JPS62215869A - Autoradiograph analysis for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To make it possible to easily confirm or correct the order of a band while determining the base sequence of nucleic acid with high accuracy, by displaying an autoradiograph as an image and simultaneously displaying a predetermined reading cursor and the base name of the band and processing the digital signal corresponding to the autoradiograph. 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 fragments each provided with a radioactive marker on a gel support by electrophoresis, an autoradiograph 11 wherein the order of band is determined is obtained using a photosensitive material or a light accumulative fluorescent sheet. A reading cursor 12 is displayed on a display picture as the folding line along the band of each lane. The digital signal corresponding to the autoradiograph is subjected to signal processing to determine the base sequence of DNA. The name of the determined base is displayed on the base name entry column 13 on the display picture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention 1] The present invention relates to autoradiographic analysis power υ for determining the base sequence of nucleic acids.

[Behind the Invention J:FO] What is the background of molecular biology that has developed rapidly in recent years? In order to elucidate the organism's internal organs and replication mechanisms, it is essential to clarify the genetic information possessed by the organism. In particular, D, which carries specific genetic information
It is essential to determine the base sequence of nucleic acids such as NA (or DNA fragments, hereinafter the same).

Maxavi-Gilb, who uses autoradiography, is a typical method for determining the 1X base sequence of nucleic acids such as DNA and RNA.
ert ) law and Sanger-Coulson (Sangc
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 binding a group containing a radioactive isotopic end of p1 and attaching a radioactive label, a base-specific bond between the constituent units (Ill! group units) of a chain molecule is created using a chemical f-stage. Cut into. 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 can only be seen). Next, this separation development pattern is visualized on, for example, a x5 film to obtain an autoradiograph thereof, and from the obtained autoradiograph and each base-specific cleavage means, it is determined that the radioactive diagonal element is a bonded chain. Bases located in a certain positional relationship from the end of the molecule are sequentially determined, thereby determining the base sequence of the entire target object.

In addition, the latter Sanger-Courlain method uses chemical means to convert DNA molecules complementary to chain molecules of DNA or DNAlfr fragments and radioactively labeled molecules into base-specific molecules. This is a method in which the base sequence is determined from the autoradiograph in the same manner as above using a mixture of base-specific DNA compounds obtained.

The applicant has developed a radiographic image conversion method using a storage phosphor sheet instead of the conventional radiographic method using a radiographic film as a method for obtaining an autoradiograph of a radiolabeled substance separated and developed on a support medium. A patent application has already been filed for the method of utilizing
(Japanese Patent Application No. 58-201231), 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, visible to Radiation energy can be emitted as photostimulated light by exciting it with electromagnetic waves (excitation light) in the infrared region. According to this method, the 'A light 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 transferred to a phosphor sheet as −μ radiation energy and then read out as a charge as photostimulated light, it can be obtained directly as a digital signal and then recorded and stored in an appropriate recording medium. I can do something.

Conventionally, the base sequence of a nucleic acid has been determined for a visualized autoradiograph by determining the base sequence of a radioactively labeled nucleic acid.
! Visually determine the separation development position t (band) of the group-specific cleavage degradation product or base-specific synthesis product (hereinafter simply referred to as base-specific fragment of nucleic acid), and compare the positions of these bands with each other. It is determined by Therefore, analysis of autoradiographs is usually performed through human vision, which requires a great deal of time and effort. Furthermore, because 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 autoradiograph as a digital signal and then subjecting this digital signal to appropriate signal processing. There is (Unexamined Japanese Patent Publication No. 1983-
126527, Japanese Patent Application Publication No. 126278-1982, Japanese Patent Application No. 60-74899-), Japanese Patent Application No. 85275-1980, etc.)
.

[9. Summary of Akira] The present invention provides an autoradiographic analysis method that allows the base sequence of a nucleic acid to be determined with high accuracy.

The present invention also provides an autoradiographic analysis method that allows the determined band order to be easily and advantageously confirmed and/or corrected.

