JPS62215868A - Autoradiograph analysis for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To make it possible to interrupt the determination of the base sequence of nucleic acid at any time and to confirm the base sequence developed and determined, by recording and preserving the informations relating to the autoradiograph of the separation and development pattern of the base sequence of nucleic acid, a predetermined reading cursor and the name of the base belong ing to a band in a respectively corresponding state. CONSTITUTION:With respect to the migration pattern obtained by separating and developing a mixture of four kinds of base (G, A, T, C) specific DNA segments each provided with a radioactive marker on a gel support by electrophoresis, an autoradiograph 11 is obtained by a radiographic method using a photosensitive material or a radiation image converting method using a light accumulative fluorescent sheet. Reading cursors 12, 13 are displayed on a display picture as the folding line along the band of each lane. Because the formed cursors 12, 13 coincide with the migration pattern, the base sequence of DNA is easily determined. The name of the determined base is displayed on the base name entry column on the display picture.

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, in order to elucidate the functions and replication mechanisms of living organisms,
It is essential to clarify the genetic information possessed by living organisms. Among other things.

DNA (or DNAItl) that carries specific genetic information
It has become essential to determine the base sequence of nucleic acids such as i-Katamono (hereinafter the same).

Maxim Gilbert (Maxa@-GilbC) uses autoradiography as a typical method for determining the #1 base A of nucleic acids such as , DNA, and RNA.
rL) method and Sanger-Coulson (SaIIge
r-Coulson) method is known. The former Maxam-Gilbert method first involves determining the DN whose base sequence is to be determined.
32 at one end of the chain molecule of A or DNA fragment.
After attaching a radioactive label to a group containing a radioactive isotope such as p, the bonds between the constituent units (salts and fu units) of the chain molecule are cleaved base-specifically using chemical means. . Next, the mixture of the obtained base-specific DNA cleavage products was separated and developed on a support medium by gel electrophoresis, and a large number of cut portions were separated from the eel by 1 spacing of the eel. A U-open pattern (however, it cannot be seen visually) is obtained. Next, this separation development pattern is visualized on, for example, an X-ray film L to obtain its autoradiograph, and from the obtained autoradiograph and each base-specific cleavage means, it is possible to determine whether the radioactive isotope is bound to a chain molecule. The bases located at a certain position from the end of the base are sequentially determined, and the base sequence of the entire target object is thereby determined.

The latter Sanger-Courlain method uses chemical means to generate a base-specific DNA compound that is complementary to a chain molecule of DNA or a DNA fragment and has been given a radioactive label. The base-specific DNA obtained by
This method uses a mixture of synthetic compounds 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 spread, and a high-sensitivity X-ray film. It is obtained on a film by superimposing them for a certain period of time and exposing the film to light. Also,
The base sequence of a nucleic acid is determined by visually determining the separated development positions (bands) of base-specific fragments of the nucleic acid from an autoradiograph visualized on a film, and by comparing the positions of these bands with each other. , and then 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 in 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 an autoradiograph is analyzed, but once the nucleotide sequence information is recorded on another medium after the analysis, the relationship between the two cannot be re-examined. It was very difficult to extract the data, 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 sequence information stored in different formats78 in different media have value as a series of information in each medium, but a one-to-one correspondence is immediately made between the two media. It was almost 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.

In addition, the present invention provides for interrupting base sequencing of nucleic acids at any time,
The present invention also provides an autoradiographic analysis method that can be used to perform an autoradiographic analysis.

Furthermore, undeveloped IJ1 also provides an autoradiographic analysis method that allows easy confirmation of the determined base sequence of the nucleic acid.

That is, the present invention provides a separation and development pattern consisting of a plurality of separation and development rows formed by separating and developing 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 read cursors passing through the bands on the autoradiograph and pointing to the relative positions of the bands; and (3) information regarding the names of the bases to which the bands on the autoradiograph were assigned. It is characterized by separating into three types consisting of (1) pattern information, (2) cursor information, and (2) cursor information and (3) nucleotide polynucleotide information, and storing them in association with each other. The present invention provides an autoradiographic analysis method for determining the base sequence of a nucleic acid.

The autoradiograph of the separation development pattern and the base sequence of the nucleic acid determined based on it can be determined in a one-to-one manner by the reading cursor passing through one band on the autoradiograph and the name of the base to which the band is assigned. They can be displayed in correspondence. In the present invention, by recording and storing information on the separated expansion pattern, reading cursor, and base name separately on a suitable recording medium, it is possible to store any recording material in a state where a one-to-one correspondence is always maintained. Alternatively, it can be displayed and recorded on a display screen such as a CRT.

