JPS62215866A - Autoradiograph analysis for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To make it possible to easily determine the base sequence of nucleic acid with high accuracy, by performing the image display of an autoradiograph in the determination of the basis sequence of nucleic acid and forming a reading cursor, which traverses separation development rows and moves along the band of each row, on the displayed image. CONSTITUTION:With respect to the migration pattern formed by electrophoretically 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 medium, an autoradiograph is obtained by radiograph method using a photosensitive material or a radiation image converting method using an accumulative fluorescent sheet. The digital signal corresponding to the autoradiograph is obtained by an image reading device. In this case, a cursor 8 formed in a folding line shape along the shape of a band is moved to the band developing direction on a picture to successively read the band.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to an autoradioclub 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 possessed by living organisms in order to elucidate the mechanisms of biological functions and replication. It is said that In particular, the DN that carries specific genetic information
It has become essential to determine the base sequence of nucleic acids such as A (or DNA1tl'i fragment, hereinafter the same).

Maxas-GilbcrL uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
) law and Sanger-Courson (Sanger-C
Oulson) method is known. The former Maxam-Gilbert method first involves determining the DNA sequence for which the J12 base is to be determined.
Alternatively, add 32 to one end of a chain molecule of a DNA fragment.
After attaching a group containing a radioactive isotope such as p to impart a radioactive label, chemical means are used to cleave the bonds between the constituent units (base units) of the chain molecule in a base-specific manner.

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 I can't even see it)
get. This separation development pattern is visualized on, for example, an X-ray film to obtain its autoradiograph, and from the obtained autoradiograph and each base-specific cutting means, the end of the chain molecule to which the radioactive isotope is bonded is determined. The bases in a certain positional relationship are sequentially determined from the base, and the base sequence of the entire target object is thereby determined.

In addition, the latter Sanger-Courlain method uses chemical means to base-specifically generate a DNA compound that is complementary to DNA or a chain molecule of DNA @ fragment and has been given a radioactive label. Synthesize.

This method uses the obtained mixture of base-specific DNA compounds to determine the base sequence from the autoradiograph in the same manner as described above.

Conventionally, the visible images of autoradiographs are based on the separation and development of base-specific cleavage degradation 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 overlapping a support medium and a high-sensitivity X-ray film for a certain period of time and exposing the film to light. And the base sequence of nucleic acid is
It is determined by visually determining the separation development position t of the base-specific fragment of the fill and comparing the separation development positions with each other.

Specifically, autoradiograph analysis involves first fixing the autoradiograph on a visualized X-ray film, and then manually moving the plastic cursor (reading guide plate) installed on the Scherkasten. While moving up and down (or left and right) on the film, use this cursor as a guide to find the radioactive label? This is done by determining the relative positional relationship between the V and V points.

A transparent plastic cursor usually has a thin straight line drawn in the center (a so-called hairline cursor).
The hairline cursor can be moved in one direction (in the direction of the film) while fixed to the cursor. Therefore, if a radiolabeled substance is ideally separated and developed, that is, a molecule! # The development rows are almost straight lines, and the radioactivity of each row can be easily used to find the relative position of the hunts (separation and development process of radioactive labeled substances) in each row, and rank each hunt. I can do it.

However, a smiling phenomenon or offset distortion often occurs in separated and developed patterns obtained by separation and development using electrophoresis or the like. The smiling phenomenon is caused by the heat dissipation effect (edge effect) during the separation development process, and is a phenomenon in which the separation development distance at both ends of the support medium becomes shorter than the separation distance at the center of the support medium. . In addition, offset distortion occurs due to differences in the shape of the sample entrance (slot), etc., and the start position and start time of the molecule a expansion of the sample differs in each row (lane). refers to the overall positional deviation of In addition to these bunt misalignments, defects in the hunt itself, meandering lanes, etc. may occur.

In such a case, the hairline cursor described above is a straight line, and since it is fixed to the device from the beginning and can only move t lines, it is difficult to judge the positional relationship of the hunt based on the cursor height. Actually, these positional deviations and defects are corrected by the analyst or IIJll, and careful attention is paid to avoid misreading the order of the bands.
was paying. Therefore, the hairline cursor was nothing more than a 1j reduction in reading.

[Summary of the Invention 1 Unreleased IJ provides an autoradiographic analysis method that allows the base sequence A of a nucleic acid to be determined easily and with high accuracy.

In other words, the present invention provides autoradiography of a plurality of separated spread arrays formed by spreading radioactively labeled base-specific DNA fragments or RNA fragments in a one-dimensional direction onto a support medium. A method for determining the base sequence of a nucleic acid by analyzing a graph, comprising: 1) displaying an image of the autoradiograph as '+tt'A based on a digital signal corresponding to the autoradiograph; and 2) Molecules on the displayed image! The present invention provides an autoradiographic analysis method for determining the base sequence of a nucleic acid, comprising the following steps: #: creating a reading cursor across the development rows and along the bands of each row.

