JPS62225956A - Signal processing for determining base sequence nucleic acid - Google Patents

Abstract

PURPOSE:To automatically determine the base sequence of nucleic acid handily and at a high accuracy, by separating a true band and a noise from a mean value for fixed sections of a digital signal obtained from an autoradiograph. CONSTITUTION:A pattern obtained by separating and developing a base specific DNA segment imparted a radioactive marker or a mixture of a base specific RNA segment on a support medium in a 1-D way is visualized on an X-rays film to obtain an autoradiograph (A). The density gradient is converted to a digital signal for each of four slots 1-4 of the graph (A) to be memorized into a memory and a primary waveform is prepared which comprises the position along the direction of migration and a signal level. A mean level value is calculated for areas having a signal level above a fixed value in a fixed section on the waveform and a true band and a noise are separated from the resulting value. Then, the base sequence is judged by the position of the band.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention 1] 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 mechanisms of biological mechanisms and replication,
It is essential to clarify the genetic information possessed by living organisms. Among other things.

It is essential to determine the base sequence of nucleic acids such as DNA (or DNA fragments, hereinafter the same) that carry specific genetic information.

Maxam-Gilber (Mδ!all-Gilber) uses autoradiography as a typical method for determining 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 cleaved in a base-specific manner 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 111i development pattern is visualized on, for example, an X-ray film to obtain its autoradiograph, and from the obtained autoradiograph and each base-specific cleavage means, it is determined that the chain molecule to which the radioactive isotope is bound is By sequentially determining the bases in a certain positional relationship from the end, it is possible to determine the base sequence of the entire target object.

The latter Sanger-Cournon 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 been filed for a method using a radiation image conversion method using a stimulable phosphor sheet (Japanese Patent Application Laid-Open No. 71459-
No. 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 (excitation light) in the 1+f to infrared region are absorbed. ) can emit radiation energy as fluorescence. 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, since the autoradiograph of a radiolabeled substance is stored in a phosphor sheet as radiant energy and then read out photoelectrically as photostimulated light, the autoradiograph obtained directly as a digital signal can also be stored in a suitable recording medium. be able to.

Conventionally, the base sequence of a nucleic acid has been determined using a base-specific cleavage degradation product or a base-specific composite of a radioactively labeled nucleic acid (hereinafter simply referred to as a base-specific fragment of a nucleic acid) in a visualized autoradiograph. ) separation deployment position (
It is determined by visually judging the bands) and comparing the positions of those bands with each other. Therefore, analysis of autoradiographs is usually performed through human vision, which requires a great deal of time and effort.

Furthermore, since it relies on the human eye, there are limits to the accuracy of base sequence information, such as the base sequence of nucleic acids obtained 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-
No. 126527, patent application No. 1989-89615, patent application No. 1983
0-226091, patent application No. 60-226092, etc.)
When using a conventional radiation film, the digital signal corresponding to the autoradiograph is first visualized on the film and then read photoelectrically using reflected or transmitted light. It can be obtained by In addition, when using a stimulable phosphor sheet,
The autoradiograph is obtained by directly reading out the phosphor sheet on which the recorded autoradiograph 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.

For example, the preparation and separation of base-specific nucleic acid fragments may be insufficient during sample preparation, or samples from other slots may be mixed in when the sample is injected into each slot of the support medium. , a band (this is called a ghost band or extra band) may appear at a position where it should not appear. Alternatively, noise may occur due to radioactive impurities being mixed into the sample or exposure to natural radiation during the exposure process. As a result, since bands are comparatively identified including these extra bands and noise, errors occur in base sequence determination and the accuracy of the information obtained decreases.

Even when such noise occurs, it is desirable 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] In a method for automatically determining the base sequence of a nucleic acid using autoradiography, the present inventor has developed a method for automatically determining the base sequence of a nucleic acid using autoradiography. Through suitable signal processing, 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. 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) a one-dimensional waveform consisting of a position along the separation development direction and a signal level for each separation development column; 2) Calculating the average level value of a region on the one-dimensional waveform that has a signal level equal to or higher than a certain value within a certain section from the search start point; 3) Determining the darkness value based on the average level value. 4) searching for whether or not there is a peak having a signal level equal to or higher than the dark value in the section; 5a) if a peak exists in the fourth step, a band is located at the position of the peak; 5b) If the peak does not exist in the fourth step,
By sequentially repeating the step of setting a position a certain distance ahead of the search start point as the next search start point, and 6) the above second to fifth steps,
Step of determining the positions of all bands at the separation development position.

