JPH0529069B2 - - 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. The applicant has utilized a radiographic image conversion method using a stimulable phosphor sheet instead of the conventional radiographic method using a radiographic film as a method of obtaining an autoradiograph of a radiolabeled substance separated and developed on a support medium. A patent application has already been filed for the method for
issue). 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,
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. Conventionally, the base sequence of a nucleic acid has been determined using a base-specific cleavage degradation product or a base-specific composite of a radioactively labeled nucleic acid (hereinafter simply referred to as a base-specific fragment of a nucleic acid) in a visualized autoradiograph. ) is determined by visually determining the separation development position (band) and comparing the positions of these bands with each other. Therefore, analysis of autoradiographs is usually performed through human vision, which requires a great deal of time and effort. Furthermore, since it relies on the human eye, there are limits to the accuracy of the information that can be obtained, such as the determined base sequence differing depending on the analyst. Therefore, the applicant has already filed a patent application for a method for automatically determining the base sequence of DNA by obtaining the above-mentioned autoradiograph as a digital signal and then subjecting this digital signal to appropriate signal processing. (Unexamined Japanese Patent Publication No. 126527, No. 126527, Unexamined Japanese Patent Publication No. 59-
No. 126278, patent application No. 1983-74899, patent application No. 1983-85275
No. etc.). [Summary of the Invention] The present invention provides an autoradiographic analysis method that can determine the base sequence of a nucleic acid with high accuracy. The present invention also provides an autoradiographic analysis method that can easily and advantageously confirm and/or correct the band order once determined. That is, the present invention provides () a base-specific DNA to which a radioactive label has been added;
In a method for determining the base sequence of a nucleic acid by analyzing an autoradiograph of a plurality of separation and development columns formed by separation and development of fragments or RNA fragments in a one-dimensional direction on a support medium, (1 ) electrically displaying the autoradiograph in which the band order has been determined as an image based on a digital signal corresponding to the autoradiograph; (2) separating the autoradiograph based on the input information for confirming the band order; Fix the reading cursor to the autoradiograph and display it on the screen so as to cross the development row and pass through the position of one band,
At the same time, displaying the base name of the band on the screen; (3) Based on the input information for confirming the order of the bands, the cursor is read so as to pass through the position of the band adjacent to the band fixed in the previous step. By repeating the step of fixing the cursor on the autoradiograph and displaying the base name of the band on the screen at the same time, and (4) the third step, the determined order of the bands can be displayed on the autoradiograph. An autoradiographic analysis method for determining the base sequence of a nucleic acid, the method comprising: a step of confirming on a graph; and () base-specific DNA to which a radioactive label has been added.
In a method for determining the base sequence of a nucleic acid by analyzing an autoradiograph of a plurality of separation and development columns formed by separation and development of fragments or RNA fragments in a one-dimensional direction on a support medium, (1 ) electrically displaying the autoradiograph in which the band order has been determined as an image based on a digital signal corresponding to the autoradiograph; (2) separating the autoradiograph based on the input information for confirming the band order; Fix the reading cursor to the autoradiograph and display it on the screen so as to cross the development row and pass through the position of one band,
At the same time, displaying the base name of the band on the screen; (3) Based on the input information for confirming the order of the bands, the cursor is read so as to pass through the position of the band adjacent to the band fixed in the previous step. fixing the cursor on the autoradiograph and displaying it on the screen and simultaneously displaying the base name of the band on the screen; (4) displaying the reading cursor and/or base name displayed in the third step on the display screen; (5) Confirm the determined order of bands by sequentially repeating the third and fourth steps. An autoradiographic analysis method for determining the base sequence of a nucleic acid, comprising the steps of: In the present invention, "determining the order of bands" refers to assigning an order to the bands based on the position of each band along the separation expansion direction, and assigning base names to the bands based on the separation expansion sequence to which the band belongs. It means to attach . According to the method of the present invention, when confirming the base sequence of a nucleic acid that has been determined once by ranking bands on an autoradiograph, the base sequence is displayed together with the autoradiograph, and the base sequence is Rather than being listed all at once as a character string, each base is displayed in correspondence with a band, so you can reliably check each band one by one. And the base name and band are linear,
