JPH0462341B2 - - Google Patents

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, it is essential to clarify the genetic information of living organisms in order to elucidate their functions and replication mechanisms. It is becoming. In particular, DNA (or DNA fragments,
It has become essential to determine the base sequence of nucleic acids such as (the same applies below).

Maxam Gilbert uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
-Gilbert method and Sanger-Coulson method are known. The former Maxam-Gilbert method first attaches a group containing a radioactive isotope such as ^32P to one end of a chain molecule of DNA or DNA fragments whose base sequence is to be determined. After using it as a radioactive labeling substance, the bonds between each constituent unit of the chain molecule are cleaved base-specifically using chemical means. Next, the mixture of base-specific DNA cleavage products obtained by this operation is separated and developed by gel electrophoresis, and the other cleavage products are separated and developed to form a separated development pattern (however, it is visually (cannot be seen). 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. sequentially determine the bases in a certain positional relationship from
This makes it possible to determine the base sequence of all objects.

In addition, the latter Sanger-Coulson method is
A DNA compound complementary to a chain molecule of DNA or a DNA fragment and to which a radioactive label has been added is synthesized in a base-specific manner using chemical means,
This method uses this mixture of base-specific DNA compounds and determines the base sequence from its autoradiograph in the same manner as above.

In order to easily and accurately determine the base sequence of the above nucleic acids, the present applicant has proposed a conventional radiographic method using photographic materials such as the above X-ray film in the autoradiograph measurement operation used therein. Instead, a patent application has been filed for a method using a radiation image conversion method using a stimulable phosphor sheet (Japanese Patent Laid-Open No. 59-83057, Japanese Patent Application No. 58-201231). Here, the stimulable phosphor sheet is made of a stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, electromagnetic waves in the visible to infrared region ( excitation light)
By exciting it, radiation energy can be emitted as fluorescent light. According to this method, the exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur. Furthermore, autoradiographs of radiolabeled substances are stored in a phosphor sheet as radiation energy and then read out in chronological order as photostimulated light, so in addition to images, they can be displayed and recorded in any format such as symbols and numbers It is possible to do so.

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, the analysis of the obtained autoradiograph is usually done 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 fact that the base sequence of a nucleic acid determined by analyzing an autoradiograph differs 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 Application No. 126527, No. 126527, Unexamined Japanese Patent Publication No. 59-
No. 126278, Patent Application No. 89615, Patent Application No. 1989-
140908 etc.). When using a conventional radiation film, the digital signal corresponding to the autoradiograph can be obtained by first making the autoradiograph into a visible image on the film and then photoelectrically reading it using reflected or transmitted light. can get. 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 is an overall positional deviation between columns (so-called offset distortion) due to differences in the start position or start time of sample separation and development in each column. Offset distortion is caused, for example, by the fact that the shapes (indentation sizes) of the numerous slots (sample injection ports) provided at the top of the support medium, such as gel media, are not completely the same and tend to vary individually. occurs. In addition, when the sample is attached to the support medium, if the attachment positions are shifted from each other, or if urea is not sufficiently washed out of the gel medium immediately before sample injection, the rate of penetration of the sample into the support medium may be different, causing distortion. It is a contributing factor to the occurrence of

FIG. 1 shows a specific example where offset distortion occurs in the separation and development pattern due to the irregular shapes of the slots. Figure 1 a shows the upper end of the support medium used for separation and development of the sample, and b
shows the pattern obtained by injecting the same sample into each slot (1) to (4) and then separating and developing it. Figure 1a
As shown in (b), as a result of the third slot having a larger recess than the other slots, only the separation/deployment row of the third slot is shifted downward as a whole, as shown in (b), and there is a misalignment between it and the other rows. (Δy).
Such relative positional deviation between columns is called offset distortion.

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,
By suitably processing the digital signal corresponding to the autoradiograph of a separation rotation pattern with offset distortion, it has been possible to automatically determine the base sequence of a nucleic acid easily and with high precision.