The present invention provides a signal processing method for determining the base sequence of a nucleic acid, the method comprising:

According to the present invention, in a digital signal corresponding to an autoradiograph of a separated 11N pattern obtained by separating and developing a mixture of base-specific fragments of nucleic acids on a support medium, noise is generated in the separated development pattern. Even in such cases, the base sequence of a nucleic acid can be obtained simply and with high precision by passing it through a signal processing circuit that has a signal processing function that can eliminate noise and detect only the true band.

Generally, the image density (digital signal level) of extra bands and noise appearing in an autoradiograph due to sample contamination, natural radiation, etc. is lower than that of the true bands. However, in methods such as the Sanger-Coursin method, as the sample base-specific fragment increases in molecular weight, it contains more radioactive isotopes and the radioactivity intensity increases. A density gradient (change in signal level) occurs in which the image density decreases as the separation development distance increases. Therefore, when determining whether a genuine band exists or not or whether a band is a genuine band, a certain darkness value is set so that the peak of the signal level appearing on the separated development pattern is higher than this threshold value. If you just uniformly select samples based on whether they are true or not, you tend to detect extra bands as true bands or, conversely, mistakenly exclude genuine bands, making it impossible to obtain accurate base sequence information. .

According to the present invention, even if the signal level differs locally depending on the separation development position, the average value of the signal level is calculated for each fixed interval, and the threshold is set based on this to differentiate the true band from the noise. By setting a suitable value that can be separated, it is possible to detect only the true band in just the right amount based on the threshold value that varies from section to section. And detected■? By comparing the band positions between separation and development columns based on the band positions, the base sequence of the nucleic acid can be determined simply and with high precision.

[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,
A base-specific DNA cleavage product mixture, which is a type of base-specific DNA fragment mixture, is obtained by base-specific cleavage and decomposition of radiolabeled DNA according to the aforementioned Maxam-Gilbert method.

Furthermore, the base-specific DNA compound mixture can be synthesized according to the Sanger-Luzon method described above using DNA as a template and a radioactively labeled deoxynucleoside triphosphate and a DNA synthesizing enzyme. 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, I by a method appropriate to these substances.
AC, 36S, 31. L? It is imparted by retaining a radioactive isotope such as hI.

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, the support medium on which the radiolabeled substance has been separated and developed is subjected to radiography using a conventional photographic material.
Alternatively, an autoradiograph thereof is obtained by a radiation image conversion method using a stimulable phosphor sheet, and then a digital signal corresponding to the autoradiograph of the radiolabeled substance is obtained via an appropriate readout system.

When using the former radiographic method, first the support medium and a photographic light-sensitive material such as an X-ray film are superimposed at low temperature or room temperature for a long time (several hours to several tens of hours) to expose the radiographic film. , and developed to produce a visible image of the autoradiograph of the radiolabeled substance on radiographic film.

Next, the autoradiograph visualized on the radiation film ball 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 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 and stores the radiation energy.
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. in particular,
For example, by scanning a phosphor sheet with a laser beam to emit radiation energy as photostimulated light, and then detecting this photostimulated light photoelectrically, an autoradiograph of a radiolabeled substance can be obtained directly without creating a visible image. Obtained as an electrical signal. Furthermore, by A/D converting this electrical signal, a digital signal corresponding to an autoradiograph can be obtained.

For details of the above-mentioned autoradiograph measurement operation and method of obtaining a digital signal corresponding to the autoradiograph, see the above-mentioned JP-A-59-83057 and JP-A-59-1.
It is described in various publications such as No. 26527 and Japanese Unexamined Patent 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 (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, readout conditions are set by inputting information about the position of each separation expansion column, band width, etc. in advance, and one or more scanning lines on each band are set in advance. By performing scanning with a light beam at a scanning line density such 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 DXF consists of coordinates (x, y) expressed in a coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (Z) at those coordinates.
One signal corresponds to one pixel.

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 in the support medium thus obtained is subjected to signal processing by the method of the present invention as described below, and the base sequence of the target nucleic acid is A decision will be made.

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.

l) Guanine (G) specific DNA compound 2) Adenine (
A) Specific DNA compound 3) Thymine (T) specific DNA
Compound 4) Cytosine (C)-specific DNA composite Here, each base-specific DNA composite is a base-specific DNA composite synthesized by the Sanger-Courlain method, that is, a DNA composite of various lengths having the same terminal base. Consists of DNA compounds.

FIG. 1 shows an autoradiograph of the electrophoresis pattern formed by electrophoresing a base-specific DNA compound of the "-mating type" into four slots, respectively. In the autoradiograph, a concentration gradient occurs along the migration direction (direction of arrow →).

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

First, a one-dimensional waveform consisting of a position along the migration direction and a signal level is created for each migration column (lane).