That is, the present invention.

(1) Analyzing the autoradiograph of a plurality of separated and developed arrays formed by separating and developing III group-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.

l) The autoradiograph in which the band order has been determined,
2) electrically displaying an image based on the digital signal () corresponding to the autoradiograph; 2) determining the position of the band across the separation development column based on the input information for confirming the order of the bands; fixing the reading cursor on the autoradiograph so that it passes through the autoradiograph and displaying it on the screen, and simultaneously displaying the base group of the band on the screen; 3) based on the input information for confirming the band order, fixing the reading cursor on the autoradiograph and displaying it on the screen so as to pass through the position of the band adjacent to the band on which the cursor is fixed, and simultaneously displaying the base group of the band on the screen; and 4. ) A step of confirming the determined band order on an autoradiograph by repeating the third step.

An autoradiographic analysis method for determining the base sequence of a nucleic acid, comprising: and (II) a radioactively labeled base-specific DNA fragment or RNA fragment is placed on a support medium in a one-dimensional direction. In a method for determining the base sequence of a nucleic acid by analyzing autoradiographs of a plurality of separated and developed arrays formed by separation and development, 1) the autoradiograph in which the band order has been determined is A step of electrically displaying an image based on a digital signal corresponding to the radiograph; 2) reading across the separation development column and passing through the position of one band based on input information for confirming the order of the bands; 3) fixing the cursor on the autoradiograph and displaying it on the screen and simultaneously displaying the base groups of the band on the screen; 3) fixing the cursor in the previous step based on input information for confirming the band order; A step of fixing the reading cursor on the autoradiograph and displaying it on the screen so as to pass through the position of [Han] ~ adjacent to the band that was selected, and simultaneously displaying the base group of the band on the screen, 4) Third step The read cursor and /
or the step of deleting, adding, or changing the base group according to the information input based on the display screen, and 5) by sequentially repeating the third and fourth steps, the determined band The process of checking and correcting the hierarchy.

An autoradiographic analysis method for determining the base sequence of a nucleic acid, the method comprising:

In the present invention, "J for determining the order of bands" refers to an order given to the bands based on the position of each band along the separation development direction, and a base group assigned to the band based on the separation development sequence to which the band belongs. I'm interested in adding .

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 Because each base is displayed in correspondence with a band instead of being listed all at once as a character string, it is possible to reliably check each band one by one. And the base group and the band are linear,
Since a pairwise 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 repeatedly, or if a band has been missed.

Also, during the confirmation, the band order (order and base base)
If you find a mistake in reading, double reading of a band, or omission of a band, you can easily correct it on the spot. Even in this modification, since each band base is displayed sequentially, it can be corrected without error.

Therefore, the reliability of the finally obtained base sequence can be significantly improved.

In particular, when the order of the first band is automatically determined by signal processing as described above, there is a high demand for the final confirmation and even correction to be carried out 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,
A nucleic acid fragment refers to a portion of a long chain molecule. For example, 1t! A base-specific DNA cleavage degradation product mixture, which is a type of base-specific DNA1fr fragment mixture, is a mixture of base-specific DNA 1fr fragments that contains radioactively labeled DNA according to the Maxam-Gilbert method described above. Obtained by group-specific cleavage and decomposition. In addition, the salt-specific DNA compound mixture is prepared by using DNA as a template (Pr type), radioactively labeled deoxynucleoside triphosphate, and DNA synthesizing enzyme according to the Sanger-Courlain method described above. It can be obtained by synthesis.

Furthermore, a mixture of base-specific RNA fragments can be obtained as a mixture of cleavage and decomposition products or as a mixture of synthetic products by the same method as described above. Note that DNA consists of four types of bases: adenine, guanine, thymine, and cytosine as its middle structure, while RNA consists of four types of bases: adenine, guanine, uracil, and cytosine.

The radioactive label is 32p,
It is given by holding radioactive isotopes such as direct AC, %s, koH, and "I".

