JPS62215867A - Autoradiograph analysis for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To make it possible to easily determine the base sequency of nucleic acid with high accuracy, by performing the image display of an autoradiograph in the determination of the base sequence of nucleic acid and displaying not only the name of the base belonging to the band of a separation development row determined using the reading cursor displayed on the display image but also the reading cursor. 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 fregments each provided with a radioactive marker on a gel support medium by electrophoresis, an autoradiograph is obtained using a radiographic method using a photosensitive material or a radiation image converting method using a light accumulative fluorescent sheet. Subsequently, the digital signal corresponding to the autoradiograph is obtained through a reading system. In this case, a reading cursor 7 is displayed on a display picture as a folding line or curve along the band of each lane on a display picture. Because the formed cursor 7 coincides with the migration pattern, the base sequence of DNA is easily determined. The name of the determined base sequence and the reading cursor are displayed.

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 is said that In particular, the DN that carries specific genetic information
Li! of nucleic acids such as A (or DNA fragments, hereinafter the same). It has become essential to define the base sequence.

Maxas-Gilbert uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
) method and Sanger-Courson (Sanger-C
Oulson) method is known. In the former Maxam-Gilbert method, first, a 32 p
0-order, in which a group containing a radioactive isotope, such as Next, the resulting mixture of base-specific DNA cleavage products was separated and developed on a support medium by gel electrophoresis, and a separated development pattern formed by a large number of cleavage products being developed separately (however, visually cannot be seen)
get. This separation development pattern is visualized on, for example, an X-ray film to obtain its autoradiograph, and the obtained autoradiograph and each salt,! ! The specific cleavage means sequentially determines the bases located in a certain positional relationship from the end of the chain molecule to which the radioactive isotope is bound, thereby determining the base sequence of the entire target object.

In addition, the latter Sanger φ Coulson method uses a chemical -L stage to convert a DNA compound that is complementary to a chain molecule of DNA or a DNA fragment and has been given a radioactive label into a salt M-specific The base-specific DNA obtained by
This method uses a mixture of synthetic compounds and determines the base sequence from the autoradiograph in the same manner as above.

Conventionally, visible images of autoradiography are based on the separation and development of base-specific cleavage products or base-specific composites (hereinafter simply referred to as base-specific fragments of nucleic acids) of radioactively labeled nucleic acids. It is obtained by overlaying a high-sensitivity X-ray film on a support medium prepared by the above-mentioned method for a certain period of time and exposing the film to light. Then, the base sequence of the nucleic acid is determined by visually determining the separated development positions (bands) of the 11 base-specific fragments of the nucleic acid from the autoradiograph visualized on film, and comparing the positions of these bands with each other. It is determined by

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

In the center of the transparent IJJ cursor of plastic acid, there is usually a
A thin straight line is drawn (a so-called hairline cursor), and the hairline cursor can be moved in the - direction (direction published in the film) with the hairline cursor fixed at the [/] mark. Using this linear hairline cursor as a guide, the relative positions of the bands in each column can be determined and the bands can be ranked.

Alternatively, an analysis method using a computer system using a digitizer board or the like is also being tried. In other words, an X-ray film with an i+f autoradiograph is fixed on a digitizer board that has a two-dimensional coordinate system and is connected online to a computer, and then the band on the film is pointed with an indicator stick. In this method, the ku base name to which the band should belong is inputted via another input means such as a keyboard. According to this method, the bands are automatically ranked and 11! according to the coordinate system input by the pointing stick. You can give it a nickname.

However, in both the above methods using Scherkasten and the method using a computer system, it is difficult to distinguish between read-processed bands and unprocessed bands. There are disadvantages such as the line of sight (directed toward the keyboard) for (input) is different, and there is a time interval between the two. Therefore, it was not possible to avoid reading errors such as reversing the order of bands, missing readings, and duplicating readings, as well as mistakes in writing base names.

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

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

That is, 1. the present invention provides a salt to which a radioactive label has been added;
(i l# By analyzing the autoradiographs of multiple separation and development rows formed by separating and developing different DNA fragments or RNA fragments in one-dimensional direction on a support medium, the base sequence of the nucleic acid can be determined. 1) electrically displaying an image of an autoradiograph based on a digital signal corresponding to the autoradiograph; 2) displaying a reading cursor on the displayed image; and 3) displaying a reading cursor on the displayed image. An autoradio for determining the base sequence of a nucleic acid, the method comprising the step of simultaneously displaying the name of the base to which the band of the minute S expansion sequence determined using the method is assigned, and the reading cursor on a display image. It provides a graph analysis method.