FIG. 2 shows a one-dimensional waveform created by extracting digital signals in the direction of the arrow shown in FIG. 1 for the first lane. In other words, FIG. 2 corresponds to the concentration sectional view of the first lane.

FIG. 3 is a partially enlarged view of FIG. 2. In FIG. 3, the left side part is a one-dimensional waveform near the electrophoresis start position, and peak A (maximum signal level) is lower than the peaks on both sides, and is clearly an extra band or noise. Further, the right part is a one-dimensional waveform of a part far from the electrophoresis start position (the migration distance is large), and peak B is a true band from comparison with other peaks on the right side. However, between peak A and peak B, peak A has a higher signal level, so it can be seen that it is not possible to simply determine whether the band is a true band or not based only on the signal level (absolute value) of each peak. . That is, it is necessary to perform a relative comparison taking into account the signal levels around the peak of interest.

FIG. 4 is a partially enlarged view of the one-dimensional waveform in FIG. 2, and is a diagram for explaining the determination of a threshold value, which is a characteristic requirement of the present invention, and the operation of determining a band based on the threshold value.

Next, the subsequent operations for band determination will be explained with reference to FIG.

First, on the one-dimensional waveform, the average level value Z1 of the signal level Z within a certain section from the search start point Xl of the band is calculated.

In FIG. 4, the position of the peak C of the true band that has already been determined is the search starting point x1.

It is usually preferable to select the starting point for the initial search at the bottom of the run where the gap between bands is wide (the position where the migration distance is maximum, the right end of Figure 2), and then move the search from the bottom to the top of the run. It is preferable to proceed in sequence. The fixed interval may be set in advance based on the electrophoresis distance (practical length of the support medium), or may be set according to an appropriate electrophoresis distance (or along the electrophoresis direction) so as to vary depending on the search position. position), and in this case, the number of peaks detected within one section can be made approximately constant regardless of the search position. Also, as shown in Fig. 4, the search direction (arrow ←) starts from the starting point Xi.
Alternatively, 1/2 L may be taken on each side with the starting point as the center point.

The average level value Z1 is obtained by calculating the average value of the signal levels for areas, that is, sections, L2 and L3, in which the signal level is equal to or higher than a certain value Za. Specifically, the average value of signals having a level of Za or higher is determined. This makes it possible to remove relatively small extra bands and noise (background noise) appearing in the entire electrophoresis pattern.

A threshold value TI is determined based on this average level value ZI.

The threshold T, for example, can be obtained by substituting ZI into a function that uses a preset average value as a variable [T+=f
('Z+)], specifically, T, = α・Z.

(However, the coefficient α is a positive number other than O). Thereby, the threshold value T can be set to a suitable value that can separate the extra band and the true band. For example, by setting the coefficient α to a constant greater than 1, even when the peak of the extra band is relatively large or when there are a large number of extra bands, it can be suitably eliminated. The function f(ZI) or the coefficient α may also be expressed as an appropriate function of migration distance so that it varies depending on the search position.

Next, a peak whose signal level is equal to or higher than the threshold value 11 is searched for within the section starting from the search start point x1.

A peak is a point on a one-dimensional waveform where the signal level is maximum. At this time, we are not talking about the whole section, but about a certain section (I Z a or more).

Search time can be shortened by searching for peaks only for ~L3.

Then, a peak whose signal level is equal to or higher than a threshold value T1 is determined to be a true band, and a peak smaller than T1 is determined to be an extra band or noise and is ignored. This results in
In FIG. 4, among the two peaks D and E within the interval ˜L3, only the peak E whose signal level exceeds the threshold TI is determined to be the true band, and the peak D, which is the ex-i-lab band, is excluded.

When the determination of the band within the interval is completed in this way, the position of the peak E (true band) whose liI value is less than or equal to T1 within the interval is determined as the next search starting point XI+4, and this The above operation is repeated for a certain section from the search starting point Xi or □. Threshold T.

If there are two or more peaks that are less than or equal to "-," select the peak closest to the search start point in the search direction, and then shift the interval little by little while taking into account slight changes in the surrounding signal level. , the true band can be determined with higher accuracy. In addition, by selecting the peak farthest from the search start point in the search direction, there is less overlap between the sections, so the number of band search operations described above can be reduced, and the processing time can be shortened.

If there is no peak (genuine band) beyond the threshold value T within the interval, the next search start point XI+ is set at a position where the search start point
1, repeat the above operation. This distance dL may be set in advance to a certain value, such as a distance corresponding to one pixel, or may be expressed as a function of migration distance and determined based on the search starting point.

