the Invention] The method of the present invention semi-automatically obtains position information of a radiolabeled substance by passing a digital signal corresponding to an autoradiograph of the radiolabeled substance through an appropriate signal processing circuit having a signal processing function. can be obtained as desired symbols and/or numerical values. Then, in the signal processing circuit, by further applying appropriate arithmetic processing and other related information to the position information represented by symbols and/or numerical values, desired information is semi-automatically obtained, for example.
It is possible to determine the base sequence of DNA.

As a result of various studies on autoradiographic measurements used for microanalysis of proteins, base sequencing of nucleic acids, etc., the present inventor discovered that a radiolabeled substance, which is a detection target, is separated and developed on a support medium. It has been found that autoradiographs are usually noisy due to various distortions or impurities. For example, distortion of the entire separation development pattern occurs due to insufficient alignment between the photosensitive material and the support medium during exposure, separation development occurs due to inconsistent separation development conditions, uneven support medium, etc. These include the occurrence of meandering and smiling phenomena in columns, the offset distortion that occurs depending on the operation of attaching the sample to the support medium, and the occurrence of noise due to radiation emitted from radioactive impurities or natural radiation.

When analyzing autoradiographs with these distortions or noises, a high-speed, large-capacity automatic computer is required to automatically recognize and correct distortions or noises on the corresponding digital image data. In addition, it is difficult to guarantee that the analysis results will necessarily have high accuracy.

According to the method of the present invention, rather than completely automatically processing and analyzing digital signals corresponding to autoradiographs obtained by reading photosensitive materials such as radiation films and stimulable phosphor sheets, By providing the necessary information about the various distortions mentioned above and performing appropriate and efficient signal processing based on this information, the time and labor required in the conventional analysis method can be significantly reduced. At the same time, it makes it possible to perform analysis with higher precision and efficiency than if the analysis were completely automated. In other words, by semi-automating analysis operations,
Desired location information can be easily obtained.

In the method of the present invention, when providing necessary information prior to processing at each stage of signal processing,
An autoradiograph based on digital signals
Since the image is displayed on a CRT or the like, the welder can judge and provide necessary information from the displayed image. In particular, the separation and development array of the radiolabeled substance on the support medium is often meandering due to separation and development conditions such as electrophoresis, individual differences in the support medium, exposure conditions, and the like. In addition, the size of each separation deployment site,
The shape, center position, etc. are also different. Therefore,
By inputting information about the separation/deployment sequence prior to digital signal processing, subsequent signal processing such as determination of the separation/deployment site can be performed with high accuracy.

Additionally, the digital image data that has been signal-processed at each stage of signal processing can be displayed overlaid on the autoradiograph before signal processing.
The analyst can make a judgment based on this displayed image and order the execution of further necessary signal processing. That is,
When a separation and development method such as electrophoresis is used, there is a phenomenon in which the separation and development distance at both ends of the support medium is generally shorter than the separation and development distance at the center of the support medium due to heat radiation during the separation and development process (smiling phenomenon). tends to occur. According to the method of the present invention, by inputting information about the position of any one separated deployment site for each separation deployment row, correction for this smiling phenomenon can be performed easily and with high precision.

Alternatively, when depositing the sample on the support medium, the deposition positions may be offset from each other or the sample penetration rate may be different, resulting in a different starting position or time point for separation and development of the sample in each column. Therefore, mutual positional deviation between columns (so-called offset distortion) may occur. According to the method of the present invention, by inputting information about the starting position of the separation and expansion row, correction for this offset distortion can be performed easily and with high precision.

In addition, the digital image data processed at each stage of signal processing can be displayed on the screen, making it possible to check at each stage of signal processing whether or not the analysis is being performed appropriately. It is possible. In particular, the analyst can compare and confirm the positional information of the radiolabeled substance obtained through signal processing and the autoradiograph displayed as an image. You will be given an opportunity to make corrections.

Therefore, in the present invention, digital signal processing for autoradiographic analysis can be semi-automated, and its processing functions can be suitably controlled and adjusted, making it possible to perform autoradiographic analysis with high reliability. can.