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 ), it is possible to record and preserve the information about the autoradiograph and the base sequence of the nucleic acid while maintaining a one-to-one correspondence.

As a result, when you want to confirm the base sequence of another person's nucleic acid in an autoradiograph that has already been analyzed, or when you want to restart the analysis of an interrupted Saiichi radiograph, you can easily confirm it at any time or add +1 from the middle. I can take a break. Specifically, in the case of confirming base sequences, it is easy to tell at a glance whether the band assignment is correct or not.
do. Furthermore, when resuming an interrupted analysis, it is possible to continue from where it was interrupted without reading from the beginning.

Furthermore, by recording and storing the pattern information, cursor information, and nucleotide polynucleotide information separately, it is possible to extract only one or two types of information.

This makes it possible to apply base number 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 constraints in autoradiographic analysis for nucleic acid base sequences are removed, and
This makes it possible to proceed with Shionoff sequencing 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 of obtaining an autoradiograph of a radiolabeled substance separated and developed on a support medium. We have already applied for a patent on the method of using
No. 3057, patent application No. 58-201231). here,
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 the radiation energy to be emitted as photostimulated light.

According to this method, it is possible to significantly shorten the photoperiod, and chemical fog, which has been a problem in the past, does not occur. Furthermore, an autoradiograph of a radiolabeled substance is stored in a phosphor sheet as radiant energy and then read out photoelectrically as photostimulated light, so it can be obtained directly as a digital signal and then recorded on a suitable recording medium. Can be saved.

[Structure of the invention] An example of a sample used in Unreleased 11 is:

Examples include mixtures of base-specific fragments of nucleic acids, such as DNA and RNA, to which radioactive labels have been added. here,
A nucleic acid fragment refers to a portion of a long chain molecule. For example, a base-specific DNA cleavage degradation product mixture, which is a type of base-specific DNA fragment mixture, is a mixture of base-specific DNA fragments.
DNA radioactively labeled according to the Gilbert method
It is obtained by base-specific cleavage and decomposition of . Also, 11! The group-specific DNA synthesis mixture is prepared by converting DNA into a template (FJ) according to the Sanger-Cournon method described above.
It can be obtained by synthesis using a radioactively labeled deoxynucleoside triphosphate and a DNA synthesizing enzyme.

Furthermore, a mixture of base-specific RNA fragments can be prepared as a mixture of cleavage products or synthesized by the same method as above.
11) can be used as a J mixture. Furthermore, DNA has adenine, guanine, thymine, and silyl as its middle constituents.
It consists of four types of bases: ~syn, while RNA consists of four types of bases: adenine, guanine, uracil, and cytosine. Consists of base.

The radioactive label can be added to 32p, I by a method appropriate to these substances.
It is given by retaining radioactive isotopes such as n(:, δS, coH, L3I, etc.).

11 of the radioactively labeled nucleic acid sample! A mixture of group-specific fragments can be prepared by electrophoresis, thin layer chromatography, or column chromatography using various known support media such as gel support media.

Separation and development are carried out on a support medium by various separation and development methods such as vapor 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.

Base-specific DNA labeled with the following four types of radioactive labels
An example of a migration pattern in which a mixture of A fragments is separated and developed on a gel support medium by electrophoresis will be explained.

■) Guanine (G) specific DNA fragment 2) Adenine (
A) Specific DNA fragment 3) Thymine (T) specific DNA
Fragment 4) Cytosine (C) specific DNA fragments, each 11
! Group-specific oNAFIi fragments are base-specifically cleaved, degraded or synthesized, i.e., terminal "i! It consists of DNAFli pieces of various lengths with the same base.

First, an autoradiograph of the migration pattern on a support medium is obtained by radiography using a conventional photosensitive material or by a radiation image conversion method using a stimulable phosphor sheet. to obtain a digital signal corresponding to the autoradio club.

When using the former radiographic method, first the support medium and the photographic material such as X-ray film are heated at a low temperature (-90 to -
The radiographic film is exposed to light by overlapping at 70° C. for a long period of time (several tens of hours), and then developed to form a visible image of the autoradiograph of the radiolabeled substance on the radiographic film. The autoradiograph visualized on the radiographic film is then read using an image reading device. for example,
By irradiating a radiation film with a light beam and photoelectrically detecting the transmitted or reflected light, an autoradiograph is obtained as an electrical signal. Furthermore, by A/D converting this electric signal, a digital signal corresponding to an autoradiograph can be obtained.

