JPH0529068B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to an autoradiographic analysis method for determining the base sequence of nucleic acids.

[Background of the invention] In the field of molecular biology, which has developed rapidly in recent years, it is essential to clarify the genetic information of living organisms in order to elucidate their functions and replication mechanisms. It has become commonplace. Above all,
It has become essential to determine the base sequence of nucleic acids such as DNA (or DNA fragments, hereinafter the same) that carry specific genetic information.

Maxam Gilbert uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
-Gilbert method and Sanger-Coulson method are known. The former Maxam-Gilbert method first attaches a radioactive label to one end of a chain molecule of DNA or DNA fragments whose base sequence is to be determined by attaching a group containing a radioactive isotope such as ³² P. Later, using chemical means, the structural units (base units) of chain molecules
base-specifically cleaves the bond between. Next, the resulting mixture of base-specific DNA cleavage products is separated and developed on a support medium by gel electrophoresis, and a separated development pattern (however visually (cannot be seen at the target). Next, this separation development pattern is visualized on, for example, an X-ray film to obtain an autoradiograph thereof, and from the obtained autoradiograph and each base-specific cleavage means, a chain molecule to which a radioactive isotope is bound is determined. The bases located in a certain positional relationship from the end of the target are sequentially determined, thereby determining the base sequence of the entire target object.

In addition, the latter Sanger-Coulson method is
Base-specific DNA synthesis obtained by base-specifically synthesizing a radioactively labeled DNA compound that is complementary to a chain molecule of DNA or DNA fragments using chemical means. This method uses a mixture of substances and determines the base sequence from the autoradiograph in the same manner as above.

Traditionally, the visible image of an autoradiograph is
A support medium in which a base-specific cleavage product or a base-specific composite product (hereinafter simply referred to as a base-specific fragment of a nucleic acid) of a nucleic acid to which a radioactive label has been added is separated and developed, and a highly sensitive X-ray film. It is obtained on a film by exposing the films to light by overlapping them for a certain period of time. In addition, the base sequence of a nucleic acid can be determined by visually determining the separated and developed positions (bands) of base-specific fragments of a nucleic acid from an autoradiograph visualized on a film, and by comparing the positions of these bands with each other. After that, it is recorded and stored on a recording material such as paper or a recording medium such as a magnetic disk. Therefore, since the information regarding the autoradiograph of the separation development pattern and the information regarding the base sequence of the nucleic acid determined based thereon are recorded and stored on completely different media, it has been difficult to associate the two.

In other words, the relationship between the separation expansion pattern and the nucleotide sequence information included in the pattern is clear when analyzing the autoradiograph, but once the nucleotide sequence information is recorded on another medium after the analysis is completed, the relationship between the two cannot be re-examined. There were problems such as it was very difficult to extract, and if you wanted to interrupt and restart the analysis, you had to start over from the beginning, and checking the analysis results took the same amount of time and effort as the analysis itself.
Pattern information and base sequence information that are stored in different formats in different media have value as a series of information in each medium, but it is almost impossible to immediately establish a one-to-one correspondence between the two media. It was impossible.

[Summary of the Invention] The present invention provides an autoradiograph analysis method that can accumulate and store separation expansion pattern information and base sequence information while maintaining a one-to-one correspondence.

The present invention also provides an autoradiographic analysis method that can interrupt and restart nucleic acid base sequencing at any time.

Furthermore, the present invention also provides an autoradiographic analysis method that allows easy confirmation of the determined base sequence of a nucleic acid.

That is, the present invention provides a separation and development pattern consisting of a plurality of separation and development rows formed by separation and development of base-specific DNA fragments or RNA fragments to which a radioactive label has been added in a one-dimensional direction on a support medium. In a method for determining the base sequence of a nucleic acid by analyzing an autoradiograph, information regarding the autoradiograph and the base sequence of the nucleic acid determined based on the autoradiograph is: (1) Information regarding the autoradiograph of the separation expansion pattern; , (2) information regarding a plurality of reading cursors that pass through the bands on the autoradiograph and point to the relative positions of the bands, and (3) information regarding the names of the bases to which the bands on the autoradiograph are assigned. and (1) pattern information, (2) cursor information, and (2) cursor information.
The present invention provides an autoradiographic analysis method for determining the base sequence of a nucleic acid, which is characterized in that (3) base name information is recorded and stored in association with each other.

The autoradiograph of the separation development pattern and the base sequence of the nucleic acid determined based on it are clearly shown in a one-to-one relationship by the reading cursor passing through one band on the autoradiograph and the name of the base to which the band is assigned. can be displayed with the following correspondence. In the present invention, by recording and storing information on the separated development pattern, reading cursor, and base name separately on a suitable recording medium, it is possible to store the information on any recording material or CRT while maintaining a one-to-one correspondence at all times. It can be displayed and recorded on a display screen such as .

That is, in addition to conventional autoradiograph (pattern) information and base sequence information, information about the reading cursor corresponding to each band on the autoradiograph is also recorded and saved, and base sequence information is stored at this cursor (i.e., band ), information about autoradiographs and nucleic acid base sequences can be stored while maintaining a one-to-one correspondence.

This makes it easy for someone else to confirm the nucleic acid base sequence of an already analyzed autoradiograph, or to restart an interrupted autoradiograph analysis at any time. can. Specifically, in the case of confirming a base sequence, it is obvious at a glance whether the band assignment is correct or not. Also,
When restarting an interrupted analysis, you can continue from where you left off without reading from the beginning.

Furthermore, by recording and storing the pattern information, cursor information, and base name information separately, it is possible to extract only one or two types of information. This makes it possible to apply base name information to another separation expansion pattern for the same nucleic acid, or to use only cursor information to a completely different separation expansion pattern.

Therefore, according to the present invention, various restrictions in autoradiographic analysis for nucleic acid base sequences can be removed and base sequence determination can be carried out more advantageously.

The present applicant has developed a radiation image conversion method using a stimulable phosphor sheet instead of the conventional radiography method using a radiation film as a method for obtaining an autoradiograph of a radiolabeled substance separated and developed on a support medium. A patent application has already been filed for the method of using
No. 201231). Here, the stimulable phosphor sheet is made of a stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, electromagnetic waves (excitation light) in the visible to infrared region are emitted. By exciting it with , radiation energy can be emitted as photostimulated light. 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, since an autoradiograph of a radiolabeled substance is once stored in a phosphor sheet as radiation energy and then read photoelectrically as photostimulated light, it is directly obtained as a digital signal and then recorded and stored on a suitable recording medium. be able to.

[Structure of the Invention] Examples of samples used in the present invention include:
Examples include mixtures of base-specific fragments of nucleic acids such as DNA and RNA that have been given radioactive labels. Here, the nucleic acid fragment means a part of a long chain molecule. For example, a base-specific DNA cleavage mixture, which is a type of base-specific DNA fragment mixture, is produced by base-specific cleavage and decomposition of radiolabeled DNA according to the Maxam-Gilbert method described above. can get. Also,
The base-specific DNA compound mixture is synthesized using DNA as a template and a radioactively labeled deoxynucleoside triphosphate and a DNA synthase according to the Sanger-Coulson method described above. can get.

Furthermore, a mixture of base-specific RNA fragments can also be obtained as a mixture of cleavage and degradation products or a mixture of synthetic products by the same method as above. Furthermore, DNA consists of four types of bases as its constituent units: adenine, guanine, thymine, and cytosine, while RNA consists of adenine, guanine, uracil,
Consists of four types of bases: cytosine.

Radioactive labels can be applied to these substances in an appropriate manner.
It is given by retaining radioactive isotopes such as ³² P, ¹⁴ C, ³⁵ S, ³ H, ¹²⁵ I, etc.

The sample, a mixture of base-specific fragments of radioactively labeled nucleic acids, is subjected to electrophoresis, thin layer chromatography, column chromatography, etc. using various known support media such as gel support media.
Separation and development are carried out on a support medium using various separation and development methods such as paper chromatography.

Information to be recorded and stored in the analysis method of the present invention can be obtained, for example, by the method described below.

An example of a migration pattern in which a mixture of base-specific DNA fragments to which four types of radioactive labels have been added are separated and developed on a gel support medium by electrophoresis will be explained.

(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 cleaved, degraded, or synthesized in a base-specific manner, that is, it consists of DNA fragments of various lengths that have the same terminal base.

First, an autoradiograph of the migration pattern on the support medium is obtained by radiography using a conventional photographic material or by a radiation image conversion method using a stimulable phosphor sheet.
A digital signal corresponding to the autoradiograph is obtained via a suitable readout system.

When using the former radiographic method, first the support medium and a photographic light-sensitive material such as an X-ray film are overlapped for a long time (several hours to several tens of hours) at low temperature or room temperature, and then the radiographic film is exposed. , and developed to produce a visible image of the autoradiograph of the radiolabeled substance 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 the 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.

Details of the above-mentioned autoradiograph measurement operation and method of obtaining a digital signal corresponding to the autoradiograph are described in the above-mentioned Japanese Patent Laid-Open No. 59-83057.

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 signal processing method of the present invention is not limited to these methods, and the signal processing 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. is possible.

Furthermore, 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. .

Furthermore, in the present invention, reading conditions are set by inputting information about the position of each electrophoresis column, band width, etc. in advance, and one or more lines on each band are set during the reading operation. By performing scanning with a light beam at a scanning line density such that the scanning lines pass through, the reading time can be shortened and necessary information can be efficiently obtained. Note that in the present invention, the digital signal corresponding to an autoradiograph includes the digital signal obtained in this manner.

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.

Next, after temporarily storing the digital signal corresponding to this autoradiograph in memory (buffer memory or nonvolatile memory such as a magnetic disk), the signal processing circuit, autoradiograph, cursor, and base name entry field are A display means such as a CRT that can be displayed simultaneously, an input means that can move the cursor and perform input for displaying base names, and a storage means that can record and save pattern information, cursor information, and base name information. The autoradiograph to be analyzed (the migration pattern of the radiolabeled substance) is visualized on a display screen.

Note that 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. For example, image processing includes
Examples include spatial frequency processing, gradation processing, averaging processing, reduction processing, and enlargement processing.

The autoradiograph displayed as an image on the screen shows the signal level (image density, i.e. the amount of radiolabeled substance) in the coordinate system set on the screen using the brightness of the color, similar to conventional photographic images. It may be an image, or it may be a binary image that is simplified and represented by binarizing the signal level in advance.

Alternatively, an autoradiograph is an image that is expressed by multiplexing multiple two-dimensional waveforms consisting of one-dimensional positions (positions along the electrophoresis direction) and signal levels at regular intervals in a direction perpendicular to the electrophoresis direction. (a so-called bird's eye view). According to this bird's-eye view, the amount of radiolabeled substance is three-dimensionally expressed as the height of the bird's-eye view, so it is possible to accurately read the peak position of the band, and it is possible to understand the positional relationship of bands between electrophoresis columns (lanes). Bands can be easily separated in a band-dense region near the migration start position. For details on how to display an autoradiograph from a bird's-eye view, please refer to Japanese Patent Application No. 60-181431 filed by the applicant.
It is stated in the specification of the No.

FIG. 1 shows an example of an autoradiograph of a migration pattern displayed as a grayscale image on a screen.

Further, FIG. 2 shows an example of an autoradiograph of a migration pattern displayed as a bird's-eye view.

Next, a basic reading cursor is displayed on the display screen, and bands are ranked (attributed) based on this cursor.

The reading cursor may be displayed as a simple straight line across the lanes, but if the migration pattern has misaligned bands or band defects due to the smiling phenomenon or offset distortion, the reading cursor may be displayed as a simple straight line across the lanes. It is preferable to create and display a polygonal line or a curved line.

Here, the smiling phenomenon is caused by the heat dissipation effect (edge effect) during the migration process, and refers to a phenomenon in which the migration distance at both ends of the support medium is shorter than the migration distance at the center. . Offset distortion is caused by differences in the sample migration start position and start time for each lane due to differences in the shape of the sample injection port (slot), etc., and the overall mutual relationship between lanes. Refers to misalignment. In addition to these band misalignments, defects in the band itself, meandering lanes, etc. may occur.

The basic polygonal line or curved reading cursor can be created, for example, as follows.

FIG. 3 is a diagram showing another example of an autoradiograph of a migration pattern displayed as a grayscale image on the screen. In FIG. 3, the electrophoresis direction is rightward, and a smiling phenomenon occurs in the electrophoresis pattern.

First, draw a vertical line from the top of the screen to the position entered based on the display screen. Preferably, the first position entered is the position of the band on the end lane (FIG. 31). The lane at the end is selected because the reading cursor is created starting from this position.

The position information can be input using a mouse cursor, a light pen, a joystick, or the like. From the viewpoint of positional accuracy, a mouse cursor is particularly preferred.

For example, define the rectangular area on the screen where the autoradiograph will be displayed with the coordinates (x _a , y _a ), (x _b , y _b )
If the position coordinates of the mouse cursor (arrow 2 on the screen) are expressed as (x _n , y _n ), the position information x _n = x ₁ , y _n = y ₁ is input by the mouse cursor. By doing so, line segment 3 (x ₁ , y _a )
(x ₁ , y ₁ ) is displayed on the screen.

Next, a straight line is drawn from the position of the starting point 4 to the input position (second position) inside the electrophoresis pattern. The position is input using a mouse cursor or the like in the same manner as described above. First of all,
A line segment (x ₁ , y ₁ ) (x _n , y _n ) is displayed between the starting point 4 (x ₁ , y ₁ ) and the position (x _n , y _n ) of the mouse cursor 2. When the mouse cursor 2 moves, this line segment disappears on the screen and a new line segment is displayed between the starting point 4 and the moved position. Note that the mouse cursor can be freely moved within the rectangular area partitioned by the above coordinates (x _a , y _a ) and (x _b , y _b ). In this way, as the mouse cursor moves, line segments connecting the starting point 4 and each movement position of the mouse cursor 2 are displayed one after another. By inputting the position where the line segment preferably crosses the lane, the next line segment connected to line segment 3 is fixed and displayed on the screen.

In this way, the line segments (x _i , y _i ) (x _i+1 , y _i+1 ) (where, i is a positive integer). Note that since the sample is an exclusive combination of four types of base-specific DNA fragments, the bands in each lane cannot exist at the same position (migration distance). Therefore, ``along the band'' means to draw a line segment along the band in a macroscopic sense so that the migration distance of each lane is equal, and each line segment strictly follows the band of each lane. It is not meant to pass above.

Next, as shown in FIG. 4, when the line segment completely crosses the electrophoresis pattern, according to the input information that the last input position is the end point, from the end point position 5 to the bottom edge of the screen. perpendicular line segment 6 (x _i ,
y _i ) (x _i , y _b ). In this manner, a reading cursor 7 consisting of a plurality of connected polygonal lines that completely traverses the electrophoresis pattern is displayed on the screen.

The reading cursor can be created in any area on the electrophoresis pattern, and may be an area above the pattern near the electrophoresis start position, or an area below the pattern where the electrophoresis distance is long.

Furthermore, the input position information may be about the number of lanes, or may be more or less than that, depending on the electrophoresis pattern. It is sufficient if at least one position is input and the starting point and ending point can be recognized. The reading cursor may be in the form of a polygonal line connecting the input positions with a straight line, or may be in the form of a curved line by subjecting the obtained polygonal line to appropriate arithmetic processing (curve approximation).

Since the created reading cursor matches the migration pattern, an analyst can accurately and easily determine the DNA base sequence using this cursor. The details of the method of creating and displaying the reading cursor are described in the specification of Japanese Patent Application No. 47924/1988 filed on March 5, 1988 by the present applicant.

Band assignment is performed while fixing this basic reading cursor to each band on the electrophoresis pattern.
At this time, by displaying the name of the base to which the band is assigned on the screen at the same time, it is possible to prevent reading errors such as omissions or duplicate readings, and entry errors such as omissions.

FIG. 5 shows an autoradiograph 11 of the migration pattern displayed on the mask, and a reading cursor 12,1.
3 and a base name entry column 14. FIG.
In FIG. 5, the electrophoresis direction is to the right, and the base name of the band already read based on the reading cursor 12 (indicated by the initial letter G, A, T, or C)
is displayed in the entry field 14.

First, according to the input information regarding cursor movement, the basic reading cursor (this is called the active cursor) 13 that will be used for reading from now on.
Translate to the left or right to match the position of the next band to be read. The cursor 12 that has already been used for reading (this is called a mark cursor) is displayed as it is on the screen together with the name of the base that has been read.

At this time, distinguish between the mark cursor and the active cursor, and also distinguish between the entry field for base names that have been read and the entry field for unprocessed base names that should be processed next (Figure 5, 15).
It is preferable to display them separately. Thereby, reading errors and writing errors can be more reliably prevented. These distinctions are made based on differences in color, pattern, brightness, etc.

If the active cursor no longer matches the migration pattern during the band assignment process, a cursor of the desired shape can be created and displayed simply by repeating the same operations as described above.

Lanes (1) to (4) to which each band belongs each have information about the terminal bases consisting of G, A, T, and C, so the name of the base corresponding to the lane to which the band belongs can be determined by keyboard operation etc. When input, the base name is immediately displayed in the unprocessed entry column 15. Of course, if the display is incorrect, it can be corrected on the spot by re-entering the information. Also,
You can also correct errors later.
At this time, since the combination of the above four types of base-specific DNA fragments is an exclusive combination,
Band assignment can be performed by utilizing the fact that two or more bands (bands in different lanes) cannot exist at the same position.

The next time an input is made to move the cursor, the active cursor and unprocessed entry field 1
The identification display using color or the like in No. 5 is determined to be used (mark cursor) and processed, and the display is changed immediately. That is, a new mark cursor is displayed at the position of the active cursor, and the base name entry field 15 is displayed in the same manner as the other processed entry fields. The reading cursor that newly moves on the screen according to the input information is an active cursor. In this way, base names can be assigned to the bands in order.

For details on how to simultaneously display the reading cursor and the base name entry field, please refer to the applicant's March 1986 document.
It is described in the specification of Japanese Patent Application No. 1983-55481 filed on May 12th (2).

After the reading is completed, a reading cursor corresponding to each band on a one-to-one basis and a series of base names written in the entry field at the bottom of the cursor remain on the screen. By connecting this series of bases in order from the end, the DNA base sequence (for example, T-G-C-A-
T-C-G-...) can be obtained.

The autoradiograph displayed on the screen through the above operations and the information regarding the base sequence of the nucleic acid are used as pattern information, which is a characteristic requirement of the present invention.
Three types of cursor information and base name information are recorded and saved.

First, only information about the autoradiograph of the migration pattern displayed on the screen is recorded and saved as pattern information. Specifically, it is recorded and saved as digital image data corresponding to a grayscale image as shown in FIG. 1, a bird's eye view as shown in FIG. 2, or a binary image. As described above, digital image data consists of two-dimensional coordinates (x, y) corresponding to one pixel and a signal level (z).

Second, only information about a large number of fixed reading cursors superimposed on the electrophoresis pattern on the screen is independently recorded and saved as cursor information.
This cursor information is data about the number of cursors corresponding to the number of assigned bands, and each cursor is assigned a number according to the order of the bands. The two-dimensional coordinates of the nodes of each polygonal line are recorded and saved as data. Specifically, data regarding a large number of reading cursors as shown in FIG. 6 is recorded and saved.

Third, only information about the names of bases to which bands are assigned is independently recorded and saved as base name information. This base name information is numbered according to the order of the bands, and specifically, character string data as shown in FIG. 7 is recorded and saved.

Therefore, pattern information and cursor information are stored in correspondence with two-dimensional coordinates fixed on the image (this is called address correspondence), and cursor information and base name information are stored in the order of bands on the electrophoresis pattern. (This is called order correspondence) and is stored.

These three types of information are stored in storage means such as a secondary storage device to form three information files. Information files that have been recorded and saved separately can be combined again at any time.
It can be reproduced on a display screen such as a CRT or on a recording material such as a photosensitive material or a heat-sensitive recording material. That is, for example, by integrating three information files in which pattern information, cursor information, and base name information corresponding to FIGS. 1, 6, and 7 are recorded and saved, a display as shown in FIG. 5 is again created. It can be expressed in form.

However, the storage means (recording medium) on which information is recorded and stored is not limited to secondary storage devices.
Any recording medium can be selected as long as the above three types of information can be stored separately while maintaining a one-to-one correspondence and can be combined and reproduced at any time. Furthermore, it is not necessarily necessary to record and save all three types of information on the same recording medium.

Furthermore, information can be recorded and saved not only after the analysis of the autoradiograph is completed, but also at any time during the analysis. If the recording medium used is one that can be written and read at any time, such as a magnetic disk, information can be repeatedly recorded on the medium once recorded and saved.

Therefore, when confirming an analyzed autoradiograph at a later date, it is possible to easily match and confirm the assigned bands and base names. Furthermore, when autoradiograph analysis is interrupted, band assignment can be continued from the point where it was interrupted.

In the above, the sample base-specific DNA
Although the case has been described in which an exclusive combination of (G, A, T, C) is used as a mixture of fragments, the analysis method of the present invention is not limited to this combination; for example, (G, G+A, T+C) , C) can be applied to various combinations. Similarly, mixtures of base-specific RNA fragments (e.g.
The method of the present invention can also be applied to combinations of G, A, U, and C).

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of an autoradiograph of a migration pattern displayed as a grayscale image on a screen. FIG. 2 is a diagram showing an example of an autoradiograph of a migration pattern displayed as a bird's-eye view on the screen. FIG. 3 and FIG. 4 are diagrams each showing an autoradiograph displayed on the screen, and are diagrams for explaining the process of creating a reading cursor. 1: Electrophoresis band, 2: Mouse cursor, 3,
6: line segment, 4: starting point, 5: ending point, 7: reading cursor. FIG. 5 is a diagram showing an example of an autoradiograph, a reading cursor, and a base name entry field displayed on the screen. 11: Autoradiograph, 12: Used cursor (mark cursor), 13: Unused cursor (active cursor), 14: Base name entry field, 15: Unprocessed entry field. FIG. 6 is a diagram showing an example of cursor information and a large number of line-shaped reading cursors that are recorded and saved.
FIG. 7 is a diagram showing an example of a series of base names recorded and saved as base name information.

[Claims]

1. By analyzing autoradiographs of multiple separation and development columns formed by one-dimensionally separating and developing base-specific DNA fragments or RNA fragments to which radioactive labels have been added, nucleic acid (1) electrically displaying an autoradiograph in which the band order has been determined as an image based on a digital signal corresponding to the autoradiograph; (2) the band order Based on the input information for confirmation, the reading cursor is fixed on the autoradiograph and displayed on the screen so as to cross the separation development column and pass through the position of one band, and at the same time, the base name of the band is displayed on the screen. (3) Based on the input information for band order confirmation, the reading cursor is fixed on the autoradiograph so as to pass through the position of the band adjacent to the band on which the cursor was fixed in the previous step. and (4) confirming the determined order of the bands on the autoradiograph by repeating the third step. For base sequencing of nucleic acids characterized by comprising

Claims (1)

An autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, characterized in that: 3. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein the pattern information, cursor information, and base name information are stored in a secondary storage device. 4. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein the pattern information, cursor information, and base name information are recorded and stored in the same recording medium. 5 The above pattern information is a grayscale image, a binary image,
Alternatively, the image consists of an image obtained by multiplexing a plurality of two-dimensional waveforms consisting of positions along the separation development direction and image densities at regular intervals in a direction perpendicular to the separation development direction. An autoradiographic analysis method for determining the base sequence of a nucleic acid as described in Section 3. 6. For determining the base sequence of a nucleic acid as set forth in claim 1, wherein the cursor information is composed of a plurality of polygonal lines or curved lines that cross the separation and expansion rows and follow the bands of each row. Autoradiograph analysis method. 7. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein the base name information consists of characters and/or symbols. 8 After the above autoradiograph is produced by overlapping a support medium and a stimulable phosphor sheet containing a stimulable phosphor and recording an autoradiograph of the radiolabeled substance on the support medium on the phosphor sheet. , the base of the nucleic acid according to claim 1, which is obtained as a digital signal by irradiating the phosphor sheet with excitation light and photoelectrically reading out the autoradiograph as photostimulated light. Autoradiographic analysis methods for sequencing. 9 The above autoradiograph is produced by overlapping a support medium and a photographic light-sensitive material, photosensitively recording an autoradiograph of a radiolabeled substance on the support medium on the light-sensitive material, and then recording the autoradiograph visualized on the light-sensitive material. By reading the graph photoelectrically,
The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein the method is obtained as a digital signal.