[Structure of the Invention] Samples to be separated and developed in the present invention include macromolecular substances derived from living organisms such as radioactively labeled proteins, nucleic acids, derivatives thereof, decomposition products thereof, and synthetic products. can be mentioned. Radioactive labels are imparted by allowing these substances to retain radioactive isotopes such as ³² P, ¹⁴ C, ³⁵ S, ³ H, ¹²⁵ I, etc. in an appropriate manner. When the sample is one of these biopolymer substances, the method of the present invention can be effectively used as a means for separating it and identifying its molecular weight, molecular structure, etc. However, the substances to be subjected to the autoradiographic analysis of the present invention are not limited to these polymer substances.

The sample radiolabeled substance is subjected to electrophoresis, using various known support media such as gelling support media.
Separation and development is carried out on a support medium using various separation and development methods such as thin layer chromatography, column chromatography, and paper chromatography.

Next, an autoradiograph of the support medium on which the radiolabeled substance has been separated and developed is obtained by radiography using a conventional photographic light-sensitive material or by a radiation image conversion method using a stimulable phosphor sheet. A digital signal corresponding to the autoradiograph of the radiolabeled substance is obtained via a readout system.

When using the former radiographic method, first the support medium and a photographic material such as X-ray film are overlaid for a long time (several tens of hours) to expose the radiographic film, and then developed to release the radiolabeled substance. The autoradiograph is visualized on radiographic film. Next, the autoradiograph visualized on the radiographic film is read using an image reading device. For example, an autoradiograph is obtained as an electrical signal by irradiating a radiation film with a light beam and photoelectrically detecting the transmitted or reflected light. Furthermore, by A/D converting this electrical signal, a digital signal corresponding to an autoradiograph can be obtained.

When using the latter radiation image conversion method,
First, the support medium and the stimulable phosphor sheet are overlapped for a short time (several seconds to several tens of minutes) at room temperature, and the radiation energy emitted from the radiolabeled substance is accumulated in the phosphor sheet. is recorded on the phosphor sheet as a kind of latent image. Here, the stimulable phosphor sheet is made of a support made of, for example, a plastic film, divalent europium-activated barium fluoride bromide (BaFBr: Eu ²⁺ )
A phosphor layer made of a stimulable phosphor such as phosphor and a transparent protective film are laminated in this order. The stimulable phosphor contained in the stimulable phosphor sheet absorbs and accumulates radiation energy when it is irradiated with radiation such as X-rays, and then accumulates when excited with light in the visible to infrared region. It has the property of emitting radiation energy as photostimulated light.

Next, the autoradiograph stored and recorded on the stimulable phosphor sheet is read out using a reading device. Specifically, for example, by scanning a phosphor sheet with a laser beam to emit radiation energy as photostimulated light, and detecting this photostimulated light photoelectrically, an autoradiograph of a radioactively labeled substance is converted into a visible image. It can be obtained directly as an electrical signal without any additional processing. Furthermore, by A/D converting this electrical signal, a digital signal corresponding to an autoradiograph can be obtained.

Note that in any of the above methods, it is not necessary to read (or read out) the autoradiograph over the entire surface of the radiation film (or stimulable phosphor sheet), and it is of course possible to read out the autoradiograph only on the image area. .

The obtained digital signal D _xy consists of coordinates (x, y) expressed in a coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (z) at the coordinates. The level of the signal represents the image density at that coordinate, ie, the amount of radiolabeled substance. Therefore, the series of digital signals (ie, digital image data) has two-dimensional positional information of the radiolabeled substance.

For details on the above-mentioned autoradiograph measurement operation and method for obtaining a digital signal corresponding to the autoradiograph, see the aforementioned Japanese Patent Application Laid-open No. 59-83057;
It is described in various publications such as JP-A-59-126527 and JP-A-59-126278.

In the above, a method using conventional radiography and radiographic image conversion methods was described as a method for obtaining a digital signal corresponding to an autoradiograph of a radiolabeled substance separated and developed on a support medium. The analysis method of the present invention is not limited to these methods, and the analysis method of the present invention can be applied to digital signals obtained by any other method as long as there is a correspondence with the autoradiograph of the radiolabeled substance. It is possible.

The digital signal corresponding to the autoradiograph of the radiolabeled substance separated and developed on the support medium thus obtained is subjected to signal processing by the method of the present invention as described below, and the autoradiograph is An analysis of the graph is performed.

The digital signal is temporarily stored in a memory (buffer memory or nonvolatile memory such as a magnetic disk) in a signal processing circuit.