When using the latter radiation image conversion method, first, the support medium and stimulable phosphor sheet are heated at room temperature for a short period of time (several seconds to
The autoradiograph is recorded as a kind of latent image on the phosphor sheet by overlapping the phosphor sheets (for several tens of minutes) and accumulating the radiation energy emitted from the radiolabeled substance in the phosphor sheet. 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
) and a transparent protective film are laminated in this order.The stimulable phosphor contained in the stimulable phosphor sheet is When irradiated with radiation, it absorbs and accumulates the radiation energy, and then, when excited with light in the visible to infrared region, it emits the accumulated radiation energy as photostimulated light.

Next, use a readout device to create a stimulable phosphor sheet. Read out the recorded autoradiograph. For example, an autoradiograph of a radioactively labeled substance is produced by scanning a phosphor sheet with a laser beam to emit stimulated light, which is radioactive energy, and detecting this stimulated light photoelectrically. It can be obtained directly as an electrical signal without any conversion. Furthermore, by A/D converting this electric signal, a digital signal corresponding to an autoradiograph can be obtained.

Details of the above-mentioned autoradiograph measurement operation and method of converting the digital signal corresponding to the autoradiograph are described in the above-mentioned Japanese Patent Laid-Open No. 59-83057, etc.
has been admonished.

In addition, 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 spread out on a support medium. , but is not limited to these methods, even digital signals obtained by any other method can be radioactive Pi.
As long as there is a correspondence with the autoradiograph of the n substance, it is appropriate to apply the signal processing method of unreleased IJJ.

Furthermore, in any of the above methods, it is not necessary to read out (or read out) the autoradiograph over the entire surface of the radiation film (or Ja-based phosphor sheet), and it is of course possible to read out the autoradiograph over only one image area. be.

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 two or more lines on each band are set in 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 the autoradiograph includes the digital signal obtained in this manner.

The obtained digital signal Dxy 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) contains 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 simultaneously processed. A display means such as a CRT capable of displaying
The data is sent to a device having an input stage capable of performing input for cursor movement 3 and base name display, and a storage means capable of recording and saving pattern information, cursor information, and base name information. A radiograph (migrating pattern of a radiolabeled substance) is visualized on a display screen.

It should be noted that the digital signal may be subjected to processing (image processing) in advance in order to obtain a visible image with excellent observation and interpretation characteristics, such as purulence and contrast.

Examples of image processing include spatial frequency processing, gradation processing, addition f-average processing, reduction processing, and enlargement processing.

The autoradiograph image displayed on the screen represents the signal level (image C degree, that is, the amount of radiolabeled substance) in the coordinate system set on one screen by the brightness of the color.
It may be a grayscale image similar to a conventional photographic image, or it may be a binary image expressed in a simplified manner by binarizing the signal level in advance.

Alternatively, the autoradiograph is -dimension 21
An image expressed by multiplexing multiple two-dimensional waveforms (positions along the electrophoresis direction) and signal levels at regular intervals in a direction perpendicular to the electrophoresis direction (so-called birdwi diagram)
It may be. According to this bird track map, the amount of radioactively labeled substances is expressed three-dimensionally as the height of the bird W & diagram.
The peak position of the band can be read accurately, the positional relationship of bands between electrophoresis rows can be grasped, and bands can be easily separated in a region where bands are dense near the electrophoresis start position. The details of the method of displaying an autoradiograph using a Σ and bird's-eye diagram are described in the specifications for Japanese Patent Application No. 181431/1983 filed by the present applicant.

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 an electrophoresis pattern displayed as a Karasu diagram.

Next, display the basic reading cursor on the display screen,
Bands are ordered (belonged) 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 hunts 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. Alternatively, it is preferable to create and display a curve.

This phenomenon is caused by the heat dissipation effect (edge effect) caused by the heat dissipation effect (edge effect), and is a phenomenon in which the migration distance at both ends of the support medium is shorter than the migration distance at the center of the support medium. 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 difference between lanes. Refers to misalignment. In addition to these band misalignments, defects in the band itself, meandering lanes, etc. may occur.

The basic polygonal or curved reading cursor is
For example, it can be created 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 to the right, and a smiling phenomenon occurs in the electrophoresis pattern.