A mixture of base-specific fragments of radioactively labeled nucleic acids as a sample is subjected to electrophoresis, thin layer chromatography, column chromatography, and vapor chromatography using various known support media such as gel support media. Molecule A is developed on a supporting medium by various methods such as S and S.

Next, radioactive! By radiography using conventional photographic materials on support media classified as 5m materials,
Alternatively, after an autoradiograph is obtained by a radiation image conversion method using a stimulable phosphor sheet, a digital signal corresponding to the autoradiograph is obtained via an appropriate readout system.

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, 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 has a support made of, for example, a plastic film,
Divalent europium activated barium fluoride bromide (BaFBr:
A phosphor layer made of a stimulable phosphor such as Eu'') and a transparent protective film are laminated in this order.The stimulable phosphor contained in the stimulable phosphor sheet is
It has the characteristic that when it is irradiated with radiation such as a ray, it absorbs and stores the radiation energy, and then when it is excited with light in the visible to infrared region, it releases the accumulated radiation energy as photostimulated light.

Next, the autoradiograph stored and recorded on the stimulable phosphor sheet is read out using a reading device. in particular,
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 radiolabeled substance can be directly obtained without the need for IIF visual imaging. obtained as an electrical signal. Furthermore, by A/D converting this electrical signal, a digital signal corresponding to an autoradiograph can be obtained.

For details of the above-mentioned autoradiograph measurement operation and method of obtaining a digital signal corresponding to the autoradiograph, see the above-mentioned JP-A-59-83057 and JP-A-59-1.
It is described in various publications such as No. 26527 and Japanese Unexamined Patent Application Publication No. 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. Not limited to these methods, even if digital signals are obtained by any other method, as long as there is a correspondence with the autoradiograph of the radiolabeled substance, the unreleased Ill signal processing method can be applied. That is the turning point.

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 perform it only on the image area. .

Furthermore, in the present invention, reading conditions are set by inputting information about the position of each separation expansion column, band width, etc. in advance, and two lines are read on each band in the reading operation. By scanning with a light beam at a scanning line density that allows the above scanning lines to pass through,
It is possible to shorten reading time and efficiently obtain necessary information. 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 Dxy consists of coordinates (x, y) expressed in a coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (2) 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, the image that appears on the autoradiograph is The ranking of all 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 terminal 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. 60-74899, Japanese Patent Application No. 85275-1982, Japanese Patent Application No. 181432-1982, and Japanese Patent Application No. 2260-1988 filed by the present applicant.
No. 91 and Japanese Patent Application No. 60-226092. For details of the latter artificial analysis method, please refer to Japanese Patent Application No. 1983 filed by the applicant on March 5, 1988 and March 12, 1988 (2).
It is described in each specification of Japanese Patent Application No. 1983.

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 includes the band position (x, y) and the band position expressed in the coordinate system fixed to the autoradiograph (i.e., the coordinate system of the digital signal). Attributed salt
This is band data consisting of a name (for example, G, A, T or C in the case of DNA).

The first method for analyzing undeveloped IJJ will be explained below, taking as an example a migration column (separation and development column) in which a mixture of the following four types of pieces is separated and developed in a gel support medium by electrophoresis.

l) Guanine (G) specific DNA fragment 2) Adenine (
A) Specific DNA II piece 3) Thymine (T) specific DN
A fragment 4) Cytosine (C)-specific DNA fragment Here, each base-specific DNA fragment is a DNApJi fragment of various lengths that has been cleaved, degraded, or synthesized in a base-specific manner, that is, has the same terminal base. consists of things.

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. Display means such as CRT that can display
After sending the data to a device that has an input means that allows input to confirm the order of bands, an autoradiograph (migration pattern of radiolabeled substance) of the analysis pair is displayed on the display screen based on the pattern information. Image. This device further includes an input means for inputting information for creating a cursor and displaying base names, pattern information, cursor information, and 11! It is desirable to have storage means that can record and store base sequence information.