First, based on this digital signal, a display device such as a CRT connected to a signal processing circuit displays the
Displays an autoradiograph (separation and development pattern of a radiolabeled substance) as an image. The digital signal may be subjected to signal processing (image processing) in advance so as to obtain a visible image with appropriate density and contrast and excellent observation and interpretation performance. Examples of image processing include spatial frequency processing, gradation processing, averaging processing, reduction processing, and enlargement processing.

Next, each separation and development column (lane) is determined, and the digital image data is made one-dimensional.

Note that if the entire separated development pattern displayed as an image at this time is tilted due to misalignment during the exposure operation (registration irregularity), as shown in FIG. By rotating the pattern, an erect image (see FIG. 1b) can be obtained.

In the present invention, the term "erect image" includes not only the case where the separation and development directions are aligned in the vertical direction, but also the case where they are aligned in the horizontal direction, as shown in FIG. 1b.

FIGS. 1a and 1b each show an example of a separation and development pattern of a radiolabeled substance displayed as an image.

To rotate a pattern, for example, the operator inputs information about the required amount of rotation (angle, direction) to a signal processing circuit based on the display screen, and then performs signal processing for pattern rotation based on this input information. It is done by doing. Information can be input by keyboard operation, joystick operation, or operation using an input member such as a light pen on the screen. A desired erect image is displayed as an original pattern based on the signal-processed digital signal.

A cursor image is displayed on the screen for this erect image, and information about the lane is input using this cursor line. First, the operator moves the displayed cursor lines so that they overlap on one lane. The cursor line can be moved, for example, by keyboard operation, joy stick operation, or touch pen operation on the screen. At this time, it is preferable that the cursor line be distinguished from the lane by decreasing the dots or displaying it in a color different from that of the image. The moved cursor line is fixed so as to match the straight line portion at the top of the lane (an area closer to the starting position of separation development than near the center of the lane). When the cursor line passes through approximately the center point of each separation development site (band) on the lane, information that a separation development column exists at the position of this fixed cursor line is input to the processing circuit by key operation. Ru.

As shown in Figure 2a, if cursor line 2 does not perfectly match the lane because lane 1 is meandering rather than straight, correct the cursor line on the screen and enter lane information. do. Note that FIGS. 2a and 2b each show an example of the separation and development pattern of the radiolabeled substance displayed as an image.

For example, when performing this by keyboard operation, first the lowest position 3 where the cursor line 2 coincides with the lane 1 is recognized, and then the cursor line segment up to this position is fixed. Next, move the cursor point 4 in the y direction on the cursor line to the mismatched position, then move the cursor point 4 to the x direction without changing the y coordinate.
Fix the cursor point after moving in the direction. By this input operation, a new cursor line segment connecting the position 3 and this fixed cursor point position with a straight line is displayed on the screen, as shown in FIG. 2b. Repeat this operation by dividing it into sections of appropriate length, and if each cursor line segment passes approximately the center point of the band over the entire lane, the separation expansion column will be placed at the position of the final cursor line. Information indicating that exists is input. Alternatively, the cursor line can be corrected by touching the stylus pen to the center point of each band in the area of mismatch on the screen.

Based on this lane information, a certain range of digital signals along each lane is extracted. Specifically, a digital signal that matches the cursor line or a digital signal that is within a certain width area from the cursor line is extracted.

Through this signal processing, a representative profile of the separated development pattern can be obtained. Digital image data that had two-dimensional information now has one-dimensional information for each lane. Note that the profile can be confirmed by displaying an image of the representative profile obtained here superimposed on the original pattern.

Next, one-dimensional signal processing is performed based on the representative profile. For example, the position of each band can be detected by creating a one-dimensional waveform (graph with the position on the lane on the horizontal axis and the signal level on the vertical axis) for the digital signal for each lane, and finding its peak. can do. Furthermore, by comparing peak positions between lanes, information about relative band positions in the entire separation development pattern can be obtained.

However, the one-dimensional signal processing method for detecting each band and determining the relative position between lanes of each band is not limited to the above method, and various calculation processes can be performed. .

If desired, by displaying the detected band peaks on the screen superimposed on the original pattern, the operator can confirm the analysis results while looking at this superimposed display screen. Furthermore, the obtained position information may be simultaneously displayed on the screen as numerical values and/or symbols. At this time, it is also possible to partially modify the analysis results if necessary.

Next, an embodiment of autoradiographic analysis using the method of the present invention will be described using DNA base sequencing as an example.