That is, the present invention provides base-specific DNA fragments or base-specific RNAs to which a radioactive label has been added.
Determining the base sequence of a nucleic acid by performing signal processing on digital signals corresponding to autoradiographs of multiple separation and development columns formed by separating and developing a mixture of fragments in one-dimensional direction on a support medium. In the method, (1) detecting at least two bands at the bottom of each separation deployment column and sequentially numbering the bands from the bottom end; (2) determining the number of the band and its separation development distance for each separation deployment column; (3) Obtaining the difference in the separation development distance between the separation development columns from the obtained correlation, and correcting the separation development position for each column by using this difference as a positional deviation between the columns. The present invention provides a signal processing method for determining the base sequence of a nucleic acid, comprising the following steps.

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, which includes: (1) detecting at least two bands at the bottom of each separation development column; , a step of assigning serial numbers to the bands in order from the bottom end; (2) a step of obtaining a correlation between the band number and its separation expansion distance for each separation expansion column; (3) a step of determining the separation expansion column from the obtained correlation. (4) detecting all the bands on each separation development column, and correcting the separation development position for each column by using this difference as the positional deviation between the columns; and (4) detecting all the bands on each separation development column, and The present invention also provides a signal processing method for determining the base sequence of a nucleic acid, which comprises the step of assigning an order to bands based on the following.

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

Generally, in the separation development pattern, the intervals between the separation development bands are sparse in the lower part (ie, the region where the separation development distance is large), while the intervals between the bands become closer as the separation development start position in the upper part is approached. Here, the term "lower part" generally refers to the area below the vicinity of the center of the support medium, and the term "upper part" generally refers to the area above the vicinity of the center. Therefore, even if offset distortion occurs between the separation and development columns and the band positions are shifted, the order of the bands can be relatively easily determined by comparing the band positions of each column in the lower region. It is possible to decide. However, since the bands are closely spaced in the upper region, positional deviations between columns make it difficult to determine the order of the bands, causing errors in the base sequence of the resulting nucleic acid.

The present inventor focused on the fact that the interval between bands is different between the upper and lower parts of the separated developed pattern, and that it is easy to determine the order of the bands even when offset distortion occurs in the lower region. We have discovered a method for appropriately and easily correcting distortion. That is, in the lower region, not only is the order of the bands easily determined, but also the correlation between the serial number of the band and its separation development distance for each column is linear.
Positional deviations between columns can be easily detected, and thereby offset distortion can be corrected all at once.

By further performing appropriate signal processing on the offset distortion-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 that have been given radioactive labels. Here, the nucleic acid fragment means a part of a long chain molecule. For example, a base-specific DNA cleavage mixture, which is a type of base-specific DNA fragment mixture, is produced by base-specific cleavage and decomposition of radiolabeled DNA according to the Maxam-Gilbert method described above. can get.

In addition, the base-specific DNA synthesis mixture is prepared using DNA as a template and a radioactively labeled deoxynucleoside triphosphate according to the Sanger-Coulson method described above.
It can be obtained by synthesis using DNA synthase.

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

Radioactive labels can be applied to these substances in an appropriate manner.
It is given by retaining radioactive isotopes such as ³² P, ¹⁴ C, ³⁵ S, ³ H, ¹²⁵ I, etc.

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

Next, an autoradiograph of the support medium on which the radiolabeled substance has been separated and developed is obtained by radiography using a conventional photographic light-sensitive material or by a radiation image conversion method using a stimulable phosphor sheet. A digital signal corresponding to the autoradiograph of the radiolabeled substance is obtained via a readout system.