Furthermore, 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 the image processing include spatial frequency processing, single gradation processing, summation equalization processing, reduction processing, and enlargement processing.

The autoradiograph displayed as an image on the screen expresses the signal level (image density, i.e., the amount of radioactive substance) in the coordinate system set on the 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 simplified by converting the signal level to 24N in advance.

Alternatively, the autoradiograph can be expressed as a −dimensional position (
It may also be an image (a so-called island map) that is displayed by multiplexing a large number of two-dimensional waveforms (positions along the electrophoresis direction) and signal levels at regular intervals in a direction perpendicular to the electrophoresis direction. This bird's eye map shows the amount of radiolabeled substance three-dimensionally as the height of the bird's track map, so it is possible to accurately read the peak position of the band, and the position of the band between the electrophoresis rows (lanes). It is possible to easily understand the relationship and separate bands in a dense region of bands near the migration start position. The details of the method for displaying an autoradiograph using a bird's-eye view are described in the specification of Japanese Patent Application No. 181431/1988 filed by the present applicant.

FIG. 2 shows an example of an autoradiograph of a migration pattern displayed as a gray scale image on the screen.

Further, FIG. 3 shows an example of an autoradiograph of a migration pattern displayed as a bird's eye diagram.

Next, based on the input information for band order confirmation,
A reading cursor is fixedly displayed on the electrophoresis pattern on the screen so as to cross the S development column and pass through the position of one band, and at the same time, the determined base name of the band is displayed on the screen.

Information for confirming the order of bands can be input by manual keyboard operations such as pressing a confirm key, for example.

According to the first key input, information about the lowest band of the electrophoresis pattern (x, ,
, , T) is extracted, a reading cursor that has been previously input as information or created on the screen is fixed to the electrophoresis pattern so as to pass through the band position (X l + y 1) and displayed on the screen.

The band information +V may also be displayed on the screen and L at the same time or by switching the screen. In that case, it is preferable to make the extracted band visible by a pointer such as an arrow.

However, if the electrophoresis pattern has band misalignment or band defects due to the smiling phenomenon or offset distortion, create and display a polygonal line or curved line along the band of each lane. It is preferable (see FIG. 6, 12).

Here, the smiling phenomenon is caused by the heat dissipation effect (edge effect) during the migration process, and the migration distance at both ends of the support medium is shorter than the migration distance layer at the center. refers to a phenomenon. Also, offset distortion is
This 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 refers to the overall positional deviation between lanes. 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 as follows, for example. Figure 4 shows another example of an autoradiograph of an electrophoresis pattern displayed as a C-light image on screen-L. FIG. In FIG. 4, the migration direction is to the right.

First, draw a vertical line from the top of the screen to the input position based on the display screen. Preferably, the first position entered is the position of the band on the end lane (FIG. 4, 1). 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 bar, a joystick, or the like.

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

For example, the rectangular area where the autoradiograph is displayed on the screen is represented by coordinates (Xa, ya), (xb, yb), and the position coordinates of the mouse cursor (arrow 2 on screen H) are represented by (xm, ym). In this case, by inputting the position information xm=x1+'I m = 3' with the mouse cursor, line segment 3 (xl, ya) (
xl, yI) are 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. Inputting the position is performed using a mouse cursor or the like in the same way as with the two feet. First of all, a line segment (x 1 , y + ) (XIn, yI, R) is displayed between the starting point 4 (xl, yl) and the position of mouse cursor 2 (Xffl+ym). , this line segment disappears on the screen, and a new line segment is displayed between the starting point 4 and the moved position.The mouse cursor is moved to the above coordinates (X
a.

ya), (Xb+yb). 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, from the top lane to the bottom lane, cross the lanes and follow the band of each lane, sequentially.
Line segment (xi, yl) (X+ or 1゜yl.1) (however,
i is a positive integer).