According to the method of the present invention, unlike the case of using a conventional Scherkasten, a reading cursor can be displayed on the screen on which the autoradiograph is visualized and can be freely moved on the screen.

Further, unlike the case where a conventional Scherkasten is used or a digitizer board is used, the reading cursor and the base name of the band determined and input based on the cursor are displayed on the screen at the same time. Therefore, since it is clear which band has been read, it is possible to prevent reverse reading of bands, omission of reading, double reading, etc. during reading as in the conventional method. In addition, the base names read and input are displayed instantly on the same screen, so you can understand the correspondence between the read band, cursor, and base name.
It is possible to proceed with the reading operation while checking the information, and it is possible to eliminate omissions (input) of base names and errors in entry due to temporal and spatial gaps as in the past.

Therefore, the Ii! of nucleic acids! The base sequence can be determined with high precision and in a short time.

In addition, mistakes made during reading and writing can be avoided by displaying the cursor used for reading and the cursor to be used in the future by distinguishing them by color, pattern, brightness, etc. This can be further prevented by displaying the location of the base name to be entered separately in the same way.

In particular, when the reading cursor is created and displayed on the screen so as to cross the molecule a, development column (lane) and follow the band of each lane, the band position shift between lanes may occur due to smiling phenomenon, offset distortion, etc. Based on the cursor, the order of the bands can be read more accurately even when a cursor occurs. Here, the smiling phenomenon refers to the heat dissipation effect (edge effect) during the separation and expansion process.
Offset distortion is a phenomenon in which the separation distance at both ends of the support medium becomes shorter than the separation distance at the center of the support medium. This is caused by differences in the sample separation and development start position and start time for each lane due to differences in shape, etc., and the overall positional deviation between lanes is the difference in the position of these bands. In addition, defects in the band itself, meandering lanes, etc. may occur.

This reading cursor can be moved not only vertically (or horizontally) but also in any direction, so even if the molecule a expansion pattern is meandering, the cursor can be moved optimally according to the pattern. This also increases the accuracy of reading.

The applicant has already filed a patent application for a method of obtaining an autoradiograph of a separated development pattern using a radiation image conversion method using a stimulable phosphor sheet instead of the conventional radiography method using a radiation film. (Japanese Patent Application Laid-Open No. 59-83057, Patent Application No. 58-201)
No. 231).

Here, the stimulable phosphor sheet is made of stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, '1 [magnetic wave ( By excitation with excitation light), radiation energy can be emitted as photostimulated light. According to this method, the exposure time can be significantly shortened,
Furthermore, chemical fog, which has been a problem in the past, does not occur. Furthermore, the autoradiograph of a radiolabeled substance is stored in a phosphor sheet as radiant energy and then read out photoelectrically as photostimulated light.
It can be obtained directly as a digital signal.

[Structure of the Invention] Examples of samples used in the present invention include radioactively labeled nucleic acids such as DNA and RNA.

Ii! Mention may be made of mixtures of group-specific fragments. Here, the nucleic acid fragment means a part of the Ii chain molecule. For example, a base-specific DNA cleavage mixture, which is a type of base-specific DNA fragment mixture, can be obtained by base-specific cleavage and decomposition of radiolabeled DNA according to the Maxam-Gilbert method described above. . In addition, the base-specific DNA compound mixture is radioactively labeled using DNA as a template (PJ type) according to the Sanger-Courlan method. It can be obtained by synthesis using deoxynucleoside triphosphate and DNA synthase.

Furthermore, a mixture of salt-specific RNA fragments can also be obtained as a mixture of cleavage and degradation products or as a mixture of synthetic products by the same method as described above. Note that DNA consists of four types of JiJ groups, adenine, guanine, thymine, and cytosine, as its constituent tp position, whereas RNA consists of four types of PA and U, adenine, guanine, uracil, and cytosine.

The radioactive label is added to 32p, l by a method appropriate to these substances.
AC, 3f+3.3 H, L2sI Natonoa 1tlt
It is attached by retaining the sexual isotope.