When using the former radiographic method, first the support medium and a photographic material such as an X-ray film are overlapped for a long time (several hours) at a low temperature (-90 to -70°C) to expose the radiation film. It is then developed to create a visible image of the autoradiograph of the radiolabeled substance on the radiographic film. Next, the autoradiograph visualized on the radiographic film is read using an image reading device. For example, an autoradiograph is obtained as an electrical signal by irradiating a radiation film with a light beam and photoelectrically detecting the transparent 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 the stimulable phosphor sheet are overlapped for a short time (several seconds to several tens of minutes) at room temperature, and the radiation energy emitted from the radiolabeled substance is accumulated in the phosphor sheet. is recorded on the phosphor sheet as a kind of latent image. Here, the stimulable phosphor sheet is made of a support made of, for example, a plastic film, divalent europium-activated barium fluoride bromide (BaFBr: Eu ²⁺ )
A phosphor layer made of a stimulable phosphor such as phosphor and a transparent protective film are laminated in this order. The stimulable phosphor contained in the stimulable phosphor sheet absorbs and accumulates radiation energy when it is irradiated with radiation such as X-rays, and then accumulates when excited with light in the visible to infrared region. It has the property of emitting radiation energy as photostimulated light.

Next, the autoradiograph stored and recorded on the stimulable phosphor sheet is read out using a reading device. Specifically, for example, by scanning a phosphor sheet with a laser beam to emit radiation energy as photostimulated light, and detecting this photostimulated light photoelectrically, an autoradiograph of a radioactively labeled substance is converted into a visible image. It can be obtained directly as an electrical signal without any additional processing. Furthermore, by A/D converting this electrical signal, a digital signal corresponding to an autoradiograph can be obtained.

For details on the above-mentioned autoradiograph measurement operation and method for obtaining a digital signal corresponding to the autoradiograph, see the aforementioned Japanese Patent Application Laid-open No. 59-83057;
It is described in various publications such as JP-A-59-126527 and JP-A-59-126278.

In the above, a method using conventional radiography and radiographic image conversion methods was described as a method for obtaining a digital signal corresponding to an autoradiograph of a radiolabeled substance separated and developed on a support medium. The signal processing method of the present invention is not limited to these methods, and the signal processing method of the present invention can be applied to digital signals obtained by any other method as long as there is a correspondence with the autoradiograph of the radiolabeled substance. is possible.

Furthermore, in any of the above methods, it is not necessary to read (or read out) the autoradiograph over the entire surface of the radiation film (or stimulable phosphor sheet), and it is of course possible to read out the autoradiograph only on the image area. .

Furthermore, in the present invention, 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, a scanning line is scanned on each band. By performing scanning with a light beam so that it passes through, the reading time can be shortened and necessary information can be efficiently obtained. 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 D _xy consists of coordinates (x, y) expressed in a coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (z) at the coordinates. The level of the signal represents the image density at that coordinate, ie, the amount of radiolabeled substance. Therefore, the series of digital signals (ie, digital image data) has two-dimensional positional information of the radiolabeled substance.

The digital signal corresponding to 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 base sequence will be determined.

The embodiment of the signal processing method of the present invention is based on base-specific DNA to which the following four types of radioactive labels have been added.
A case will be explained in which the electrophoresis column (separation and development column) is formed by a combination of fragments.

(1) Guanine (G)-specific DNA fragment (2) Adenine (A)-specific DNA fragment (3) Thymine (T)-specific DNA fragment (4) Cytosine (C)-specific DNA fragment Here Each base-specific DNA fragment is base-specifically cleaved, degraded, or synthesized, that is, it consists of DNA fragments of various lengths that have the same terminal base.

FIG. 2 shows an autoradiograph of the electrophoresis pattern obtained by electrophoresing the above-mentioned four types of base-specific DNA fragments into four slots. Second
As shown in the figure, offset distortion occurs in the obtained autoradiograph.

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

First, two or more bands are detected for each electrophoresis column (lane) and their order is determined.

For example, after extracting a digital signal within a certain area along the migration direction of each lane, a one-dimensional waveform consisting of the position (y) of the extracted signal and the level (z) of that signal is created for each lane. . If the digital signal is detected by scanning in the electrophoresis direction at a scanning line density that covers each band as described above, the one-dimensional waveform for each lane can be directly created. can do.