According to the method of the present invention, the reading cursor is not fixed to a device such as a Scherkasten as in the past, but can be created arbitrarily on the display screen on which the autoradiograph is visualized. In other words, even if there is a deviation in hunt position between lanes of a separation development pattern due to the smiling phenomenon, offset distortion, etc., the cursor can be created in any shape (broken line, curved line, etc.) along the band. You can create an optimal cursor for each pattern and display it on the screen. Moreover, it can be automatically created in a short time based on the position information input along the hunt for each lane. This also makes it easy to recreate the cursor if the cursor no longer follows the hunt while it is being created.

Furthermore, the cursor displayed on the screen can be moved not only up and down (or left and right), but also in any direction. This allows the cursor to be moved optimally to the pattern even if the separation expansion pattern is meandering.

Therefore, the analyst can directly read the relative positions of the bands based on the cursor while moving the created reading cursor along the separation gC opening direction and assign the order as is. As before, using the linear cursor as a guide,
There is no need for the analyst to correct the position of a bunt that has misaligned in one lesson, to supplement a defective hunt, or to memorize and read the positional relationship before and after the hunt.・It can prevent the reversal of reading order, missing bands, and duplication, and can definitely avoid the troubles of reading from number 1 to number 1.
:^11- can be determined with high accuracy.

In addition, because the hunt can be ordered directly based on the reading cursor, it is not only more accurate than using traditional shearkasten and hairline cursors, but also requires less attention to detail or skill than traditional reading cursors. Bands can be easily ordered and the base sequence can be determined in a short time without the need for additional steps.

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, JP-A No. 60-101)
No. 74, Japanese Patent Application No. 173393/1982). 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 , it can be emitted as stimulated light, which is radiation energy. According to this method, 'A
The light time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur. Further, the autoradiograph of the radiolabeled substance can be directly obtained as a digital signal because it is stored in the phosphor sheet as radiant energy and then read out photoelectrically as photostimulated light.

[Structure of the Invention] Examples of samples used in the present invention include a mixture 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,
The base-specific DNA cleavage/degradation 5iIs mixture, which is a type of base-specific DNA 1tli fragment mixture, is a mixture of base-specific DNA cleavage and decomposition 5iIs, which is a type of base-specific DNA 1tli fragment mixture.
It is obtained by base-specific cleavage and decomposition of NA.

In addition, the base-specific DNA compound mixture can be synthesized using DNA as a template and a radioactively labeled deoxynucleoside triphosphate and a DNA synthase according to the Sanger-Courlin method described above. It will be done.

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

The radioactive label is added to 32P, 1 using a method appropriate for these substances.
It is given by retaining radioactive isotopes such as 4c, ``S, 3H, ``1''.

The sample, a mixture of base-specific fragments of radioactively labeled nucleic acids, is subjected to electrophoresis, 181-layer chromatography, column chromatography, and vapor chromatography using various known support media such as gel support media. It is separated and developed on a support medium by various separation and development methods such as.

Next, autoradiography using the method of the present invention
IF iG ffi ”-i7 tit fffi i
T? z n N A M 14f Sweet IQ Km J
h”: I will explain this for 64M.

First, salts labeled with the following four types of radioactive labels7.
The electrophoretic pattern obtained by spreading a mixture of ti-specific DNA fragments on a gel support medium by electrophoresis can be determined by radiography using conventional photosensitive materials or by radiation image conversion using a stimulable phosphor sheet. The method obtains the autoradiograph and then obtains the digital signal corresponding to the autoradiograph via a suitable readout system.

l) Guanine (G) specific DNA fragment 2) Adenine (
A) Specific DNA! N piece 3) Thymine (T) specific DN
A fragment 4) Cytosine (C) specific DNA fragment Here, each Ii
! Group-specific DNA fragments are base-specifically cleaved, degraded, or synthesized, that is, the terminal 11! It consists of DNA fragments of various lengths that share the same group.

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 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 the stimulable phosphor sheet are brought together at room temperature for a short period of time (several seconds to several tens of minutes) to allow the phosphor sheet to absorb the radiation energy emitted from the radiolabeled substance. The graph is recorded as a kind of latent image on a phosphor sheet. Here, the 7i multilayer phosphor sheet includes 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
and a transparent protective film are layered 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 X-rays, it absorbs and accumulates the radiation energy, and then, when 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 obtained directly without creating a visible image. obtained as an electrical signal. Furthermore, by A/D converting this electric 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 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. 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 any digital signal obtained by any other method as long as it has a correspondence with the autoradiograph of the radiolabeled substance. is possible.