Comes with a radioactive label that is a sample! The mixture of base-specific fragments of the nucleic acid obtained by the reaction is subjected to electrophoresis, thin layer chromatography,
1 liter per minute on a support medium using various separation and development methods such as column chromatography and paper chromatography.
l is expanded.

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

Comes with the following four types of radioactive labels! base-specific D
The electrophoretic pattern obtained by separating and developing a mixture of NA fragments on a gel support medium by electrophoresis can be automatically determined by radiography using conventional photosensitive materials or by radiation image conversion using a stimulable phosphor sheet. A radiograph is obtained and then a digital signal corresponding to the autoradiograph is obtained via a suitable readout system.

l) Guanine (G) specific DNA fragment 2) Adenine (
A) Specific DNA fragment 3) Thymine (T) specific DNA
Fragment 4) Cytosine (C) specific DNA fragment, where each ii
Single-base-specific DNA fragments are base-specifically cleaved, degraded, or synthesized, that is, they consist of DNA fragments of various lengths that have the same terminal base.

When using the former radiographic method, first the support medium and the photosensitive material such as X-ray film are heated at a low temperature (-90 to
After exposing the radiographic film to light by overlapping it for a long time (several tens of hours) at -70°C), it is developed to create a visible autoradiograph of the radiolabeled substance on the radiographic film.6 Next, using an image reading device, The autoradiograph visualized on the radiographic film is read. 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 electric signal, a digital signal corresponding to an autoradiograph can be obtained.

When using the latter radiation image conversion method, first, the support medium and stimulable phosphor sheet are heated at room temperature for a short period of time (several seconds to
The autoradiograph is recorded as a kind of latent image on the phosphor sheet by overlapping the phosphor sheets (for several tens of minutes) and accumulating the radiation energy emitted from the radiolabeled substance in the phosphor sheet. Here, the stimulable phosphor sheet includes, for example, a support made of a plastic film, divalent europium-activated barium fluoride bromide (BaFBr:Eu")
A phosphor layer made of a stimulable phosphor such as phosphor and a transparent protective film are laminated in this order. When the phosphor is irradiated with radiation such as X-rays, it absorbs and accumulates the radiation energy, and then it is emitted from the 11f to infrared region. When excited with light, it emits 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, specifically,
For example, by scanning a phosphor sheet with a laser beam to emit radiation energy as photostimulated light, and then detecting this photostimulated light photoelectrically, an autoradiograph of a radiolabeled substance can be obtained directly without creating a visible image. By converting this electrical signal (1) into an electrical signal (1), it is possible to obtain a digital signal corresponding to an autoradiograph.

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

In addition, in the above, a method using conventional radiography and radiographic image conversion methods was described as a method for obtaining digital credibility corresponding to an autoradiograph of a radiolabeled substance separated and developed on a support medium. However, digital signals obtained by any other method may be used in the analysis method of the present invention as long as they correspond to the autoradiograph of the radiolabeled substance. ji) It is capable.

Furthermore, in any of the above methods, it is not necessary to read out (or read out) the autoradiograph over the entire surface of the radiation film (or stimulable phosphor sheet), and it is of course possible to read out only one 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 in the reading operation, information on each band is set. By scanning with a light beam at a scanning line density such that two or more scanning lines 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 I)
), and the signal level represents the image density at that coordinate, ie, the amount of radiolabeled substance. Therefore, the series of digital signals (ie, digital image data) contains information about the two-dimensional position of the radiolabeled substance.

First, the digital signal corresponding to this autoradiograph is once stored in memory (eight buffer memory or non-volatile memory such as a magnetic disk), and then the signal processing circuit, autoradiograph, cursor and base name The autoradiograph to be analyzed (radiolabeled substance (separated development pattern) is visualized on the display screen.

Note that the digital signal may be subjected to signal processing (image processing) in advance to obtain a visible image with appropriate density and contrast, and excellent observation and reading comprehension. Examples of image processing include, for example, spatial Frequency processing, gradation processing,
Examples include averaging processing, reduction processing, and enlargement processing.

The autoradiograph displayed as an image on the screen expressed the signal level (image density, ie, the amount of radiolabeled substance) in the coordinate system set on the screen by the lightness of the color.

It may be a C-light image similar to a conventional photographic image, or it may be a binary image simplified by pre-binarizing the signal level.