Since a one-to-one correspondence is established using a reading cursor in the form of a polygonal line, it is possible to judge at a glance whether there is an error in the order of the bands, if a band has been read twice, or if a band has been missed. Furthermore, if an error in the order of bands (order and base name), duplicate reading or omission of bands is discovered during confirmation, it can be easily corrected on the spot. Even in this correction, since each band is displayed sequentially for each base, corrections can be made without error. Therefore, the reliability of the finally obtained base sequence can be significantly increased. In particular, when the ordering of the first band is automatically determined by signal processing as described above, there is a strong demand for final confirmation and further correction by the researcher himself. It can be said that the method of the present invention is a very effective method. [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. A mixture of base-specific fragments of radioactively labeled nucleic acids, which is a sample, 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 autoradiograph of the support medium on which the radiolabeled substance has been separated and developed is obtained by radiography using a conventional photographic light-sensitive material or by a radiation image conversion method using a stimulable phosphor sheet. A digital signal corresponding to the autoradiograph is obtained via a 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 characteristic 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 the autoradiograph can be obtained. For details on the above-mentioned autoradiograph measurement operation and method for obtaining a digital signal corresponding to the autoradiograph, see the aforementioned Japanese Patent Application Laid-open No. 59-83057;
It is described in various publications such as JP-A-59-126527 and JP-A-59-126278. In the above, a method using conventional radiography and radiographic image conversion methods was described as a method for obtaining a digital signal corresponding to an autoradiograph of a radiolabeled substance separated and developed on a support medium. The 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, 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 performing 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. In addition, in the present invention, the digital signal corresponding to autoradiograph also 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. The digital signal corresponding to this autoradiograph is once stored in a memory (buffer memory or non-volatile memory such as a magnetic disk) and then sent to a signal processing circuit. Next, by performing signal processing on the obtained digital signal, or by visually judging the autoradiograph that is electrically displayed based on the digital signal, everything that appears on the autoradiograph can be analyzed. The order of the bands is determined. The former method of automatic analysis using signal processing basically involves determining the position of the band by detecting the position where the signal level is at its maximum, and then assigning an order to the bands based on this position. Since the base is defined, this can be done by assigning a base name to the band based on the separation and development sequence to which the band belongs. The latter artificial analysis on the display screen is performed, for example, by creating a reading cursor on the screen and using this to assign base names to bands in an orderly manner. For details of the former automatic analysis method, please refer to Japanese Patent Application No. 1986-74899 and Japanese Patent Application No. 60-85275 filed by the applicant.
No. 60-181432, Japanese Patent Application No. 60-226091, and Japanese Patent Application No. 60-226092. For details of the latter artificial analysis method, please refer to Japanese Patent Application No. 61-47924 filed on March 5, 1988 and Japanese Patent Application No. 61-55481 filed on March 12, 1988 (2). It is stated in each specification of the issue. The obtained band order and autoradiograph are recorded and stored in a suitable recording medium as band information and pattern information, respectively. Specifically, as shown in Figure 1, the band information is based on the band position (x, y) expressed in the coordinate system fixed to the autoradiograph (i.e., the coordinate system of the digital signal) and the band information. This is band data consisting of the name of the assigned base (for example, G, A, T, or C in the case of DNA). Below, the first analysis method of the present invention will be described in detail below. Explanation will be given using an example of an expansion column. (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, pattern information about the autoradiograph of the electrophoresis pattern consisting of the four electrophoresis columns (lanes) mentioned above and band information about the determined band order are displayed simultaneously in the signal processing circuit, autoradiograph, cursor, and base name. The autoradiograph to be analyzed (radiolabeled substance The electrophoresis pattern) is visualized as a visible image on the display screen. This device is further equipped with an input means for creating a cursor and displaying base names, and a storage means for storing pattern information, cursor information, and base sequence information. desirable. Note that the digital signal corresponding to the autoradiograph, which is stored as pattern information, is subjected to signal processing (image processing) in advance to obtain a visible image with appropriate density and contrast and excellent observation and interpretation performance. You can leave it there. Examples of image processing 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. 