FIG. 3 shows a one-dimensional waveform consisting of a signal position (y) and a signal level (z) for each lane. In FIG. 3, the position of the vertical axis (y=y ₀ ) indicates the reference origin on the digital image data.

On the right side of each one-dimensional waveform in Figure 3 (area where y is large), for example, by finding the point where the sign of the signal level difference value is reversed (changes from + to -), the signal level can be determined. Find the position of maximum. The position where this maximum value is obtained is defined as the band position. The number of bands to be detected varies depending on the total number of bands on the electrophoresis pattern and the condition of the pattern, but for example, if the total number of bands is 150 to 200,
It is preferable to detect about 10 bands for each lane.

For all of the obtained bands, serial numbers (n) are assigned in order of the electrophoresis position (y) farthest from the reference origin. In the lower region of the electrophoresis pattern, as is clear from the one-dimensional waveform in Figure 3, the band spacing is sparse, so even if offset distortion occurs, the band positions may not be reversed between lanes. Therefore, the order of bands can be easily determined.

Next, for each lane, the correlation between the band number and its migration distance is determined.

For example, by creating a graph in which the horizontal axis represents the band number (n) and the vertical axis represents the migration distance (y'), a regression line as shown in FIG. 4 is obtained. Note that FIG. 4 shows a regression line consisting of the band number (n) and migration distance (y') for each lane. straight line 1
4 corresponds to the slot number.

Here, the migration distance (y') represents the distance from the reference origin ( _y0 ) to the position of each band (y'=y- _y0 ). The reference origin can be, for example, the position of the slot. Therefore, when there is a positional shift between the lanes, the distance y' from the common reference origin is not necessarily the true migration distance. Furthermore, the regression line for each lane is expressed by the general formula: y'=an+b. Here, a and b are each constants, and b represents the y' intercept.

Usually, there is a local linear relationship between the band number and the migration distance in the lower region of the migration pattern, which can be approximated by a regression line as shown in FIG. Therefore, if offset distortion had not occurred, the detected bands would have existed on one regression line. In other words, the four regression lines obtained for each lane should have overlapped into one.

Note that the method of expressing the correlation between band numbers and their migration positions is not limited to the regression line described above; for example, the correlation can be expressed with higher precision by approximating an appropriate higher-order curve to create a regression curve. Relationships can also be determined.

Next, the difference in migration distance between the lanes is determined from the obtained correlation, and the migration position of each lane is corrected based on this difference.

In Figure 4, the difference in migration distance between lanes is
It appears as the difference in the y′ intercept when the migration distance y′ at any point on the horizontal axis is determined. For example, the difference in migration distance between the first slot and the second slot is _b12 .
This difference in y' intercept corresponds to the positional deviation between lanes due to offset distortion. If the slope of each regression line (a value in the above general formula) is different, the average value of the difference in y' at any point on the horizontal axis, or the difference in y' at a suitable point on the horizontal axis is calculated as the migration distance. It can be the difference. Also, when the correlation is expressed by a regression curve, the difference in migration distance,
That is, positional deviation between lanes can be determined.

Based on the difference in migration distance obtained, shift the position (y) of the signal on each lane toward the top or bottom.
For example, for the second slot lane, the fourth
By shifting the entire one-dimensional waveform 2 shown in the figure by b ₁₂ in the direction of the origin on the y-axis (subtracting the value of b ₁₂ with respect to y), the migration position can be corrected all at once. In this way, positional deviations between lanes can be corrected for each lane at once.
Offset distortion of the migration pattern can be corrected.

For example, by detecting all the positions where the level of the signal appearing on the waveform is maximum for the one-dimensional waveform of each lane whose migration position has been corrected,
The positions of all bands on each lane can be detected.