Alternatively, the autoradiograph can be expressed as a −dimensional position (
It may be an image (a so-called rut diagram) that is expressed by multiplexing a large number of two-dimensional waveforms consisting of position along the electrophoresis direction) and signal level in a certain period of time in a direction perpendicular to the electrophoresis direction. According to this bird w1 map
．． ) is expressed three-dimensionally as χ of rtX1Z, the peak position of the band can be read accurately, and the positional relationship of the bands between the electrophoresis rows (lanes) can be grasped, as well as the position near the electrophoresis start position. Separation of the band in the cloth region of the band can be easily performed. The details of the method of displaying an autoradiograph using a bird track map are described in the specifications for Japanese Patent Application No. 181431/1980 filed by the present applicant.

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

Further, FIG. 2 shows an example of an autoradiograph of a migratory pattern displayed as a bird track diagram.

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

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

A polygonal or curved reading cursor can be created and displayed on the screen, for example, as follows.

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

First, draw a vertical line from the top of the screen to the input position based on the display screen, preferably the first position input is the position of the band on the edge lane (Figure 3 1) , because the reading cursor is created starting from this position.

Location information can be entered using a mouse cursor, light pen, joystick, etc.8
From the viewpoint of positional accuracy, a mouse cursor is particularly preferred.

For example, if the rectangular area on the screen where the autoradiograph is displayed is expressed by the coordinates (Xa, 7a), (xb + Vb), and the position coordinates of the mouse cursor (arrow 2 on the screen) are expressed by (xm, ym). Then, by inputting the positional information xm=X++Vm=yI with the mouse cursor, line segment 3 (X+, Va)
(X+, V+) is displayed on the screen.

Next, the input of the 0 position, in which a straight line is drawn from the position of the starting point 4 to the input position (second position) inside the migration pattern, is performed using a mouse cursor or the like in the same manner as described above. First things first.

When the starting point 4 (X+, V+) and the mouse cursor 2 move, 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 is at E coordinates (Xa.

Ya), (Xb, Vb) can freely move within the rectangular area defined by νJ. thus.

As the mouse cursor moves, line segments connecting the starting point 4 and each movement position of the mouse cursor 2 are displayed one after another. By inputting the position where the line segment preferably crosses the lane, the next line segment connected to line segment 3 is displayed fixedly on the screen.

In this way, sequentially across the lanes from the top lane to the bottom lane and along the band of each lane,
Line segment (X+, Y+) (X+ or 1°Y++t) (However,
i is a positive integer).

Note that since the sample is an exclusive combination of four types of base-specific DNA fragments, the bands in each lane are at the same location.
Therefore, 1 along the r band means drawing a line segment along the band in a macroscopic sense so that the migration distance of each lane becomes dim. This does not strictly mean that each line segment passes over the band of each lane.

Next, as shown in Fig. 4, according to the input information that the last input position is the end point when the line segment completely crosses the electrophoresis pattern, the vertical line from position 5 of the end point to the bottom edge of the screen is Line segment 6(x,.

V+) (X+, yb). In this way, a reading cursor 7 consisting of a plurality of connected polygonal lines that completely traverses the electrophoresis pattern is displayed on the screen.

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.

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

The created reading cursor matches the migration pattern, so the analyst can use this cursor to accurately and easily read D.
NA's 11! The base sequence can be determined. For details on how to create and display the reading cursor, please refer to Japanese Patent Application No. 1986-511 filed in March 1988 by the present applicant.
It is stated in the specification of the No.

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

FIG. 5 is a diagram showing an example of the autoradiograph 11 of the migration pattern, the reading cursor 12, 13, and the base name entry column 14 displayed on the screen 1-. In FIG. 5, the electrophoresis direction is to the right, and the base name (indicated by the initial letter G, A, T, or C) of the band that has already been read based on the reading cursor 12 is displayed in the entry field 14. .

First, according to the input information regarding the movement of the reading cursor, the reading cursor 13 to be used for reading (this is called an active cursor) is moved seven lines to the left or right to match the position of the next band to be read.

At this time, not only the base name that has already been read, but also the cursor 12 used for reading (this is called a mark cursor) may be displayed on the screen as it is. A pairwise correspondence can be clearly expressed.

