JPS61233369A - Signal processing method for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To determine automatically base sequence with high accuracy by determining one sepn. development row as a reference row, determining the relative position based on the inclination of the band of the other sepn. development row, determining the ratio of the sepn. development distance from the relative position and expanding or contracting the sepn. development distance in accordance with the obtd. ratio. CONSTITUTION:The point where the code of the level difference with respect to a one-dimensional waveform 21 inverts is determined and the point 22 where the signal level is max. is found out. A regression straight line 23 is obtd. with one band and an inclination 24 of the line 23 relative to the migration direction is determined, by which the inclination of the band is determined. The reference band 31 detected of the inclination on the reference row with the 2nd slot as the reference row is extended up to an intermediate point 32 with the adjacent row and the intersected point 34 thereof is determined as the relative position of the band 31. The ratio of the sepn. development distance from the relative position is determined with respect to each row except the reference row and the primary wave of the scanning line is extended or contracted at this ratio to correct collectively the migration distance.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to a signal processing method for determining the base sequence of a nucleic acid.

[Background of the Invention] In the field of molecular biology, which has developed rapidly in recent years, in order to elucidate the functions and replication mechanisms of living organisms,
It is essential to clarify the genetic information possessed by living organisms. In particular, DNA that carries specific genetic information
It has become essential to determine the base sequence of nucleic acids such as (or DNA fragments, the same shall apply hereinafter).

Maxazen-Gilber uses autoradiography as a typical method to determine the base sequence of nucleic acids such as DNA and RNA.
t) method and Sanger-Coulson (Sanger-
Coulson) method is known. The former Maxam
In the Gilbert method, first, a group containing a radioactive isotope such as 32p is attached to one end of a chain molecule of the DNA or DNA fragment whose base sequence is to be determined, thereby radioactively labeling the target. After making the substance, the bonds between each constituent unit of the chain molecule are base-specifically cleaved using chemical means.Next, the mixture of base-specific DNA cleavage products obtained by this operation is Separation and development is performed by gel electrophoresis, and a separation and development pattern (however, this cannot be seen visually) is obtained by separating and developing a large number of cleavage products. This separation development pattern is visualized on, for example, an X-ray film to obtain an autoradiograph thereof, and from the obtained autoradiograph and each base-specific cutting means, the end of the chain molecule to which the radioactive element is bound is determined. By sequentially determining the bases in a certain positional relationship, it is possible to determine the base sequence of the entire target object.

The latter Sanger Coulson method uses chemical means to base-specifically synthesize a DNA compound that is complementary to a chain molecule of DNA or DNA fragments and has been given a radioactive label. Then, using this mixture of base-specific DNA compounds, the base sequence is determined from the autoradiograph in the same manner as above.

With the aim of determining the base sequence of the above nucleic acids simply and with high precision, the present applicant has proposed a conventional radiographic method using a photosensitive material such as the above X-ray film in the autoradiograph measurement operation used therein. Instead, a patent application has already been filed for a method using a radiation image conversion method using a stimulable phosphor sheet (Japanese Patent Laid-Open No. 59-830).
57, Japanese Patent Application No. 58-201231), where the stimulable phosphor sheet is made of stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, By exciting it with electromagnetic waves (excitation light) in the visible to infrared region, radiation energy can be emitted as fluorescence. According to this method, exposure time can be significantly shortened and chemical fog, which has been a problem in the past, does not occur.Furthermore, autoradiographs of radioactively labeled substances can be produced by Since the light is stored in a phosphor sheet and then read out in time series as photostimulated light, it is possible to display and record it in any form such as symbols and numbers in addition to images.

Conventionally, those attempting to determine the base sequence of a nucleic acid have been asked to analyze a visualized autoradiograph with a base-specific cleavage degradation product or a base-specific composite (hereinafter simply referred to simply as a base-specific synthesis product) of a radioactively labeled nucleic acid. The base sequence of the nucleic acid is determined by visually determining the separation and development position of each of the separated and development columns (referred to as specific fragments) and comparing the separated and development columns with each other. Therefore, analysis of the obtained autoradiograph is usually performed through human vision, which requires a great deal of time and effort.