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. It is.

Furthermore, in the present invention, reading (reading) conditions are set manually by inputting information such as ++' for the position of the r/meg # expansion column and the width of the band.
In operation, by scanning with a light beam at a scanning line density such that two or more scanning lines pass over each band, the readout time can be shortened and the necessary information can be obtained efficiently. . 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 DXF is a signal level consisting 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. represents the image density at that coordinate, that is, the amount of radioactive label a substance. Therefore, the series of digital signals (ie, digital image data) contains two-dimensional positional information of the radiolabeled substance.

After the digital signal corresponding to this autoradiograph is temporarily stored in a memory (buffer memory or non-volatile memory such as a magnetic disk), a signal processing circuit and a CRT or other device that can display the autoradiograph and the curve at the same time are used. The autoradiograph to be analyzed (separated development pattern of radioactive label II) is visualized as a visible image on the display screen.

Note that the digital signal may be subjected to signal processing (image processing) in advance so as to obtain a visible image with appropriate density and contrast and excellent observation and interpretation properties. Examples of image processing include spatial frequency processing, gradation processing,
Examples include addition processing, reduction processing, and enlargement processing.

The autoradiograph displayed as an image on the screen expresses the signal level (image density, or amount of radiolabeled substance) in the coordinate system set on the screen by the brightness of the color.
There may be a ci tanryo image similar to a conventional photographic image,
Alternatively, it may be a binary image that is simplified and represented by binary-forming the signal level in advance.

Alternatively, the autoradiograph can be expressed as a −dimensional position (
It may also be an image (a so-called bird track diagram) that is displayed by multiplexing a large number of two-dimensional waveforms consisting of positions (along the migration direction) and signal levels at regular intervals in a direction perpendicular to the migration direction. According to this track map, the amount of radiolabeled substance is expressed three-dimensionally as the height of the bird track chart, so the peak position of the hunt can be read accurately, and the position of the band between the electrophoresis columns (lanes) can be expressed three-dimensionally. It is possible to easily understand the relationship and separate bands in a region where bands are dense near the migration start position. The details of the method for displaying an autoradiograph using a bird track map are described in Japanese Patent Application No. 181431/1983 filed by the present applicant.

FIG. 1 is a diagram showing an autoradiograph of a migration pattern displayed as a gray scale image on one screen. In FIG. 1, the migration direction is to the right. It should be noted that a smiling phenomenon appears in the migration pattern.

Second, create a read cursor on the display screen.

A reading cursor that traverses the electrophoresis column (lane) and follows the band of each lane can be created on the screen, for example, as follows.

First, draw a vertical line from the top of one 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. 1). The lane at the end is selected because the read cursor is created starting from this position.

Positional information can be entered using a mouse cursor, light rail, joystick, or other means. From the viewpoint of positional accuracy, a mouse cursor is particularly preferred.

Input using a mouse cursor involves using a mouse cursor that is displayed as an arrow (←) on the screen and can be moved freely on the screen (Fig. 1, 2), and the mouse cursor can be moved freely and the position of the cursor can be adjusted. This is performed using a human power member (not shown) that can input information.

For example, the coordinates of the rectangular area where the autoradiograph is displayed on the screen are (Xa+ya), (Xb+yb)
The position coordinates of the mouse cursor are expressed as (Xm, 3/m
), then X m with the mouse cursor
By inputting position information such as "X, + 3/m = 'l +," the line segment (X,.

3/a) (X r + Y + ) is displayed on the screen (Fig. 1, 3).

Next, the input of the 0 position, in which a straight line is drawn from the starting point position 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.

As shown in FIG. 2, first, the starting point 4(x,

y + ) and the position of mouse cursor 2 (xfi, ym)
A line segment (x,, y+) (Xm, ym) is displayed between. When the mouse cursor 2 moves, this line segment disappears from the screen and a new line segment is displayed between the starting point 4 and the moved position. Note that the mouse cursor is located at the above coordinates (Xa
, 3/a), (Xb.

You can move freely within the rectangular area divided by 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. Input the position where the line segment preferably crosses the lane Xm=x2. By performing yffl=y2, line segment 5(x,) connected to line segment 3, as shown in FIG.

y+)(X21 y2) is displayed fixedly on the screen.

In this way, sequentially across the lanes from the northmost lane to the bottommost lane and along the band of each lane,
Line segment - ←
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 cannot exist at the same position (migration distance). Therefore, 1 along the r bunt means drawing a line segment along the hunt in a macroscopic sense so that the migration distance of each lane is equal,
This does not strictly mean that each line segment passes over the hunt of each lane.