Note that since the sample is an exclusive combination of four types of base-specific DNA Fti pieces, the bands in each lane cannot exist at the same position (migration distance R1). 1 means that a line segment is drawn along the band in a macroscopic sense so that the migration distance of each lane is equal, and it means that each line segment strictly passes over the band of each lane. It's not a thing.

Next, as shown in FIG. 5, according to the input information that the line segment completely traverses the electrophoresis pattern and the last input position is the end point, the vertical line from position 5 of the end point to the bottom edge of the screen is Line segment 6 (X + +y+) (xl, yb)
pull. In this way, a reading cursor 7 consisting of a plurality of connected polygonal lines completely crossing the electrophoresis pattern is displayed on the screen.

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

Further, 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 digit δ 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).

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). Preferably, it is converted.

Since the created reading cursor matches the migration pattern, the analyst can accurately and easily confirm the order of bands using this cursor. For details on how to create and display a reading cursor, see the above-mentioned 1986
It is described in the specification of Japanese Patent Application No. 1986 filed on March 5, 2013.

In addition, if the band order is determined visually using an autoradiograph or a reading cursor by the analyst on the displayed screen, the reading cursor used should be recorded and saved in advance as cursor information. This allows you to instantly display the optimal cursor for each band. Details of this method of storing the reading cursor are described in the specification of Japanese Patent Application No. 1988 (3) filed on March 12, 1986 by the present applicant.

On the screen, at the same time as the reading cursor is displayed, the determined base group (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, a 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 group of the band are displayed.

In this way, it is possible to confirm whether or not the order of the bands is correct while displaying the cursor and base bases for the bands for which the r/m order has been determined. In addition, in this case, since the combination of the four types of base-specific DNAff1 pieces described above is an exclusive combination, it is possible to take 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 bands.

FIG. 6 is a diagram showing an example of the autoradiograph 11 of the migration pattern, the reading cursor 12, and each base column 13 displayed on the screen during the confirmation process. In addition, in FIG. 6, the electrophoresis direction is rightward.

At this time, the reading cursor that has undergone the confirmation process and the reading cursor that has not been processed may be distinguished, and each base column that has undergone the confirmation process and the unprocessed base name column may be distinguished and displayed. This makes it easier to discover reversal of band order, duplicate reading, or omission of reading. These can be distinguished by differences in color, pattern, brightness, etc. In addition, the unconfirmed cursor and base columns are
The next key input determines that the process has been completed and immediately changes the display. Then, a cursor with an unprocessed identification is newly displayed on the screen.

If, during the confirmation process, 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 bands can be modified according to the information entered based on the display screen.

The band order can be corrected by removing the cursor and base group corresponding to the band from the display screen according to the input information using the key input, for example, when a band is read repeatedly. If there are any missed readings, this is done by adding and displaying a new cursor and base group corresponding to the band on the screen.

In addition, if the order of bands is reversed, change the cursor and base group corresponding to the band on the screen (
That is, by deleting and adding).

In this way, the cursor and base groups are sequentially displayed for the bands whose order has been determined in advance, and the cursor and base groups are deleted, added, or changed as necessary, and the order of the bands is determined to be correct. can be checked and corrected.

After confirmation of the band order and furthermore correction operations are completed, the electrophoresis pattern and the reading cursor and base base corresponding to each band on a one-to-one basis are displayed and remain on the screen. By connecting this series of bases in order from the end, a DNA base sequence (for example, 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 corresponds to a grayscale image as shown in Figure 1, a bird track diagram as shown in Figure 2, or a binary image. Record and save as digital image data. As described above, digital image data consists of two-dimensional coordinates (x, y) corresponding to one pixel and a signal level (z).

Also, information about the large number of reading cursors superimposed and fixed on each band of the electrophoresis pattern (cursor information)
cursor data as shown in the seventh UA. The cursor information is data about the number of cursors corresponding to the number of bands that have been sequenced, and each cursor is numbered according to the order of the bands. The two-dimensional coordinates of the nodes of each polygonal line are recorded and saved as data.