Additionally, because it relies on the human eye, there are limits to the accuracy of the information that can be obtained, such as the base sequence of nucleic acids determined by analyzing autoradiographs differing depending on the analyst.

Therefore, the applicant has already filed a patent application for a method for automatically determining the base sequence of DNA by obtaining the above-mentioned autoradiograph as a digital signal and then subjecting this digital signal to appropriate signal processing. (Unexamined Japanese Patent Publication No. 59-
128527, Japanese Patent Application Publication No. 126278/1982, Japanese Patent Application No. 89615/1989, Japanese Patent Application No. 140908/1987, etc.)
If a conventional radiation film is used, the digital signal corresponding to the autoradiograph can be read out photoelectrically using reflected or transmitted light after the autoradiograph is first visualized on the film. It is obtained by When a stimulable phosphor sheet is used, an autoradiograph can be obtained by directly reading out the phosphor sheet on which the stimulable phosphor sheet has been stored.

However, various distortions and noises tend to occur in separation and development patterns obtained by actually separating and developing radiolabeled substances on a support medium by electrophoresis or the like.

A typical example of this is the phenomenon (smiling phenomenon) in which the separation distance at both ends of the support medium is shorter than the separation distance at the center of the support medium. It occurs due to such things. Even when such distortion occurs, it is desired to efficiently process the digital signal corresponding to the autoradiograph and automatically determine the base sequence of the nucleic acid with high precision.

[Summary of the Invention] The present inventor has proposed a method for automatically determining the base sequence of a nucleic acid using autoradiography, in which a digital signal corresponding to the autoradiograph can be obtained even if the separation pattern has a smiling phenomenon. By appropriately processing the signals, we have achieved automatic determination of the base sequence of nucleic acids with ease and high accuracy.

That is, the present invention provides a plurality of separation and development arrays formed by separation and development of a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in a one-dimensional direction on a support medium. A method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to an autoradiograph, comprising the steps of: 1) detecting the slope of at least one band with respect to the separation development direction for each separation development column; ) The step of using one separation development column as a reference column and determining the relative position of a band whose slope has been detected on this reference column in each other separation development column based on the slope of the band in each other separation development column. , and 3) For each separation expansion column other than the reference column, calculate the ratio of separation expansion distance from the relative position,
The present invention provides a signal processing method for determining the base sequence of a nucleic acid, which includes a step of expanding or contracting the separation distance of the line array based on this ratio.

The present invention also provides a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to the autoradiograph, including: 1) for each separation and development column, at least one band in the direction of separation and development; 2) One separation development column is used as a reference column, and for the band whose slope has been detected on this reference column, each of the other separations is detected based on the slope of the band in each of the other separation development columns. 3) determining the relative position in the developed row, 3) determining the ratio of the separated developed distance from the relative position for each separated developed row other than the reference row, and expanding or contracting the separated developed distance of the line array based on this ratio; 4) A signal processing method for determining the base sequence of a nucleic acid is also provided, which includes the step of detecting all bands on each separation and development column and ranking the bands based on their positions. It is something.

According to the present invention, even if a smiling phenomenon occurs in the separation development pattern obtained by electrophoresing a mixture of base-specific fragments of nucleic acids on a support medium, a digital signal corresponding to the autoradiograph can be obtained. By passing the signal through an appropriate signal processing circuit having a signal processing function for smile correction, the base sequence of the nucleic acid can be obtained easily and with high precision.

Normally, in a separation pattern in which the smiling phenomenon occurs, each band (separation development region long in the width direction) on a separation development row with a short migration distance at both ends is perpendicular to the migration direction (i.e. horizontally ), but has a slope depending on the degree of the smiling effect.The present inventor paid attention to the slope of this band and discovered a method for suitably and easily correcting the smiling effect. Then, by further performing appropriate signal processing on the smiling-corrected digital signal, the base sequence of a nucleic acid can be determined easily and with high precision.