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

When the reading cursor is fixed on a band and the base name of the band is read, the mark cursor used for reading and the unused active cursor are distinguished, and the base name entry field for which reading has been processed and the unprocessed base name are distinguished. It is preferable to display the entry fields separately from each other, thereby further preventing reading errors and entry errors. The mark cursor and the active cursor can be distinguished, for example, by changing the color of the cursor, by distinguishing between a solid line and a dotted line, or by changing the brightness of the cursor. Further, the processed entry field and the unprocessed entry field can be distinguished by, for example, displaying only the unprocessed entry field 15 inverted or blinking, as shown in FIG. .

Also, if the reading cursor no longer matches the migration pattern during the process of band assignment.

By repeating operations similar to those described above, a cursor of a desired shape can be easily created and displayed.

Lanes (1) to (4) to which each band belongs are respectively (
Since it has information about the terminal bases consisting of G), (A), (T), and (C), when the name of the base corresponding to the lane where the band bends is entered by keyboard operation, the unprocessed entry is immediately entered. The base name is displayed in column 15.

Of course, if the 1 display is incorrect, it can be corrected on the spot by inputting +lG. At this time, since the combination of the four types of salt-specific DNA fragments mentioned above is an exclusive combination, it should be noted that two or more bands (bands in different lanes) cannot exist at the same position. It can be used to determine the ranking.

In this way, base names can be assigned to bands sequentially from the bottom of the electrophoresis pattern. Identification by color etc. of unused reading cursor and unprocessed base name entry field is 1.
Next, when an input is made to move the cursor, it is determined that the device has been used and has been processed, and the display is immediately changed. The cursor that is newly moved on the screen according to the input information is identified as an unused active cursor. In Figure 7, the mark cursor 12°,
A newly displayed active cursor 13' and an entry 41115° marked as read are shown.

After the reading is completed, a pair of reading cursors corresponding to each band and the names of the bases of the series written in the entry field at the bottom of the cursor are displayed on the screen. Therefore, it is possible to easily compare and confirm the assigned bands and base names after completion or during an intermediate step. By connecting this series of bases in order from the end, the DNA base sequence (
For example, T-G-C-A-T-C-G-...) can be obtained.

In the above, a case has been described in which an exclusive combination of (G, A, T, C) is used as a mixture of base-specific DNA fragments as a sample, but the analysis method of the present invention is limited to this combination. For example, (G, G+A
, T+C, C). Similarly, a mixture of base-specific RNA fragments (
For example, for the combination of G, A, U, C), the unreleased Il
1 method can be applied. Furthermore, the method of the present invention is not limited to the separation and development array of base-specific fragments of a set of nucleic acids, but the base name entry column is applied to all the fraction GL development arrays that are simultaneously separated and developed on a support medium. It is common practice to apply them simultaneously by displaying them separately.

Information about the base sequence of the obtained nucleic acid is stored in a storage device such as a magnetic disk or magnetic tape via a signal processing circuit, or is further stored in a display screen such as a CRT, a photosensitive material, or a heat-sensitive recording material. Can be displayed and recorded.

[Brief explanation of drawings]