Next, as shown in FIG. 4, according to the manual information that the last input position is the end point when the line segment completely crosses the electrophoresis pattern, the east direction from position 6 of the end point to the bottom edge of the screen is Draw a straight line segment 'y (x + *χ-ritsu-)' 2 knee 14-y 2□9-.In this way, on the screen,
A reading cursor 8 consisting of a plurality of connected polygonal lines that completely traverses the electrophoresis pattern is displayed.

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

In addition, generally position information is input for each lane, that is, the number of positions corresponding to the number of lanes is input, or the number of positions input depending on the electrophoresis pattern may be greater than that, or It may be less. It is sufficient if at least the - position is input and the starting point and ending point can be recognized.

The reading cursor may be in the shape of a polygonal line connecting the manually operated 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.
The base sequence of NA can be determined.

Specifically, while moving the reading cursor in parallel to the left or right on the screen, the hunt is read sequentially in the order closest to the cursor. Generally speaking, the farther down the electrophoresis (to the right of the screen) the closer the bands are to each other. Since it is open, it is preferable to order the bands from the bottom of the migration pattern. In addition.

At this time, since the combination of the above four types of base-specific DNA fragments is an exclusive combination, taking advantage of the fact that two or more hunts (bands in different lanes) cannot exist at the same position, You can decide the order. Lanes (1) to (4) to which each band belongs are (G) and (A), respectively.
), (T), and (C), Ii corresponding to the lane to which each hunt belongs
! By replacing with a group, a DNA base sequence (for example, A-C-C-G-A-G-T-...) can be obtained.

Note that by setting the movement distance and movement start position of the reading cursor in advance, it is possible to automatically move the cursor at regular intervals with a simple operation. Furthermore, the cursor can be moved not only in parallel to the left and right directions of the screen, but also in diagonal directions at any angle. Therefore, even if the lane is meandering or the entire pattern is sloped, the cursor can be moved along it. Of course, it is also possible to set the direction of movement of the cursor in advance.

If the reading cursor no longer matches the migration pattern during the process of ranking the bands, a cursor of the desired shape can be simply created by repeating the same operations as described above.

In the above, we have explained the case where an exclusive combination of (G, A, T, C) is used as a mixture of salt-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, a mixture of base-specific RNA fragments T
The method of the present invention can also be applied to h (for example, a combination of G, A, U, and C). Furthermore, the method of the present invention is not limited to the separation and development array of a set of base-specific fragments of nucleic acids, but can be applied simultaneously to all separation and development arrays that are simultaneously separated and developed on the support medium. It is possible to use it.

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

[Brief explanation of drawings]

1 to 4 are diagrams showing autoradiographs displayed on the screen, respectively, and are diagrams for explaining the process of creating a reading cursor according to the present invention. l: Electrophoresis hunt, 2: Mouse cursor, 3.5.7: Line segment, 4: Starting point, 6: Ending point, 8: Reading cursor Patent applicant Fuji Photo Film Co., Ltd. Agent
Patent attorney Yasushi Yanagawa Younger brother 1 Figure 3 Figure 2 Figure 4 Ministry of Procedure 11 Sho 7 [2 April 10, 1988 Commissioner of the Patent Office Kuro
Missing (1, = Showing the item 1986 Patent Application No. 47924 2, Name of the invention Autoradiograph analysis method for determining the base sequence of nuclear intoxicants 3, Person making the amendment/Relationship with 19 cases Patent application Person name (5
20) 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 supplementary JIE None 7,
"Detailed Description of the Invention" column of the specification to be amended. We will amend the "Detailed Description of the Invention" column in the specification as follows. -Note 1 (1) r from the last line of page 14 to the first line of page 15 of the specification
For a long time (several tens of hours) at low temperatures (-90 to -70°C)!
l for a long time (several hours to tens of hours) at low temperature or room temperature.
Correct it with J. (2) “Two or more” on page 18, lines 2 to 3 of the specification
More than one r, L,,! Correct it with I. −1 above

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 the autoradiograph based on a digital signal corresponding to the autoradiograph, and 2) displaying on the displayed image, 1. An autoradiographic analysis method for determining the base sequence of a nucleic acid, comprising the step of creating a reading cursor across the separation and expansion rows and along the bands of each row. 2. In the second step, a polygonal or curved cursor is created by drawing a straight line or arc passing through at least one position based on input information regarding the position. An autoradiographic analysis method for determining the base sequence of a nucleic acid according to item 1. 3. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 2, wherein in the second step, position information is input using a pointing device. 4. The autoradiographic analysis method for determining the base sequence of a nucleic acid according to claim 3, wherein the pointing device is a mouse cursor. 5. 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. 6. 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.