[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, to which radioactive labels have been added. here,
A nucleic acid fragment refers to a portion of a long chain molecule. For example, a base-specific DNA cleavage degradation product mixture, which is a type of base-specific DNA fragment mixture, is a mixture of base-specific DNA fragments.
DNA radioactively labeled according to the Gilbert method
It is obtained by base-specific cleavage and decomposition of .

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 synthesizing enzyme according to the Sanger Fist-Luzon method described above. It is obtained by

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 can be added to 32p, + by a method appropriate to these substances.
AC,31iS,KOH,"Isu,!'c7) It is given by retaining a radioactive isotope.

The sample, a mixture of base-specific fragments of radioactively labeled nucleic acids, is subjected to electrophoresis, thin layer chromatography, column chromatography, and paper chromatography using various known support media such as gel support media. Separation and development are carried out on a support medium using various separation and development methods.

Next, an autoradiograph of the support medium on which the radiolabeled substance has been separated and developed is obtained by radiography using a conventional photosensitive material or by a radiation image conversion method using a stimulable phosphor sheet. A digital signal corresponding to the autoradiogram of the radiolabeled substance is then obtained via a suitable 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℃ for a long time (several tens of hours), and then developed to create 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 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 stimulable phosphor contained in the stimulable phosphor sheet is irradiated with radiation such as X-rays, it absorbs the radiation energy and accumulates it.
It has the characteristic that when excited with light in the visible to infrared region, the accumulated radiation energy is released 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 directly obtained without creating a visible image. 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. 28527 and JP-A-59-126278.

In the above, a method using conventional radiography and radiographic image conversion methods was described as a method for obtaining a digital signal corresponding to an autoradiograph of a radiolabeled substance separated and developed on a support medium. The signal processing method of the present invention is not limited to these methods, and the signal processing method of the present invention can be applied to 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 (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 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) has two-dimensional positional information of the radiolabeled substance.

The digital signal corresponding to the autoradiograph of the radiolabeled substance separated and developed on the support medium obtained in this way is subjected to signal processing by the method of the present invention as described below, and the target nucleic acid is The nucleotide sequence of the nucleotide sequence is determined.

An embodiment of the signal processing method of the present invention will be described with reference to a case where the electrophoresis array (separation and development array) is formed by a combination of base-specific DNA fragments to which the following four types of radioactive labels have been added.

1) Guanine CG) specific DNA fragment 2) Adenine (
A) Specific DNA fragment 3) Thymine (T) specific DNA
Fragment 4) Cytosine (C)-specific DNA fragment Here, each base-specific DNA fragment is a DNA 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.

FIG. 1 shows a partial autoradiograph of the electrophoresis pattern obtained by electrophoresing the above-mentioned four types of base-specific DNA fragments into four slots, respectively.

A digital signal corresponding to this autoradiograph is temporarily stored in a memory (a discrete memory or a nonvolatile memory such as a magnetic disk) in a signal processing circuit.

First, the inclination of at least one band on each electrophoresis column (lane) with respect to the electrophoresis direction is detected.

If the digital signal is detected by scanning along each lane at a scanning line density such that at least two scanning lines are applied for each band as described above, then (
(See FIG. 1, 1: migration band, 2: scanning line), it is possible to directly create a -dimensional waveform consisting of position (y) and signal level (2) for each scanning line. Furthermore, when the entire autoradiograph is read, a signal is extracted along each lane by scanning the digital image data in the same manner as described above, and then a -dimensional waveform is created.

FIG. 2 is a diagram schematically showing the process of detecting the band inclination.