2 shows an example of an autoradiograph of a migration pattern displayed as a gray scale image on the screen. FIG. 3 also shows an example of an autoradiograph of the migration pattern displayed as a bird's eye view. Next, based on the input information for confirming the order of the bands, the reading cursor is fixed and displayed on the electrophoresis pattern 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 determined The base name of the band is displayed on the screen. Information for confirming the order of bands can be input by simple keyboard operations such as pressing a confirm key. In accordance with the first key input, for example, information (x ₁ , y ₁ , T) about the lowest band of the electrophoresis pattern is extracted from the predetermined band information as shown in Figure 1. Thereafter, a reading cursor that has been previously input as information or created on the screen is fixed to the electrophoresis pattern and displayed on the screen so that it passes through the band position (x ₁ , y ₁ ). Band information may also be displayed on the screen at the same time or by switching screens, and in that case it is preferable to make the extracted bands visible at a glance using a pointer such as an arrow. 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 (see FIG. 6, 12). 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. A polygonal or curved reading cursor can be created on the screen, for example, as follows. FIG. 4 is a diagram showing another example of an autoradiograph of a migration pattern displayed as a gray scale image on the screen. In FIG. 4, the migration direction is to the right. 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. 41). 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 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 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 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 ) (sequentially , 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 Figure 5, 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). When the reading cursor is fixedly displayed on the screen, the band position coordinates included in the band information are added to the cursor information (if the cursor is a polygonal line, a set of coordinates of the joints of the polygonal line). It is preferable to convert. Since the created reading cursor matches the migration pattern, the analyst can accurately and easily confirm the order of bands using this cursor. The details of how to create and display the reading cursor are described in the specification of Japanese Patent Application No. 47924/1988 filed on March 5, 1985. Additionally, if the analyst visually determines the order of bands using a reading cursor on the screen displaying the autoradiograph, the reading cursor used should be recorded and saved in advance as cursor information. By doing this, the most suitable cursor for each band can be displayed immediately. Details of this reading cursor storage method are described in Japanese Patent Application No. 1982-55482 filed March 12, 1985 (3) by the present applicant. On the screen, at the same time as the reading cursor is displayed, the determined base name (T) for the lowest band is displayed below the cursor. Next, when information for confirming the band order is input (key input, etc.), the second band from the bottom is extracted from the band information. Specifically, on the screen where band information is displayed, the pointer points to the band, and on the screen where the migration pattern is displayed, a reading cursor passing through the position of the band and the base name of the band are displayed. In this way, it is possible to confirm whether or not the order of the bands is correct while sequentially displaying the cursor and base names for the bands whose order has been determined in advance. In addition, at this time, the above four types of base-specific
Since the combination of DNA fragments is an exclusive combination, taking advantage of the fact that two or more bands (bands in different lanes) cannot exist at the same position,
You can check the order of the band. FIG. 6 is a diagram showing an example of the autoradiograph 11 of the migration pattern, the reading cursor 12, and the base name column 13 displayed on the screen during the confirmation process. In addition, in FIG. 6, the electrophoresis direction is rightward. At this time, a reading cursor that has undergone confirmation processing and a reading cursor that has not been processed may be distinguished, and a base name field that has undergone confirmation processing and a base name field that has not been processed may be displayed separately. This makes it easier to discover reversal of band order, duplicate reading, or omission of reading. These distinctions can be made based on differences in color, pattern, brightness, and the like. Furthermore, the cursor and base name columns that have not been confirmed are judged to have been processed by the next key input, and the display is immediately changed. Then, a cursor with an unprocessed identification is newly displayed on the screen. Note that in the confirmation process, if the set reading cursor no longer matches the migration pattern, a cursor of the desired shape can be easily created and displayed by repeating the same operations as described above. Furthermore, according to the second method of the present invention, if it is found in the confirmation process that there is an error in the ordering, such as reversing the order of the bands, reading them repeatedly, or missing them, the process is performed before the next key input. The order of the bands can be modified according to the information entered based on the display screen. For example, when a band is read multiple times, the order of the bands is corrected by deleting the cursor and base name corresponding to the band from the display screen in accordance with input information such as key input. If the band is misread,
This is done by adding and displaying a new cursor and base name corresponding to the band on the screen.