Further, only the information about the obtained base sequence A (11!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 11! It will be composed of base sequence information.

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 sequence information are stored as sequential numbers of bands on the electrophoresis pattern. They are stored in correspondence (this is called order correspondence). These pieces of information are stored 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. This allows easy comparison and confirmation of bands and base groups.

Note that information can be recorded and saved not only after the autoradiograph analysis (base sequence confirmation and correction) is completed, but also at any time during the process. For details on how to record and save analysis results as the above three types of information, see
It is described in the specification of Japanese Patent Application No. 1988, filed March 120, 1983 (3).

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.The analysis method of the present invention is limited to this combination. For example, (G, G+
It can be applied to various combinations such as A, T+C, and C). Similarly, for a mixture of base-specific RNA fragments (for example, a combination of G, A, U, and C), the -h method of the present invention can also be used.

[Brief explanation of drawings]

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 gray scale image on one screen. FIG. 3 is a diagram showing an example of an autoradiograph of a migration pattern displayed as a bird track diagram on the screen. FIGS. 4 and 5 are diagrams each showing an autoradiograph displayed on the screen, and are diagrams for explaining the process of creating a reading cursor. l: 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 one screen, the reading cursor, and 11! It is a figure which shows the example of a base name column. 11: Electrophoresis pattern, 12: Reading cursor 13: Base name column FIG. 7 is a diagram showing an example of a large number of polygonal line-shaped reading cursors that are recorded and saved as cursor information. Figure 8 shows 1'! ! FIG. 3 is a diagram showing an example of a series of base names recorded and saved as base sequence information. Patent applicant Fuji Photo Film Co., Ltd. Representative
Person Patent Attorney Yasushi Yanagawa Younger brother 1 Figure 2 Figure 4 Figure S Figure 6 GAGATTCCCATGCAGG...
・・・・3F Tsukune City Official Book April 10, 1988 Patent Application No. 55483 2, Title of Invention Autoradiographic analysis method for determining the base sequence of nucleic acids 3, Person making the amendment Relationship to the case Patent applicant name (520) Fuji Photo Film Co., Ltd. 4, agent address Mitsuya Yotsuya Building 8, 2-14 Yotsuya, Shinjuku-ku, Tokyo
Floor 6: Number of inventions increased by amendment None 7: "Detailed description of the invention" column of the specification to be amended. We will amend the "Detailed Description of the Invention" column of the specification as follows. (1) "Low temperature (-9
0 to -70°C) for a long time (several tens of hours) is corrected to V for a long time (several hours to several tens of hours) at low temperature or room temperature. (2) “Two or more j
is corrected to "-tree or more". (3) "Patent Application No. Sho 61-479" on page 25, line 7 and page 35, line 2 of the specification.
Corrected with No. 24 J. (ll) On page 25, line 8 of the specification, “Patent Application 1986-
(5) In the 9th line of page 29 of the specification, after ``according to the first keystroke,'' there should be ``as shown in No. 11!''. Insert the predetermined J. (6) Page 35, lines 10 to 11 and 4 of the specification.
"Ir Patent Application No. 1983" on page 1, line 10 is amended to "Japanese Patent Application No. 61-55482." (7) "As shown in FIG. 1" on page 39, lines 4 and 5 of the specification is corrected to "as shown in FIG. 2." (8) Page 39, line 5 of the specification, “j shown in Figure 2
"As shown in Figure 3 (9) Page 40, line 12, 1 of the specification"
Fixed J to the screen is corrected to J fixed to one image. −1 or more

Claims (1)