In FIG. 2, for a -dimensional waveform 21, for example, by finding the point where the sign of the difference in signal levels is reversed, the position 22 where the signal level is maximum is found. The regression line 23 is obtained by connecting the positions of the maximum values, and the migration direction of this regression line (
That is, by determining the slope (θ) 24 with respect to the direction of the scanning line, the slope of the band can be detected. This operation is performed for at least one band per lane. From the point of view of improving accuracy, the smiling effect is noticeable, and therefore the slope is detected for the band at the bottom where the slope is large (region where the electrophoresis distance is relatively large). It is preferable to carry out the analysis, and it is also preferable to carry out the analysis on adjacent bands (bands having the same migration distance) in each lane.

Next, a reference column (reference lane) is determined, and the relative position of the band on the reference lane on other lanes is determined based on the obtained band inclination.

The reference lane is the lane with the least smiling effect,
In other words, it is preferable to use the lane with the smallest slope.

FIG. 3 is a diagram schematically showing the process of determining the relative position on the lane.

First, the second slot is set as a reference lane, and as partially shown in FIG. midpoint 32 with the third slot)
Next, from this intermediate point as a base point, a straight line (a straight line parallel to the band 33) having the same slope as the detected band 33 on the adjacent lane, which is located close to the reference band 31, is drawn on the adjacent lane. extend to. The intersection point 34 between this extension line and the adjacent lane is the relative position of the reference band! Decided to be lyco.

Similar operations are performed for the remaining lanes using bands whose inclinations have been detected to determine the relative positions y8 and y4 of the reference band on each lane. When determining the relative position of a reference band on a lane that is not directly adjacent to the reference lane, the relative position may be determined directly between the reference lane and this lane, or first, the relative position may be determined between the reference lane and the adjacent lane. After determining the relative position between lanes, the operation of sequentially determining the relative position between adjacent lanes may be repeated.In this way, each lane is equally spaced with reference to the reference band on the reference lane. You can find the position reached in time.

Next, the position is corrected for each lane based on the obtained relative position on each lane.

For example, for the third slot, the migration distance V's from the migration start point to the relative position y3 on the lane, and the migration pressure Ill3''2 from the migration start point to the reference band position y2 on the reference lane were determined. Later, the ratio y't/V
's is calculated. By performing the same operation for other slots, the ratio of migration distance (
At the same time, it is also possible to calculate the ratio of migration speeds.

- When a ratio is calculated using the slopes of two or more bands for a lane, or when a ratio is calculated using two or more reference bands, the average ratio is taken as the ratio for the lane. The ratio of the obtained migration distances represents the degree of the smiling effect of each lane.

Based on this ratio, the migration distance for each lane other than the standard is expanded or contracted. For example, for the third slot, the migration distance can be corrected all at once by extending the one-dimensional waveform of the scanning line at a ratio of Y't/V's. The electrophoresis distances of the remaining lanes are also expanded or contracted all at once based on the ratio of their respective electrophoresis distances. In this way, the smiling effect can be corrected for all lanes.

If the reference lane is the central lane where the smiling effect does not occur, the positions obtained by correcting each lane indicate the positions where the electrophoresis would have occurred if the smiling effect had not occurred. be.

In this way, even if a smiling phenomenon occurs in the electrophoresis pattern, the smiling effect can be corrected by performing signal processing for correcting the electrophoresis distance for each slot.

Furthermore, for the one-dimensional waveform of the scanning line whose migration distance has been corrected, for example, by detecting all positions where the level of the signal appearing on the waveform is maximum, the positions of all bands on each lane can be determined. can be detected. The position of the band may be the position of the maximum value of a scanning line passing approximately at the center of the lane, or the position of the maximum value of each scanning line of the lane may be averaged.

Based on the position of the detected band, all bands are ranked in order from the bottom of the electrophoresis pattern. By changing lanes and taking advantage of the fact that two or more bands are not detected at the same position, the ranking can be easily determined. Since the slots (1) to (4) above each have information about the terminal bases consisting of (G), (A), (T), and (C), they are replaced with the base corresponding to the slot to which each band belongs. By doing so, the DNA base sequence (e.g. A-G-C-T-A-A-
G-...) can be obtained.