First, input based on the display screen from the top of the screen - [11
Naichino! 1M-1 Niashi+G old t〉ku1)'re mountain/
The position of Yamauchi 1h÷*zIJtkn is preferably the hunt position on the end lane (Fig. 3, 1). The lane at the end is selected because the reading cursor is created starting from this position.

Input of position information can be performed using an L stage such as a mouse cursor, a light ben, or a joystick.

From the viewpoint of positional accuracy, a mouse cursor is particularly preferred.

For example, the rectangular area on the screen where the autoradiograph is displayed is given the coordinates (Xa, ya), (X b * Y
b) and press the mouse cursor (arrow 2 on the screen)
If the position coordinates of are expressed as (xm*'/m), then the mouse cursor moves X, = x8. yIn=yI
Line segment 3 (x+
+3'a) (X+, V+) is displayed on the screen.

Next, a straight line is drawn from the starting point 4 to the input position (second 21) 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, start point 4 (x + , y
+ ) and mouse cursor 2 digit 1 (X m + 3
'm) and the line segment ('X++3/+) (xm, y
m) will be displayed. When the mouse cursor 2 moves, it disappears on this line separation screen and a new line segment is displayed between the starting point 4 and the moved position. Note that the mouse cursor has the above coordinates (X a + ya), (X b + 3/ b
) You can move freely within the rectangular area divided by the squares. 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 displayed fixedly on the screen.

In this way, sequentially across the lanes from the top lane to the bottom lane and along the band of each lane,
Line segment (X + + 3' +) (X 1011y
, or) (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, 1 along the f 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 is strictly 1 for each lane. - does not mean passing through.

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, the vertical direction from position 5 of the end point to the bottom edge of the screen is displayed. Draw a line segment 6 (x1, V+) (X++ yb). In this way, a reading cursor 7 consisting of a plurality of connected polygonal lines that completely traverses the electrophoresis pattern is displayed on the screen.
will be 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 shaped like a polygonal line connecting the input positions with a straight line, or may be shaped like a curved line by subjecting the obtained polygonal line to appropriate arithmetic processing (curve approximation).

The created reading cursor matches the migration pattern, so the analyst can use this cursor to accurately and easily read D.
The base sequence of NA can be determined. For details on how to create and display a reading cursor, please refer to the patent application filed 3J1511 in 1986 by the present applicant.
It is stated in the specification of the No.

Band assignment is performed while fixing this basic reading cursor to each band of the electrophoresis pattern.

At this time, by displaying the name of the 1X group to which the band is assigned on the screen at the same time, it is possible to prevent reading errors such as one omission or duplicate reading, and entry errors such as omissions.

FIG. 5 is a diagram showing an example of an autoradiograph 11 of an electrophoresis pattern displayed on one screen, a reading cursor 12, 13, and base name entry fl14. In FIG. 3, the electrophoresis direction is to the right, and the base name (indicated by the initial letter G, A, T, or C) of the band that has already been read based on the reading cursor 12 is displayed in the entry field 14. .

First, in accordance with input information regarding cursor movement, the basic reading cursor (this is called an active cursor) 13 to be used for reading is moved f lines 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 a mark cursor fixed to a pattern that has been used for reading and an unused movable active cursor, and also distinguish between a base name entry field that has been read and an unprocessed entry field. It is preferable to display it. 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 hunt assignment process, a cursor of a desired shape can be created and displayed in the cylinder by repeating the same operations as described above.

Lanes (1) to (4) to which each hunt belongs are (
, G), (A), (T), and (C), so when the name of the base corresponding to the lane to which the hunt belongs is entered by keyboard operation, the unprocessed The base name is displayed in the entry field 15. Of course, if the display is incorrect, it can be corrected on the spot by re-entering. Furthermore, errors can be corrected later. In addition, at this time, the above four types of a! Utilizing the fact that two or more bands (bands in different lanes) cannot exist at the same position because the combination of all one group-specific DNA pieces is an exclusive combination,
You can perform band attribution.

In this way, base names can be assigned to the hunts in order. The unused active cursor and the unprocessed base name entry column are identified by color, etc., and the next input to move the cursor is determined to be used (mark cursor) and processed, and the display is immediately displayed. be converted. The reading cursor that newly moves on the screen according to the input information is identified as an active cursor.