Regarding a sample obtained by separating and developing a mixture of base-specific DNA fragments with the following four types of radioactive labels on a gel support medium by electrophoresis,
The autoradiograph is stored in a memory in a signal processing circuit as a digital signal by a radiation image conversion method using a stimulable phosphor sheet or by a radiography method using a photosensitive material.

(1) Guanine (G)-specific DNA fragment (2) Adenine (A)-specific DNA fragment (3) Thymine (T)-specific DNA fragment (4) Cytosine (C)-specific DNA fragment Here Each base-specific DNA fragment is base-specifically cleaved, degraded, or synthesized, that is, it consists of DNA fragments of various lengths that have the same terminal base.

First, based on the obtained digital signal, an autoradiograph to be analyzed is created as shown in Figure 3.
Visualize the image on CRT.

FIG. 3 shows an autoradiograph of the electrophoresis pattern obtained by electrophoresing the above-mentioned four types of base-specific DNA fragments into four slots.

Next, based on the input of lane information for each float determined using the cursor line on this display image as described above, a certain range of digital signals along each lane is extracted (representative). profile). Specifically, only the digital signal D' _xy having certain (x, y) coordinates based on the lane information is extracted. The extracted digital signal D' _xy can be expressed as a signal D' _oy consisting of the slot number (n) and the migration coordinate (y').
That is, once the slot is determined, it can be expressed only by the migration coordinate (y'), and the two-dimensional information consisting of (x, y) has been made one-dimensional.

Note that if the entire migration pattern is tilted, it is preferable to rotate the pattern before lane determination. That is, the digital signal D _xy is first converted into a digital signal D″ _xy by performing coordinate rotation processing (x→x″, y→y″) based on the input information.
On the screen, a separated development pattern based on the digital signal D″ _xy subjected to this pattern rotation is displayed as an original pattern.

Next, one-dimensional signal processing is performed on the extracted digital signal D' _oy for each slot.
For example, as described above, all bands _B'oy on the electrophoresis pattern are detected by searching for peaks in the one-dimensional waveform of each lane. Here, band B′ _oy is the migration coordinate at the nth slot.
It has information consisting of y′ and band intensity z′.

This detected band on the original pattern
By superimposing and displaying B′ _oy , the signal processing results can be confirmed. In addition, when the original pattern is meandering, the image of the band may be displayed with the lane meandering as it is, or the meandering of the lane may be corrected and the image may be displayed with the band straight.

At this time, if a smiling phenomenon occurs in the migration pattern as shown in FIG. 3, it can be corrected as follows, for example. The presence or absence of the smiling phenomenon can be determined by the fact that the length of the lanes at both ends of the displayed original pattern is shorter than the length of the lane at the center, and the bands of lanes at both ends where smiling has occurred (especially at the bottom where the migration distance is long). This can be determined from the fact that the band) is not perpendicular (that is, horizontal) to the migration direction (see Figure 3).

To correct the smiling, first, the peak B′ _oy of each band is displayed as a point or a simulated band (a band perpendicular to the electrophoresis direction) on the above screen in a superimposed manner. Next, the cursor is placed on the migration start point of the lane where smiling occurs to recognize the position. Next, the cursor point is placed on an arbitrary band position on this lane to make the position recognized, and then the cursor point is moved downward along the lane direction and fixed at an appropriate position. With this operation,
Correction information is input so that the band position becomes the position where the cursor is fixed (that is, the position where the base-specific DNA fragment would have originally migrated if the smiling phenomenon did not occur).

Specifically, the migration coordinate y' of the band can be approximated by the equation ().

y′=at+b () (where a represents the migration speed of each band, and t
( represents the migration time, and b represents the coordinates of the migration start point) The smiling phenomenon occurs when the migration speed a of each band differs depending on the slot when a difference in gel temperature occurs due to the edge effect of the gel support medium. It happens because it comes. By substituting the above input information, that is, the actual migration coordinate and the virtual migration coordinate, into the equation (), the actual migration speed a and the virtual migration speed a′ (the migration speed when the smiling phenomenon did not occur) can be calculated. By calculating the ratio of and correcting the migration coordinates of all bands in the slot,
The entire band on the lane can be stretched downward at a constant rate.

By superimposing this smile-corrected band B″ _oy on the original pattern, the operator can check the smile correction result and use it as the final input information.At this time, the smile correction is not yet complete. (Insufficient or excessive) By repeating the above operation again, the desired information can finally be input.