In this way, the base sequence of one chain molecule of DNA can be determined. Note that the information regarding the DNA base sequence is not limited to the above display format, but may be displayed as desired.

At the same time, it is also possible to display the intensity (2') of each band as the relative amount of the radiolabeled substance. Furthermore, DNA
It is also possible to display the base sequences of both two chain molecules.

Alternatively, it is also possible to display an image based on a digital signal subjected to the above-mentioned smiling phenomenon.

At the same time, it is also possible to display the original autoradiograph as a visible image. In this case,
The final base sequence can be determined by the analyst himself based on this displayed image.

In the above, the case where an exclusive combination of (G, A, T, C) is used as a mixture of base-specific DNA fragments as a sample has been described, but the signal processing method of the present invention It is not limited to combinations, for example (
It can be applied to various combinations such as G, G+A, T+C, and C). Similarly, the signal processing method of the present invention can be applied to a mixture of base-specific RNA fragments (for example, a combination of G, A, U, and C). Furthermore, the correction of the smiling phenomenon is not limited to the separation and development array of base-specific fragments of nucleic acids of the set, but can be performed for all separation and development arrays that are simultaneously separated and developed on the support medium. be.

The base sequence information obtained in this way can also be subjected to genetic linguistic information processing, such as comparing it with the base sequences of other nucleic acids that have already been recorded and preserved.

The information about the base sequence of the nucleic acid determined by the signal processing described above is output from the signal processing circuit and then stored in a recording device directly or, if necessary, via a storage means such as a magnetic disk or magnetic tape. transmitted to.

Recording devices include, for example, those that optically record by scanning a photosensitive material with a laser beam, etc., those that record symbols and numbers displayed on a CRT etc. on a video printer, etc., and those that record using heat rays. Recording devices based on various principles can be used, such as those that record on material.

[Brief explanation of the drawing]

FIG. 1 is a partial diagram showing an example of a separation development pattern in which a smiling phenomenon occurs. FIG. 2 is a diagram schematically showing the process of detecting the band inclination. FIG. 3 is a diagram schematically showing the process of determining the relative position on the lane. 1: Electrophoresis band, 2: Scanning line, 21: Two one-dimensional waveform, 22: Two maximum values, 23: Regression line, 24: Band slope θ, 31: Reference band, 32: Midpoint, 33: Detected slope Band, ゛34: Relative position,

Claims (1)