FIG. 1 is a diagram showing a complete radiograph of an electrophoresis pattern displayed as an e-light image on the screen. FIG. 2 is a diagram showing an autoradiograph of the migration pattern displayed as a bird track diagram on the screen. FIG. 3 and FIG. 4 are diagrams each showing an autoradiograph displayed on the screen, and are diagrams for explaining the process of creating a reading cursor. 1: Electrophoresis z<nd, 2: Mouse cursor. 3.6: line segment, 4: starting point, 5: ending point. 7: Reading cursor FIGS. 5 to 7 are diagrams each showing an autoradiograph displayed on the screen, and explain the process of simultaneously displaying the reading cursor and the kM basal rate entry field according to the present invention.
This is a diagram. 11 Neoradiograph, 12.12°: Used cursor (mark cursor), 13.13': Unused cursor (active cursor), 14: Base name entry field, 15: Unprocessed entry field, 15°: Processed field Patent applicant Agent: Tomigami Photo Film Co., Ltd.
Yasuo Yanagikawa Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 Procedure 7 City New Year April 10, 1988 Patent Application No. 55481 2, Name of Invention Base of Nucleic Acid Autoradiograph analysis method for sequence determination 3, relationship with the amended case Applicant name (520) Fuji Photo Film Co., Ltd. 4, agent address Miya Yotsuya Pill, 2-14 Yotsuya, Shinjuku-ku, Tokyo 8
Floor 6, Number of inventions increased by amendment None 7, Supplement i
Subject of E (1) "Detailed Description of the Invention" column of the specification. (L) As shown in the attached sheet. (1) The "Detailed explanation of the invention" column of the specification will be amended as follows. CI) IJ] A1+1'19 (7) Page 16 [row ~
Line 17) tr Low temperature (-90 to -70°C) for a long time (
(for several tens of poems) J at low temperature or room temperature for a long time (several hours ~
Number 11 hours) Correct as J. (2) "V 2 or more lengths" on page 19, lines 17 to 18 of the specification is corrected to 1 or more r. (3) The last line of page 26 of the specification r patent application 1986-
``Patent Application No. Sho 61-47924'' shall be amended. (4) On page 28, lines 11 to 13 of the specification, "The reading cursor is in the hand...Delete the used J." (5) [9I In the specification, page 28, lines 13 to 15 Line 11 r Unused active...Distinguish from the entry field" to "
The active cursor is distinguished from J and corrected. (6) Delete r and entry error J on line 16 of line 28 of the specification. (7) Page 29 of the specification, lines 1 to 5. Also, the processed... can be done. ” to be deleted. (8) In the second line of page 30 of the 4-sheet specification, "The ranking can be determined. Next to J, break a line and insert the following sentence. "An unprocessed entry field that displays the base name. 15 is preferably displayed inverted or blinking as shown in Figure 6 to distinguish it from other base name entry fields.This can further prevent entry errors. .J (9) Delete "r in this way, ... can be added" on page 30, lines 3 to 4, line 11 of the specification. (10) Page 30, line 4 to line 8, line 11 of the specification: r unused...display will be converted. J to "Next, when an input for moving the cursor is made, the active cursor 13 and unprocessed input vAts in FIG. "It will be corrected." (11) “And,” on page 30, lines 8 and 9 of the specification.
Insert the following communication before 1. ``In other words, a new mark cursor is displayed at the position of the active cursor 13, and the base name entry 815 is displayed in the same manner as other processed entry fields.J (12) Page 30, line 10 of the specification ~ The r cursor in line 11 is identified as an unused active cursor.'' 1 cursor is an active cursor. ” he corrected. (1:1) Delete "newly displayed" on page 30, line 12 of the II specification. (14) r15° is shown on page 30, line 14 of the specification. ”, then insert the following sentence on a new line. ``In this way, you can assign base names to the hands in order from the bottom of the electrophoresis pattern.

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 based on a digital signal corresponding to the autoradiograph; 2) placing a reading cursor on the displayed image. and 3) simultaneously displaying the name of the base to which the band of the separation expansion sequence determined using the reading cursor is assigned, and the reading cursor on the display image. An autoradiographic analysis method for determining the base sequence of nucleic acids. 2. In the second step, the reading cursor is displayed as a polygonal line or a curved line that crosses the separation and expansion rows and follows the bands of each row. Autoradiograph analysis method for. 3. In the second step, a reading cursor created by drawing a straight line or an arc passing through at least one position based on input information regarding the position is displayed. An autoradiographic analysis method for determining the base sequence of nucleic acids. 4. The autoradio for determining the base sequence of nucleic acids according to claim 1, characterized in that in the third step, the name of the base is displayed as letters and/or symbols at the end of the reading cursor. Graph analysis method. 5. In the third step, the reading cursor that has already been used for band assignment and the reading cursor to be used next are distinguished and displayed at the same time. An autoradiographic analysis method for determining the base sequence of nucleic acids. 6. In the third step, the name of the base to which a band has already been assigned and the name of the base to which a band is to be assigned next are distinguished and displayed at the same time. An autoradiographic analysis method for determining the base sequence of nucleic acids. 7. The autoradiograph analysis method for determining the base sequence of a nucleic acid according to claim 1, wherein in the first step, the autoradiograph is displayed as a grayscale image or a binary image. 8. In the first step, an autoradiograph is created as an image by multiplexing a plurality of two-dimensional waveforms consisting of positions along the separation development direction and signal levels at regular intervals in a direction perpendicular to the separation development direction. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 1, characterized in that: 9. 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. 10. In the above 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.