Furthermore, if the order of bands is reversed, this is done by changing (ie, deleting and adding) the cursor and base name corresponding to the band on the screen. In this way, the cursor and base names are displayed one after another for the bands whose order has been determined in advance, and the cursor and base names are deleted, added, or changed as necessary, and the order of the bands is determined to be correct. can be checked and corrected. After confirming the order of bands and correcting them, the screen remains displaying the electrophoresis pattern, the reading cursor and base name corresponding to each band on a one-to-one basis. By connecting this series of bases in order from the end, the DNA base sequence (e.g. T-G-C-A
-T-C-G-...) can be obtained. It is preferable that information regarding the electrophoresis pattern, reading cursor, and base sequence on the screen be recorded and stored separately as pattern information, cursor information, and base sequence information in association with each other. In other words, only the information (pattern information) about the electrophoresis pattern displayed as an image on the screen is transferred to the second
It is recorded and saved as digital image data corresponding to a grayscale image as shown in the figure, a bird's eye view as shown in FIG. 3, 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). Further, only information (cursor information) regarding a large number of reading cursors fixed and superimposed on each band of the electrophoresis pattern is independently recorded and saved as cursor data as shown in FIG. The cursor information is data about the number of cursors corresponding to the number of bands sequenced,
Each cursor is numbered according to the order of the bands, and if the reading cursor is in the form of a polygonal line, for example, the two-dimensional coordinates of the nodes of each polygonal line are recorded and saved as data. Further, only information about the obtained base sequence (base sequence information) is independently recorded and saved as character string data as shown in FIG. This base sequence information is numbered according to the order of the bands. As a result, the autoradiograph analysis information, which was composed of pattern information (for example, digital image data corresponding to each pixel) and band information at the stage when the band order was determined, is changed to pattern information after this confirmation and correction operation. (digital image data corresponding to the displayed image), cursor information, and base sequence information. Pattern information and cursor information are stored in correspondence with two-dimensional coordinates fixed to the image (this is called address correspondence), and cursor information and base sequence information are stored as sequential numbers of bands on the electrophoresis pattern. Corresponding (this is called ordinal correspondence)
Saved. These pieces of information are recorded and saved in storage means such as a secondary storage device to form three information files. These information files can be recombined at any time and 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, so if someone else wants to check the analysis results at a later date. You can easily match and confirm bands and base names. Note that information can be recorded and saved not only after the analysis of the autoradiograph (confirmation and correction of the base sequence) but also at any time during the analysis. For details on how to record and save analysis results as the above three types of information, please refer to the application filed on March 12, 1988 (3).
It is described in Japanese Patent Application No. 1982-55482. 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, the method of the present invention can be applied to mixtures of base-specific RNA fragments (for example, a combination of G, A, U, and C).

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of band information regarding the determined order of bands. FIG. 2 is a diagram showing an example of an autoradiograph of a migration pattern displayed as a grayscale image on a screen. Figure 3 shows
FIG. 3 is a diagram showing an example of an autoradiograph of a migration pattern displayed as a bird's-eye view on a screen. FIG. 4 and FIG. 5 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. Figure 6 shows the migration pattern displayed on the screen.
FIG. 3 is a diagram showing an example of a reading cursor and a base name column. 11: Migration pattern, 12: Reading cursor, 1
3: Base name column. FIG. 7 is a diagram showing an example of a large number of line-shaped reading cursors recorded and saved as cursor information. FIG. 8 is a diagram showing an example of a series of base names recorded and saved as base sequence information.

[Claims]

1. Compatible with autoradiographs of multiple separation and development columns formed by separation and development of a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments on a support medium in one-dimensional direction. In a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal, the steps include: (1) obtaining a one-dimensional waveform consisting of a position along the separation development direction and a signal level for each separation development column; (2) a step of calculating an average level value of a region having a signal level of a certain value or more within a certain interval from a search reference point on a one-dimensional waveform; (3) a step of determining a threshold value based on the average level value; (4) a step of searching for whether or not a peak having a signal level equal to or higher than a threshold value exists within the section; (5a) if a peak exists in the fourth step, determining that a band exists at the position of the peak; (5b) If the peak does not exist in the fourth step, a position advanced by a certain distance from the search reference point is set as the next search reference point. a step of

Claims (1)

A method for analyzing autoradiographs. 2. A method for determining the base sequence of a nucleic acid according to claim 1, wherein the order of the bands is determined by performing signal processing on a digital signal corresponding to an autoradiograph. Autoradiograph analysis method. 3. A patent claim characterized in that the band on which the cursor is fixed in the second step is the lowest band in the separation and development row, and the order of the bands is confirmed in order from the lowest band in the second to fourth steps. An autoradiographic analysis method for determining the base sequence of a nucleic acid according to item 1. 4. Nucleic acid base sequencing according to claim 1, wherein in the second and third steps, the input information for confirming the band order is based on the determined band order. Autoradiographic analysis methods for. 5. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein in the second and third steps, the reading cursor is displayed as a polygonal line or a curved line. 6 The autoradiograph of the separated development pattern displayed in the above image, the multiple reading cursors fixed on the autoradiograph after confirmation, and the order of the confirmed bands are shown as (1) pattern information, (2) cursor information, and (3) Separate as base sequence information,
and (1) and (2) and (2) and (3) are recorded and stored in association with each other.