[Claims] 1. A plurality of separations formed by separating and spreading a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in one-dimensional direction on a support medium. In a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to an autoradiograph of a column, the steps include: 1) detecting the slope of at least one band with respect to the separation development direction for each separation development column; , 2) One separation expansion column is used as a reference column, and for the band whose slope has been detected on this reference column, the relative position in each other separation expansion column is determined based on the slope of the band in each other separation expansion column. and 3) calculating the ratio of the separation development distance from the relative position for each separation development column other than the reference column,
A signal processing method for determining the base sequence of a nucleic acid, comprising the step of expanding or contracting the separation distance of the column based on this ratio. 2. Obtain the above digital signal by scanning the autoradiograph so that each band has at least two scanning lines, and in the first step, find the position where the signal level in each scanning line is maximum. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that the slope is detected based on this position. 3. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that, in the first step, the slope of the band at the bottom of each separation and expansion column is detected. 4. Signal processing for determining the base sequence of a nucleic acid as set forth in claim 1, wherein in the second step, a separation and development column having the smallest inclination with respect to the band separation and development direction is used as a reference column. Method. 5. In the second step, the band of the reference column is extrapolated to the other column from the slope of the band of the reference column and the band of the other separation development column, and the relative position of the band in the other column is determined. A signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein the signal processing method comprises determining the base sequence of a nucleic acid according to claim 1. 6. The mixture of the base-specific DNA fragments includes (1) a guanine-specific DNA fragment, (2) an adenine-specific DNA fragment, (3) a thymine-specific DNA fragment, and (4) a cytosine-specific DNA. The method is characterized in that the separation and development column consists of four separate and development columns formed by separating and developing these four types of base-specific DNA fragments on a support medium, respectively. A signal processing method for determining the base sequence of a nucleic acid according to claim 1. 7. 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. Claim 1, wherein the autoradiograph is obtained by accumulating and recording on a sheet and then photoelectrically reading out the autoradiograph as photostimulated light by irradiating the phosphor sheet with excitation light. A signal processing method for determining the base sequence of nucleic acids. 8. A digital signal corresponding to the autoradiograph is transferred onto the photosensitive material after the autoradiograph of the radiolabeled substance on the support medium is photosensitively recorded on the photosensitive material by superimposing the support medium and the photosensitive material. 2. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein the signal processing method is obtained by photoelectrically reading an autoradiograph visualized in . 9. An autoradiograph of multiple separation and development columns formed by separation and development of a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in one-dimensional direction on a support medium. A method for determining the base sequence of a nucleic acid by performing signal processing on a corresponding digital signal, comprising: 1) detecting the slope of at least one band with respect to the separation development direction for each separation development column; 2) one separation 3) a step of determining the relative position of a band whose slope has been detected on the standard column in each of the other separation expansion columns based on the slope of the band in each of the other separation expansion columns; 3) the standard; 4) detecting all bands on each separation development column; determining the ratio of the separation development distance from the relative position for each separation development column other than the column; and expanding or contracting the separation development distance of the column based on this ratio; and 4) detecting all bands on each separation development column. 1. A signal processing method for determining the base sequence of a nucleic acid, the method comprising the step of: and ranking the bands based on their positions. 10. Obtain the digital signal by scanning the autoradiograph so that each band has at least two scanning lines, and in the first step, find the position where the signal level in each scanning line is maximum. 10. The signal processing method for determining the base sequence of a nucleic acid according to claim 9, characterized in that the slope is detected based on this position. 11. The signal processing method for determining the base sequence of a nucleic acid according to claim 9, characterized in that, in the first step, the slope of the band at the bottom of each separation and expansion column is detected. 12. Signal processing for determining the base sequence of a nucleic acid as set forth in claim 9, wherein in the second step, a separation and development column having the smallest inclination with respect to the band separation and development direction is used as a reference column. Method. 13. In the second step, the band of the reference column is extrapolated to the other column from the slope of the band of the reference column and the band of the other separation development column, and the relative position of the band in the other column is determined. 10. The signal processing method for determining the base sequence of a nucleic acid according to claim 9, which comprises determining the base sequence of a nucleic acid. 14. In the fourth step, all bands on each separation expansion column are detected by determining the position where the signal level in the scanning line is maximum. A signal processing method for determining the base sequence of nucleic acids. 15. The mixture of the base-specific DNA fragments is (1) a guanine-specific DNA fragment, (2) an adenine-specific DNA fragment, (3) a thymine-specific DNA fragment, and (4) a cytosine-specific DNA fragment. A patent characterized in that the separation and development array consists of four separation and development arrays formed by separating and developing these four types of base-specific DNA fragments on a support medium, respectively. A signal processing method for determining the base sequence of a nucleic acid according to claim 9. 16. 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. Claim 9, characterized in that it is obtained by accumulating and recording on a sheet, irradiating the phosphor sheet with excitation light, and photoelectrically reading out the autoradiograph as photostimulated light. A signal processing method for determining the base sequence of nucleic acids. 17. A digital signal corresponding to the autoradiograph is transferred onto the photosensitive material after the autoradiograph of the radiolabeled substance on the support medium is photosensitively recorded on the photosensitive material by overlapping the support medium and the photosensitive material. 10. The signal processing method for determining the base sequence of a nucleic acid according to claim 9, wherein the signal processing method is obtained by photoelectrically reading an autoradiograph visualized in .