Based on the position of the detected band, all bands are ranked in order from the bottom of the electrophoresis pattern. At this time, the four types of base-specific
Since the combination of DNA fragments is an exclusive combination, the sequence can be easily determined by changing lanes and taking advantage of the fact that two or more bands are not detected at the same position. The slots (1) to (4) above each have information about the terminal bases consisting of (G), (A), (T), and (C), so replace them with the base corresponding to the slot to which each band belongs. By this,
DNA base sequence (e.g. A-G-C-T-A
-A-G-...) can be obtained.

In the present invention, if smiling development occurs in the electrophoretic pattern, the smiling correction may be performed before the above-described offset distortion correction is performed on the digital signal.

Smiling development is a development in which the migration distance in the slots at both ends of the support medium is shorter than the migration distance in the slot in the center of the support medium, and is caused by the heat dissipation effect (so-called edge effect) during the migration process. be.

Smiling correction can be performed, for example, as follows.

In the electrophoresis pattern where smiling development occurs, the band (long strip in the width direction) is usually not perpendicular to the electrophoresis direction but at an angle depending on the degree of the smiling effect. Detect the slope of at least one band for each lane. For example, the slope can be determined by scanning digital image data so that each band has at least two scanning lines, extracting a digital signal, creating a one-dimensional waveform for each scanning line, and finding the position of its maximum value. It can be determined from the regression line obtained by connecting the lines. Alternatively, the digital signal as described above may be detected in advance in the autoradiograph reading process.

Next, one band (reference band) on one lane (reference lane) with the smallest degree of smiling effect is determined, and from the slope of this band and the slope of the nearest band on the other lanes, the standard band is determined. is extrapolated to the other lane to determine the relative position of the reference band in the other lane. Next, the ratio of migration distances for each lane is determined from this relative position and the position on the reference lane. The obtained ratio represents the degree of the smiling effect of each lane, and the migration distance of each lane is collectively expanded or contracted based on this ratio. In this way, smiling can be corrected for all lanes.

Alternatively, the migration distance can be corrected for each band individually by detecting the slope of all bands and extrapolating each band on a lane other than the reference lane to the reference lane based on its slope. can.

For details on smile correction using the above method, please refer to Japanese Patent Application No. 1986-74899 filed by the present applicant.
No. [Application filed on April 9, 1985 (1)] and patent application 1988-
It is stated in each specification of No. 74900 [filed on April 9, 1985 (2)].

By the method described above, the base sequence of one chain molecule of DNA can be determined. In addition, information about the base sequence of DNA is
The display format is not limited to the above display format; for example, it is also possible to simultaneously display the intensity (z') of each band as the relative amount of the radiolabeled substance, if desired. Furthermore, it is also possible to display the base sequences of both DNA strands.

Alternatively, DNA base sequence information can be displayed as an image based on a digital signal that has been corrected for offset distortion (and smile correction) as described above. At the same time, the original autoradiograph may be visualized and displayed. In this case, it is possible for the analyst himself to determine the final base sequence based on this displayed image.

In addition, in the above, the sample base-specific
As a mixture of DNA fragments (G, A, T, C)
Although the case where exclusive combinations of (G, G+A, T+
It can be applied to various combinations such as 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, correction of offset distortion is not limited to the separation and development array of a set of base-specific fragments of nucleic acids, 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 above-mentioned signal processing is output from the signal processing circuit and then sent to a recording device directly or, if necessary, via a storage means such as a magnetic disk or magnetic tape. transmitted.

Examples of recording devices include those that optically record by scanning a photosensitive material with a laser beam or the like;
Recording devices based on various principles can be used, such as those that record symbols and numbers displayed on a CRT or the like on a video printer or the like, and those that record on a heat-sensitive recording material using heat rays.

[Brief explanation of the drawing]

FIG. 1a is a partial view showing the shape of a slot provided at the upper end of the support medium, and FIG. 1b is a view showing an example of a separation development pattern in which offset distortion occurs. FIG. 2 is a diagram showing an example of a migration pattern in which offset distortion has occurred. FIG. 3 is a diagram showing a one-dimensional waveform consisting of a signal position (y) and a signal level (z) for each lane. FIG. 4 is a diagram showing a regression line consisting of band number (n) and migration distance (y') for each lane. Straight lines 1 to 4 correspond to slots (1) to (4), respectively.