An autoradiographic analysis method for determining the base sequence of a nucleic acid as described in Section 3. 7 In the above first step, the autoradiograph is multiplexed as a grayscale image, a binary image, or a plurality of two-dimensional waveforms consisting of positions and image densities along the separation development direction 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, characterized in that the image is displayed as an image. 8 In the above first step, the 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. 9 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. 10 Base-specific DNA with radioactive label
In a method for determining the base sequence of a nucleic acid by analyzing an autoradiograph of a plurality of separation and development columns formed by separation and development of fragments or RNA fragments in a one-dimensional direction on a support medium, (1 ) electrically displaying the autoradiograph in which the band order has been determined as an image based on a digital signal corresponding to the autoradiograph; (2) separating the autoradiograph based on the input information for confirming the band order; (3) fixing the reading cursor on the autoradiograph and displaying it on the screen so as to cross the development row and passing through the position of one band, and at the same time displaying the base name of the band on the screen; Based on the input information for order confirmation, the reading cursor is fixed on the autoradiograph and displayed on the screen so as to pass through the position of the band adjacent to the band to which the cursor was fixed in the previous step, and at the same time a step of displaying the base name on the screen; (4) deleting the reading cursor and/or base name displayed in the third step in accordance with the input of information based on the display screen, if any;
a step of adding or changing; and (5) a step of confirming and correcting the determined order of bands by sequentially repeating the third and fourth steps. Autoradiographic analysis methods for. 11. For determining the base sequence of a nucleic acid according to claim 10, wherein the order of the bands is determined by performing signal processing on a digital signal corresponding to an autoradiograph. Autoradiograph analysis method. 12 A patent characterized in that the band on which the cursor is fixed in the second step is the lowest band in the separation and development row, and the order of the bands is confirmed and corrected in the second to fifth steps starting from the lowest band. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 10. 13. Nucleic acid base sequencing according to claim 10, wherein in the second and third steps, the input information for confirming the band order is based on the determined band order. Autoradiographic analysis methods for. 14. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 10, wherein in the second and third steps, the reading cursor is displayed as a polygonal line or a curved line. 15 The autoradiograph of the separated development pattern displayed in the image above, the multiple reading cursors after confirmation correction fixed on the autoradiograph, and the order of the confirmation and correction bands are shown as (1) pattern information, (2) cursor, respectively. (1) and (3) base sequence information, and (1) and (2) and (2) and (3) are recorded and stored in correspondence with each other, according to claim 10. An autoradiographic analysis method for determining the base sequence of nucleic acids. 16 In the first step, the autoradiograph is multiplexed as a grayscale image, a binary image, or a plurality of two-dimensional waveforms consisting of positions and image densities along the separation development direction at regular intervals in a direction perpendicular to the separation development direction. 11. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 10, characterized in that the image is displayed as an image. 17 In the 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 generate an autoradiograph of the radiolabeled substance on the support medium. After accumulating and recording on the phosphor sheet,
The base sequence of a nucleic acid according to claim 10, which is obtained by irradiating the phosphor sheet with excitation light and photoelectrically reading out the autoradiograph as photostimulated light. Autoradiographic analysis methods for determination. 18 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 photosensitive material, The autoradiograph analysis for determining the base sequence of a nucleic acid according to claim 10, which is obtained by photoelectrically reading an autoradiograph visualized on the photosensitive material. Method.