Details of the method for simultaneously displaying the reading cursor and the base name entry column are described in the specification of Japanese Patent Application No. 1986-L3 filed on March 12, 1986 (2) filed by the present applicant.

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

1-1 1- rA IA & νr recognition? il;
i? ii l-L:”Edited 'Jj -k h
Pattern information, which is a characteristic requirement of the present invention, includes information regarding f-s-k 4 geographs and base sequences of nucleic acids.

Three types of cursor information and base number 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 crow's track chart 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 the information about the multiple ρ reading cursor fixed and 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 numbered according to the order of the bands. is recorded and saved as secondary -j, seat binding data of the cylinder mouth of each polygonal line. 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 screen (this is called address correspondence), and cursor information and base name information are stored in correspondence with two-dimensional coordinates fixed on the screen. They are stored in a numerically correlated manner (this is called an ordinal correspondence).

These three types of information are recorded and saved in a storage means such as a secondary storage device, and the three types of information are stored in a storage device such as a secondary storage device. files are formed. The separately recorded information files can be combined again at any time and reproduced on the display screen of a CRT shade or on a recording material such as a photosensitive material or a heat-sensitive recording material. That is, for example, the three information files recorded and saved, such as the pattern information, cursor information, and salt name information corresponding to FIGS. 1, 6, and 7, respectively, are integrated, and the data is rewritten as shown in FIG. It is possible to express the information in a different display format.

However, the storage stage (recording medium) on which information is recorded and saved is not limited to secondary storage devices, and the above three types of information can be stored separately while maintaining a one-to-one correspondence, and can be combined at any time. Any recording medium can be selected as long as it can be reproduced. 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, a case has been described in which an exclusive combination of (G, A, T, C) is used as a mixture of base-specific DNA fragments as a sample, but the analysis method of the present invention is limited to this combination. For example, (G, G+A
, T+C, C). Similarly, a mixture of base-specific RNA fragments (
For example, the method of the present invention can also be applied to combinations of G, A, U, and C).

[Brief explanation of drawings]

FIG. 1 is a diagram showing an example of an autoradiograph of a migration pattern displayed as an e-light image on one screen. Figure 52 is a diagram showing an example of an autoradiograph of a migration pattern displayed as an island track diagram 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. l: electrophoretic hunt, 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. Autoradiograph 12: Used cursor (mark cursor) 13: Unused cursor (active cursor) 1411J Base name entry field, 15: Unprocessed entry field Figure 6 is recorded and saved as cursor information FIG. 3 is a diagram illustrating an example of a large number of polygonal line-shaped reading cursors. :JS7 rg is a diagram showing an example of a series of base names recorded and saved as base name information. Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 GAGATTCCCATGCAGG... A... Mountain procedure amendment April 10, 1988

Claims (1)

[Claims] 1. Separation and development 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 of a pattern, information regarding the autoradiograph and the base sequence of the nucleic acid determined based on the autoradiograph is: (1) an 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. The information is separated into three types, consisting of (1) and (2), cursor information, and (2), cursor information and (3), base name information, and are recorded and stored in association with each other. An autoradiographic analysis method for determining the base sequence of characterized nucleic acids. 2. The nucleic acid according to claim 1, wherein the pattern information and the cursor information are associated with each other by a coordinate system, and the cursor information and the base name information are associated with each other by an order. Autoradiographic analysis method for base sequencing. 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 recorded and stored in a secondary storage device. 4. The autoradiographic analysis method for determining the base sequence of a nucleic acid as set forth in claim 1, wherein the pattern information, cursor information, and base name information are recorded and stored on the same recording medium. 5. The pattern information is formed by displaying a grayscale image, a binary image, or a plurality of two-dimensional waveforms each consisting of a position along the separation development direction and an image density at regular intervals in a direction perpendicular to the separation development direction. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, which comprises an image. 6. 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 for. 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. In the above autoradiograph, a support medium and a stimulable phosphor sheet containing a stimulable phosphor were superimposed, and an autoradiograph of a radiolabeled substance on the support medium was accumulated and recorded on the phosphor sheet. After that, the phosphor sheet is irradiated with excitation light and the autoradiograph is photoelectrically read out as photostimulated light, thereby obtaining a digital signal. Autoradiographic analysis method for base sequencing. 9. The above-mentioned autoradiograph is produced by superimposing 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. 2. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein the autoradiographic analysis method for determining the base sequence of a nucleic acid is obtained as a digital signal by photoelectrically reading the radiograph.