In order to reduce the number of operations described above, it is preferable that the arbitrarily selected band be the lowest band. This is because the amount of electrophoresis delay due to smiling is greatest at the bottom, and the cursor point should be fixed at a position horizontal (or almost horizontal) to the position of the final band of the lane in the center where almost no smiling occurs. This is because by doing so, correction can be easily performed with high accuracy.

In this way, smiling correction is performed for each slot in sequence. Based on the smiling-corrected digital signal, each band _B''oy is compared between slots with respect to its migration coordinate y'', and each band is rearranged in order of migration coordinate. The slots (1) to (4) above each have information about the terminal bases consisting of (G), (A), (T), and (C), so replace the slot number of each band with the corresponding base. By doing so, the base sequence of DNA (for example, A-G-C-T
-A-A-G-...) can be obtained.

Alternatively, as shown in FIG. 4, if offset distortion occurs in the separated and expanded pattern, it can be corrected, for example, as follows. FIG. 4 shows an autoradiograph in which the four types of base-specific DNA fragments mentioned above are separated and expanded into four slots.

The presence or absence of offset distortion can be determined from the fact that the entire lane is vertically shifted uniformly in the displayed original pattern. Offset distortion occurs because the shapes (sizes of depressions) of many slots (sample injection ports) provided at the upper end of a support medium, such as a gel medium, are not completely the same but differ from one another. In addition, the rate at which the sample enters the gel may vary due to incomplete operation when injecting the sample into each slot of the gel medium or insufficient washing of urea from the support medium immediately before sample injection. It happens because it happens.

To correct the offset, first, as in the case of the above-mentioned smiling correction, the band peak _B'oy is detected and superimposed and displayed on the screen as a point or a simulated band. Next, place the cursor point on any band position on the lane where offset distortion has occurred to recognize that position, and then move the cursor point upward or downward along the lane direction with respect to the basic lane. and secure it in place. Through this operation, correction information is input so that the band position becomes the cursor-fixed position (that is, the position where the base-specific DNA fragment would have originally migrated if no offset distortion occurred).

Specifically, by substituting this input information, that is, the actual migration coordinates and the virtual migration coordinates, into the above equation ( The entire band on the lane can be moved upward or downward in parallel by calculating the difference from the electrophoresis start position (if there is no electrophoresis) and correcting the electrophoresis coordinates for all the bands in the slot. This offset-corrected band B is placed on the original pattern.
By superimposing and displaying _oy , the result of offset correction can be confirmed and used as the final input information. At this time, if the offset correction is not yet complete (insufficient or excessive), the desired information can finally be input by repeating the above operation again.

In order to reduce the number of operations described above, it is preferable that the arbitrarily selected band be the topmost band. This is because by setting the position where the cursor point should be fixed to a position horizontal to the migration start position of the lane where no offset has occurred (basic lane), correction can be easily made without being influenced by the smiling phenomenon etc. This is because it can be done.

In this way, offset correction is performed for each slot in sequence. Based on the offset-corrected digital signal, each band _Boy is compared between slots with respect to its migration coordinate y, and by performing appropriate rearrangement, a desired DNA base sequence can be obtained.

Furthermore, the analysis results for the DNA base sequence are as follows:
The present invention is not limited to the above display format; for example, it is also possible to simultaneously display the intensity (z') of each band as the relative amount of the separated product, if desired. At the same time, it is also possible to display it as an image together with the visible image of the autoradiograph.

Furthermore, the information obtained in this way can be used in other ways, such as in other archives.
It is also possible to perform genetic linguistic information processing such as matching with DNA base sequences.

The analysis results of the DNA base sequence determined by the above-mentioned signal processing are output from the signal processing circuit, and then stored in a recording device either directly or, if necessary, via a storage means such as a magnetic disk or magnetic tape. transmitted to.

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 can be used, such as those that record symbols and numbers displayed on a CRT or the like on a video printer or the like, and those that record on a heat-sensitive recording material using heat rays.

In addition, although the above explanation has been made using DNA base sequencing as an example, the autoradiographic analysis method of the present invention is not limited to the sample being a base-specific DNA fragment, and can be applied using various separation and development means. It can be applied to a separation and development pattern of a radiolabeled substance separated and developed in a one-dimensional direction on a support medium. In particular, it can be suitably used for protein trace analysis and gene screening.