[Claims] 1. An autoradiograph of a plurality of separated and developed arrays formed by separating and developing base-specific DNA fragments or RNA fragments to which a radioactive label has been added in one-dimensional direction on a support medium. A method for determining the base sequence of a nucleic acid by analyzing 1) electrically displaying an image of an autoradiograph in which the order of bands has been determined based on a digital signal corresponding to the autoradiograph; 2) Based on the input information for confirming the order of bands, the reading cursor is fixed on the autoradiograph and displayed on the screen so as to cross the separation/development column and pass through the position of one band, and at the same time 3) Based on the input information for confirming the band order, the reading cursor is automatically moved to pass the position of the band adjacent to the band fixed with the cursor in the previous step. The determined order of bands is confirmed on the autoradiograph by repeating the step of fixing it on the graph and displaying it on the screen and simultaneously displaying the base name of the band on the screen, and 4) the third step. An autoradiographic analysis method for determining the base sequence of a nucleic acid, comprising the steps of: 2. 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 characterized in that the band on which the cursor is fixed in the second step is the lowest band in the separation and expansion row, and the order of the bands is checked 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 claim 1. 4. In the second and third steps, the input information for confirming the band order is based on the determined band order, the base sequence of the nucleic acid according to claim 1. Autoradiographic analysis methods for determination. 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 image above, the multiple reading cursors fixed on the autoradiograph after confirmation, and the order of the confirmed bands, respectively, (1) pattern information, (2) cursor information and (3) separated as base sequence information, and (1) and (2)
The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, characterized in that (2) and (3) are recorded and stored in association with each other. 7. In the first step, the autoradiograph is converted into 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 a multiplexed image. 8. In the first step, the digital signal corresponding to the autoradiograph is transmitted to the autoradiograph of the radiolabeled substance on the support medium by superimposing the support medium and the stimulable phosphor sheet containing the stimulable phosphor. A patent characterized in that the autoradiograph is obtained by accumulating and recording a graph on the phosphor sheet, then 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 claim 1. 9. In the first step, after the digital signal corresponding to the autoradiograph is photosensitively recorded on the photosensitive material by superimposing the support medium and the photographic light-sensitive material to record the autoradiograph of the radiolabeled substance on the support medium on the photosensitive material. , an autoradiograph 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. analysis method. 10. By analyzing an autoradiograph of a plurality of separated and developed columns 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, A method for determining the base sequence of a nucleic acid includes: 1) electrically displaying an autoradiograph in which the order of bands has been determined based on a digital signal corresponding to the autoradiograph; 2)
Based on the input information for confirming the order of bands, the reading cursor is fixed on the autoradiograph and displayed on the screen so as to cross the separation and development column and pass through the position of one band, and at the same time 3) Based on the input information for confirming the band order, moving the reading cursor on the autoradiograph so as to pass through the position of the band adjacent to the band to which the cursor was fixed in the previous step. 4) fixing and displaying the base name of the band on the screen at the same time; 4) the reading cursor and/or the base name displayed in the third step;
or the step of deleting, adding, or changing the base name according to the information input based on the display screen, if any, and 5) repeating the third and fourth steps in sequence, the determined band An autoradiographic analysis method for determining the base sequence of a nucleic acid, the method comprising the steps of: confirming and correcting the order of the base sequence of a nucleic acid. 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. 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. An autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 10. 13. The base sequence of the nucleic acid 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 determination. 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, respectively, (1) pattern information, (2) Separated as cursor information and (3) base sequence information, and (1)
11. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 10, wherein records of ) and (2) and (2) and (3) are stored in association with each other. 16. In the first step, the autoradiograph is converted into 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 a multiplexed image. 17. In the first step, the digital signal corresponding to the autoradiograph is transmitted to the autoradiograph of the radiolabeled substance on the support medium by superimposing the support medium and the stimulable phosphor sheet containing the stimulable phosphor. A patent characterized in that the autoradiograph is obtained by accumulating and recording a graph on the phosphor sheet, then irradiating the phosphor sheet with excitation light and photoelectrically reading out the autoradiograph as photostimulated light. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 10. 18. In the first step, after the digital signal corresponding to the autoradiograph is photosensitively recorded on the photosensitive material by superimposing the support medium and the photographic light-sensitive material to record the autoradiograph of the radiolabeled substance on the support medium on the photosensitive material. , an autoradiograph 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. analysis method.