Claims (1)

[Scope of Claims] 1. A plurality of separated and developed arrays formed by separating and developing a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in a 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, (1) detect at least two bands at the bottom of each separation development column, and sequentially form the bands from the bottom. (2) obtaining a correlation between the band number and its separation distance for each separation expansion column; and (3) determining the separation distance between the separation expansion columns from the obtained correlation. 1. A signal processing method for determining a base sequence of a nucleic acid, the method comprising: obtaining a difference between the columns, and correcting the separation development position for each column by using this difference as a positional shift between columns. 2. A patent characterized in that in the first step, a band is detected by extracting a digital signal along the separation development direction of each column and then determining the position where the level of the extracted signal in each column is maximum. A signal processing method for determining the base sequence of a nucleic acid according to claim 1. 3. A signal for determining the base sequence of a nucleic acid according to claim 1, wherein in the second step, the correlation between the band number and its separation distance is obtained as a regression line or a regression curve. Processing method. 4. For determining the base sequence of a nucleic acid as set forth in claim 3, wherein in the third step, the difference in separation distance between the separation and development columns is obtained as a difference in the intercept of a regression line or a regression curve. signal processing method. 5. Before the first step, by detecting the inclination of at least one band with respect to the separation and development direction for each separation and development row, and then determining the relative position of the band in other separation and development rows based on this inclination, The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that the separation distance of each band is corrected. 6 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 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 on the phosphor sheet. Claim 1, characterized in that the autoradiograph is obtained by accumulating and recording the phosphor sheet, and then photoelectrically reading out the autoradiograph as photostimulated light by irradiating the phosphor sheet with excitation light. A signal processing method for base sequencing of the described nucleic acid. 8 A digital signal corresponding to the above-mentioned 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 a visualized autoradiograph. 9 Compatible with autoradiographs of multiple separation and development columns formed by separation and development of a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments on a support medium in one-dimensional direction. In a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal, the steps include: (1) detecting at least two bands at the bottom of each separation and development column and serially numbering the bands from the bottom; (2) Obtaining the correlation between the band number and its separation expansion distance for each separation expansion column; (3) Obtaining the difference in separation expansion distance between the separation expansion columns from the obtained correlation; and calculating this difference. (4) detecting all bands on each separation development column and ranking the bands based on their positions; A signal processing method for determining the base sequence of a nucleic acid, characterized in that: 10 In the first and fourth steps, after extracting the digital signal along the separation development direction of each column, the band is detected by finding the position where the level of the extracted signal in each column is maximum. A signal processing method for determining the base sequence of a nucleic acid according to claim 9. 11. A signal for determining the base sequence of a nucleic acid according to claim 9, wherein in the second step, the correlation between the band number and its separation distance is obtained as a regression line or a regression curve. Processing method. 12. In the third step, the difference in separation and development distance between the separation and development rows is obtained as a difference in the intercept of a regression line or a regression curve. signal processing methods for. 13. Before the above first step, by detecting the inclination of at least one band with respect to the separation and development direction for each separation and development row, and then determining the relative position of the band in other separation and development rows based on this inclination, 10. The signal processing method for determining the base sequence of a nucleic acid according to claim 9, wherein the separation distance of each band is corrected. 14 The mixture of the above 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. , and 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 item 9. 15 A 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 the autoradiograph is obtained by accumulating and recording the phosphor sheet, and then photoelectrically reading out the autoradiograph as photostimulated light by irradiating the phosphor sheet with excitation light. A signal processing method for base sequencing of the described nucleic acid. 16 A digital signal corresponding to the autoradiograph is transferred onto the photosensitive material after superimposing the support medium and the photographic light-sensitive material to photosensitively record the autoradiograph of the radiolabeled substance on the support medium on 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 a visualized autoradiograph.