[Brief explanation of the drawing]

In FIG. 1, a is a diagram showing an example of a separated development pattern in which rotational distortion of the pattern has occurred, and b is a diagram showing an example of a separated development pattern in which rotational distortion of the pattern has occurred.
FIG. 3 is a diagram illustrating an example of a separated development pattern that has been subjected to pattern rotation processing. FIG. 2A is a diagram showing an example of a separation expansion pattern in which a distortion of the separation expansion row has occurred, and FIG. 2B is a diagram showing an example of a separation expansion pattern in which the separation expansion row is determined by a cursor line. FIG. 3 is a diagram showing an example of a separated development pattern in which a smiling phenomenon occurs. FIG. 4 is a diagram showing an example of a separated expansion pattern in which offset distortion occurs. In Figure 2 a, 1: Separation and development column (lane);
2: Cursor line, 3: Bottom position where the cursor line and lane match, 4: Cursor point.

Claims (1)

[Claims] 1. Positional information of a radiolabeled substance separated and developed on a support medium is obtained by performing signal processing on a digital signal corresponding to an autoradiograph having two-dimensional positional information of a radiolabeled substance. The autoradiograph analysis method comprises: (1) electrically displaying an image of the autoradiograph based on digital signals; (2) radioactivity determined from the displayed image; A step of extracting a signal in a certain range along the separation and development column based on input information regarding the separation and development column of the labeled substance, and (3) performing one-dimensional signal processing on the extracted signals to determine the separation and development site. An autoradiographic analysis method comprising: a step of detecting. 2 After the first step and before the second step, the pattern is rotated as a whole based on input information regarding the inclination of the separation and development pattern of the radiolabeled substance determined from the displayed image, and then the image is displayed. An autoradiographic analysis method according to claim 1, characterized in that: 3. After the third step, the position information of the radiolabeled substance obtained is confirmed by superimposing the position information on the autoradiograph displayed as an image. Autoradiograph analysis method described in section. 4 A digital signal corresponding to the autoradiograph is transmitted to the autoradiograph of the radiolabeled substance on the support medium by superimposing the support medium and a stimulable phosphor sheet containing a stimulable phosphor on the phosphor sheet. The autoradiograph according to claim 1 is obtained by accumulating and recording on the phosphor sheet, irradiating the phosphor sheet with excitation light, and photoelectrically reading out the autoradiograph as photostimulated light. Radiograph analysis method. 5. The digital signal corresponding to the autoradiograph is transferred onto the photosensitive material after superimposing the support medium and the photographic light-sensitive material to photosensitively record the autoradiograph of the radiolabeled substance on the support medium on the photosensitive material. The autoradiograph analysis method according to claim 1, wherein the autoradiograph analysis method is obtained by photoelectrically reading a visualized autoradiograph. 6. The autoradio according to claim 1, wherein the radiolabeled substance is a radiolabeled biopolymer group, a derivative thereof, a decomposition product thereof, or a composite thereof. Graph analysis method. 7. A patent claim characterized in that the biopolymer substances described above are nucleic acids, their derivatives, their decomposition products, or their composites, and the positional information obtained by signal processing represents their base sequences. The autoradiograph analysis method according to item 6. 8 By performing signal processing on the digital signal corresponding to the autoradiograph having two-dimensional positional information of the radiolabeled substance separated and developed on the support medium, the positional information of the radiolabeled substance can be expressed as symbols and/or numerical values. The autoradiograph analysis method comprises: (1) electrically displaying an image of the autoradiograph based on digital signals; (2) separating and developing arrays of radiolabeled substances determined from the displayed image; (3) Perform one-dimensional signal processing on the extracted signals to detect the separation development region, and then display the superimposed display. and (4) correcting the separation deployment distance based on input information regarding the position of one separation deployment site determined from the display image for the signal corresponding to the separation deployment site. Autoradiograph analysis method. 9 By performing signal processing on the digital signal corresponding to the autoradiograph having two-dimensional positional information of the radiolabeled substance separated and developed on the support medium, the positional information of the radiolabeled substance can be expressed as symbols and/or numerical values. The autoradiograph analysis method comprises: (1) electrically displaying an image of the autoradiograph based on digital signals; (2) separating and developing arrays of radiolabeled substances determined from the displayed image; (3) Perform one-dimensional signal processing on the extracted signals to detect the separation development region, and then display the superimposed display. and (4) correcting the starting position of separation deployment based on input information regarding the position of one separation deployment site determined from the displayed image, regarding the signal corresponding to the separation deployment site. An autoradiograph analysis method characterized by: