JPH0529067B2 - - 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. In the former Maxam-Gilbert method, a group containing a radioactive isotope such as ^32P is attached to one end of a chain molecule of DNA or DNA fragments whose base sequence is to be determined, thereby attaching a radioactive label. 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). 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 cutting means, the end of the chain molecule to which the radioactive isotope is bound is determined. The bases in a certain positional relationship are sequentially determined from the base, and the base sequence of the entire target object is thereby determined.

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 radioactively labeled nucleic acid has been separated and developed, and a highly sensitive X-ray film. It is obtained by exposing the films to light by overlapping them for a certain period of time. The base sequence of the nucleic acid is then determined by visually determining the separated development positions (bands) of the base-specific fragments of the nucleic acid from the autoradiograph visualized on the film, and by comparing the positions of these bands with each other. has been done.

Specifically, autoradiograph analysis involves first fixing the X-ray film on which the autoradiograph is visualized on a shear caste, and then using a plastic cursor (reading guide plate) installed on the shear caste. This is done by manually moving the film vertically (or horizontally) and visually determining the relative positional relationship of the bands using this cursor as a guide.

A thin straight line is usually drawn in the center of a transparent plastic cursor (so-called hairline cursor), and the hairline cursor can be moved in one direction (parallel to the film) while being fixed to the shear caster. can. Using this linear hairline cursor as a guide, the relative positions of the bands in each column can be determined and the bands can be ranked.

Alternatively, an analysis method using a computer system using a digitizer board or the like is also being tried. In other words, an X-ray film on which an autoradiograph is visualized is fixed on a digitizer board that has a two-dimensional coordinate system and is connected online to a computer. In this method, the name of the base to which the band should be assigned is input via another input means such as . According to this method, an order and a base name can be automatically assigned to bands according to the coordinate system input by the instruction stick.

However, in both the above-mentioned method using Shearkasten and the method using a computer system, it is difficult to distinguish between read-processed bands and unprocessed bands, and the line of sight for reading the bands on the X-ray film and the base name Fill in (input)
There are disadvantages such as the line of sight (directed toward the keyboard) and the keyboard are different, and there is a time gap between the two. For that, reversing the order of the bands,
It was not possible to avoid reading errors such as missed readings or duplicate readings, and errors in writing base names.

[Summary of the Invention] The present invention provides an autoradiographic analysis method that can easily determine the base sequence of a nucleic acid with high accuracy.

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 analyzes autoradiographs of a plurality of separation and development rows formed by separation and development of base-specific DNA fragments or RNA fragments to which radioactive labels have been added in a one-dimensional direction on a support medium. A method for determining the base sequence of a nucleic acid by: (1) electrically displaying an image of an autoradiograph based on a digital signal corresponding to the autoradiograph; (2) placing a reading cursor on the displayed image. The process of displaying;
and (3) a step of simultaneously displaying the name of the base to which the band of the separation expansion sequence determined using a reading cursor is assigned, and the reading cursor on a display image. It provides an autoradiographic analysis method for determination.

According to the method of the present invention, unlike the case of using a conventional shearkasten, a reading cursor can be displayed on the screen on which the autoradiograph is visualized and can be freely moved on the screen.
Further, unlike the case where a conventional shearkasten is used or a digitizer board is used, a reading cursor and the base name of the band determined and input based on the cursor are displayed on the screen at the same time. Therefore, since it is obvious at a glance which band has been read, it is possible to prevent reverse reading of bands, omission of reading, double reading, etc. during reading as in the conventional method. In addition, since the base names read and input are displayed instantly on the same screen, you can proceed with the reading operation while understanding and checking the correspondence between the read band, cursor, and base name. It is possible to eliminate omissions in entry (input) of base names and errors in entry due to physical and spatial gaps.

Therefore, the base sequence of a nucleic acid can be determined with high accuracy and in a short time.

In addition, mistakes made when reading or filling out the
By distinguishing and displaying the cursor used for reading and the cursor to be used from now on by color, pattern, brightness, etc., it is also possible to distinguish between the location of the base name that has been entered and the location of the base name that is about to be entered. By similarly distinguishing and displaying
This can be further prevented.

In particular, the read cursor can be separated into expanded columns (lanes).
If you create a band across each lane and display it on the screen, the smiling phenomenon,
Even if the bands are misaligned between lanes due to offset distortion or the like, the order of the bands can be read more accurately based on the cursor. Here, the smiling phenomenon is caused by the heat dissipation effect (edge effect) during the decomposition and expansion process, and the separation and expansion distance at both ends becomes shorter than the separation and expansion distance at the center of the support medium. refers to a phenomenon. In addition, offset distortion is caused by differences in the sample separation and development start position and start time for each lane due to differences in the shape of the sample injection port (slot), etc., and the overall difference between lanes. This refers to misalignment. In addition to these band misalignments, defects in the band itself, meandering lanes, etc. may occur.

This reading cursor can be moved not only vertically (or horizontally) but also in any direction, so even if the separation development pattern is meandering, the cursor can be moved optimally according to the pattern. This also makes it possible to improve the reading accuracy.

The applicant has already filed a patent application for a method of obtaining an autoradiograph of a separated development pattern using a radiation image conversion method using a stimulable phosphor sheet instead of the conventional radiography method using a radiation film. (Unexamined Japanese Patent Publication No. 1983-
No. 83057, Patent Application No. 1983-201231). 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, an autoradiograph of a radiolabeled substance can be obtained directly as a digital signal because it is once stored in a phosphor sheet as radiation energy and then read out photoelectrically as photostimulated light.

[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 decomposition products or as a synthesis mixture by the same method as above. DNA has four types of constituent units: adenine, guanine, thymine, and cytosine. The bases of RNA are 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.

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

A mixture of base-specific DNA fragments labeled with the following four types of radioactive labels is separated and developed on a gel support medium by electrophoresis. The autoradiograph is obtained by a radiation image conversion method using a stimulable phosphor sheet, and then a digital signal corresponding to the autoradiograph is obtained via a suitable readout system.

(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.

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 storage phosphor sheet are overlapped at room temperature for a short period of time (several seconds to several tens of minutes) to allow the phosphor sheet to store the radiation energy emitted from the radiolabeled substance, thereby creating an autoradiograph. It is recorded as a kind of latent image on the phosphor sheet. Here, the stimulable phosphor sheet includes 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. Digital signals obtained by any other method are not limited to these methods, and can be used in the analysis method of the present invention as long as they have a correspondence with the autoradiograph of the radiolabeled substance. It is.

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, the reading conditions are set by inputting information about the position of each separation expansion column and the width of the band in advance. By scanning with a light beam at a scanning line density such that the above 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.

First, the digital signal corresponding to this autoradiograph is once recorded in a memory (buffer memory or non-volatile memory such as a magnetic disk), and then the signal processing circuit and the autoradiograph.
The autoradiograph to be analyzed is sent to a device that has a display means such as a CRT that can display a cursor and a base name entry field at the same time, and an input means that can perform input for moving the cursor and displaying the base name. (separation and development pattern of radiolabeled substance) is visualized on the 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.

Second, a reading cursor is displayed on the display screen.

The reading cursor may be displayed as a simple straight line across the lanes, but if the migration pattern has a smiling phenomenon or offset distortion, it is preferable to display it as a broken line or curved line along the band of each lane.

A polygonal or curved reading cursor can be created and displayed on the screen in the following manner, for example.

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 read 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 is 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 starting point 4 to the input position (second position) inside the migration 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 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, line segments (x _i , y _i ), (x _i+1 , y _i+1 ) ( However, 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 do not 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. vertical line segment 6 (x _i , y _i )
Subtract (x _i , y _b ). In this way, on the screen,
A reading cursor 7 consisting of a plurality of connected polygonal lines that completely traverses the electrophoresis pattern is displayed.

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 shape of a polygonal line connecting input positions with a straight line, or may be curved 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.

Third, the reading cursor and the base name of the band determined using the cursor are simultaneously displayed on the display screen.

FIG. 5 shows an autoradiograph 11 of the electrophoresis pattern displayed on the screen, and reading cursors 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 the movement of the reading cursor, the reading cursor (this is called the active cursor) 13 that is about to be used for reading
Translate to the left or right to match the position of the next band to be read. At this time, not only the base names that have already been read, but also the cursor 12 used for reading (this is called a mark cursor) may be displayed on the screen as is. This makes it possible to clearly represent the one-to-one correspondence between bands and base names.

Note that by setting the movement start position, movement direction, and movement distance of the reading cursor in advance,
It is possible to move the cursor at regular intervals with simple input operations. The cursor can be moved not only horizontally in parallel on the screen, but also diagonally at an arbitrary angle. Therefore,
Even if the lane is meandering or the entire pattern is sloped, the cursor can be moved along it.

It is preferable to display the mark cursor and the active cursor separately. Thereby, reading errors can be more reliably prevented. The mark cursor and the active cursor can be distinguished, for example, by changing the color of the cursor, by using a solid dotted line, or by changing the brightness of the cursor.

In addition, if the reading cursor no longer matches the migration pattern during the process of band assignment,
By repeating operations similar to those described above, a cursor of a desired shape can be easily created and displayed.

Lanes (1) to (4) to which each band belongs are respectively
Since it has information about the terminal bases consisting of (G), (A), (T), and (C), when the name of the base corresponding to the lane to which the band belongs is entered by keyboard operation, the The base name is displayed in the processing entry column 15. Of course, if the display is incorrect, it can be corrected on the spot by re-entering the information. In addition,
At this time, since the combination of the above four types of base-specific DNA fragments is an exclusive combination, two or more bands at the same position (bands in different lanes)
It is possible to determine the order by taking advantage of the fact that there cannot exist.

It is preferable that the unprocessed entry column 15 in which the base name is displayed be displayed inverted or blinked as shown in FIG. 6 to distinguish it from other base name entry columns. This makes it possible to more reliably prevent entry errors.

Next, when an input is made to move the cursor, the active cursor 13 and the color identification in the unprocessed entry field 15 in FIG. 6 are determined to be used and processed, and the display is immediately changed. Ru. That is, a new mark cursor is displayed at the position of the active cursor 13,
The base name entry field 15 is displayed in the same manner as the other processed entry fields. A cursor that newly moves on the screen according to input information is an active cursor. In FIG. 7, the mark cursor 12',
An active cursor 13' and entry field 15' are shown as read and processed.

In this way, base names can be assigned to bands sequentially from the bottom of the electrophoresis pattern.

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. Therefore, it is possible to easily match and confirm the assigned bands and base names at the end or in the middle of the process. 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.

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). Furthermore, the method of the present invention is not limited to the separation and development array of a set of base-specific fragments of nucleic acids, but the base name entry field is provided for all separation and development arrays that are simultaneously separated and developed on the support medium. It is possible to apply them simultaneously by displaying them separately.

The obtained information about the line base sequence of the nucleic acid can be stored in a storage medium such as a magnetic disk or magnetic tape via a signal processing circuit, or it can be stored in another storage medium such as a magnetic disk or magnetic tape.
It can be displayed and recorded on display screens such as CRT, photosensitive materials, and heat-sensitive recording materials.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an autoradiograph of an electrophoresis pattern displayed as a grayscale image on a screen. FIG. 2 is a diagram showing an autoradiograph of the 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 Figures 5 to 7 are diagrams showing autoradiographs displayed on the screen, respectively, and the reading cursor and base name according to the present invention. It is a figure explaining the process of simultaneously displaying entry fields. 11: Autoradiograph, 12,12': Used cursor (mark cursor), 13,1
3': Unused cursor (active cursor),
14: Base name entry field, 15: Unprocessed entry field, 1
5': Processed entry field.

[Claims]

1 Analyzing the autoradiograph of a separation pattern consisting of multiple separation and development rows formed by one-dimensional separation and development of radioactively labeled base-specific DNA fragments or RNA fragments on a support medium. In the method for determining the base sequence of the acid, 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) (3) 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 name of the base to which the band on the autoradiograph is assigned. and (1) pattern information, (2) cursor information, and (2) cursor information.
(3) An autoradiographic analysis method for determining the base sequence of a nucleic acid, characterized in that the base name information of (3) is recorded and stored in association with each other. 2 The above pattern information and cursor information are correlated by a coordinate system, and the cursor information and base name information are correlated by order.

Claims (1)

The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 2, characterized in that a reading cursor created by drawing an arc is displayed. 4. The autoradiograph for determining the base sequence of a nucleic acid as set forth in claim 1, wherein in the third step, the name of the base is displayed as letters and/or symbols at the end of the reading cursor. analysis method. 5. The nucleic acid according to claim 1, wherein in the third step, a reading cursor that has already been used for band assignment and a reading cursor to be used next are distinguished and displayed at the same time. An autoradiographic analysis method for nucleotide sequencing. 6. In the third step, the name of the base to which a band has already been assigned and the name of the base to which a band is to be assigned next are distinguished and displayed at the same time. An autoradiographic analysis method for determining the base sequence of nucleic acids. 7. The autoradiograph analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein in the first step, the autoradiograph is displayed as a grayscale image or a binary image. 8 In the first step, the autoradiograph is displayed as an image by multiplexing a plurality of two-dimensional waveforms consisting of positions along the separation development direction and signal levels 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 according to claim 1, characterized in that: 9 In the above first step, a digital signal corresponding to the autoradiograph is generated by superimposing the support medium and the stimulable phosphor sheet containing the stimulable phosphor to produce an autoradiograph of the radiolabeled substance on the support medium. 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. An autoradiographic analysis method for determining the base sequence of a nucleic acid according to item 1. 10 In the first step, after the digital signal corresponding to the autoradiograph is superimposed on the support medium and the photographic light-sensitive material and the autoradiograph of the radiolabeled substance on the support medium is photosensitively recorded on the light-sensitive material, The autoradiographic analysis for determining the base sequence of a nucleic acid according to claim 1, which is obtained by photoelectrically reading an autoradiograph visualized on the photosensitive material. Method.