The above operations are repeated in sequence up to the electrophoresis start point (left end in FIG. 2) to detect all genuine bands on the first lane. Furthermore, by performing the same operation for the remaining three lanes, all genuine bands on the electrophoresis pattern can be detected.

In addition, swim! In addition to the occurrence of noise such as extra bands in the pattern, if various distortions and noises such as smiling phenomenon, offset distortion, or band fusion occur, please check the above before or during the band determination. Signal processing for these corrections may be performed.

Here, the smiling phenomenon is a phenomenon in which the migration distance of the slots at both ends of the support medium is shorter than the migration distance of the slots at the center of the support medium, and is caused by the heat dissipation effect (so-called edge effect) during the migration process. arise. Offset distortion refers to the overall positional deviation between lanes.
This occurs because the start position and start time of sample electrophoresis differ for each slot due to differences in the shape of the slots. In addition, band fusion refers to two or three bands joining together to form one wide band due to insufficient electrophoresis, generally in the region near the electrophoresis start position of the second part of the pattern. It is likely to occur in

For details of these corrections by signal processing, see Japanese Patent Application No. 1988-74899 and Japanese Patent Application No. 60-74 filed by the same applicant.
No. 900, Japanese Patent Application No. 85275/1983, Japanese Patent Application No. 85/1983
No. 5276, Japanese Patent Application No. 1986-111 i86, Japanese Patent Application No. 60-111
It is described in each specification of No. 0-111187.

By comparing the positions of authentic bands determined between lanes with each other, bands can be immediately ranked. At this time, since the combination of the four types of base-specific DNA compounds described above is an exclusive combination, taking advantage of the fact that two or more bands (bands in different lanes) cannot exist at the same position, The ranking can be easily determined. The slots (1) to (4) above are (G) and (
Since it has information about the terminal bases consisting of A), (T), and (C), by replacing the bases with the bases corresponding to the slots to which each band belongs, the DNA base sequence (for example, A
-G-C-T-A-AG-...) can be obtained.

In this way, the base sequence of one chain molecule of DNA can be determined. Note that information about the DNA base sequence is not limited to the above display format; for example, if desired, the information about the intensity of each band (2
') can also be expressed as the relative amount of radiolabeled substance. Furthermore, it is also possible to display the base sequences of both two stranded molecules of DNA.

Alternatively, the DNA base sequence information can be displayed as an image based on a digital signal that has been subjected to the above signal processing. That is, the corrected position of each band can be visualized and displayed together with the original autoradiograph. In this case, it is possible for the analyst himself to determine the final base sequence based on this displayed image.

In addition, although the case where an exclusive combination of (G, A, T, C) is used as a mixture of base-specific DNA compounds as a sample has been described above, the signal processing method of the present invention applies to this combination. For example, it is not limited to (
It can be applied to various combinations such as G, G+A, T+C, and C). Furthermore, the method of the present invention is not limited to base-specific DNA composites obtained by the Sanger-Cournon method, and can be applied to base-specific DNA fragments prepared by any method.

Similarly, the method of the present invention can also be applied to a mixture of base-specific RNA fragments (for example, a combination of G, A, U, and C). Furthermore, the band determination is not limited to the separation and development array of the base-specific fragments of the nucleic acid set, but can be performed for all the separation and development arrays that are simultaneously separated and developed on the support medium. .

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.

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 drawings]

FIG. 1 is a diagram showing an example of an autoradiograph of a migration pattern in which a concentration gradient occurs. FIG. 2 is a diagram showing a one-dimensional waveform along the arrow in FIG. 1 for the first lane. FIG. 3 is a partially enlarged view of the one-dimensional waveform in FIG. 2. FIG. FIG. 4 is a partially enlarged view of the one-dimensional waveform in FIG. 2,
It is a figure explaining the processing method of this invention. x: search starting point, L constant interval, Za mini-value, Tl: threshold. Peak C, E: True band, Peak D 22 Straband

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, 1) a primary sequence consisting of a position along the separation development direction and a signal level for each separation development column; obtaining the original waveform; 2) calculating the average level value of an area on the one-dimensional waveform having a signal level equal to or higher than a certain value within a certain interval from the search start point; 3) determining a threshold value based on the average level value. 4) searching for whether or not a peak having a signal level equal to or higher than a threshold exists in the section; 5a) if a peak exists in the fourth step, a band is located at the position of the peak; 5b) If the peak does not exist in the fourth step,
By sequentially repeating the step of setting a position a certain distance ahead of the search start point as the next search start point, and 6) the above second to fifth steps,
A signal processing method for determining the base sequence of a nucleic acid, comprising the step of determining the positions of all bands on a separation and development column. 2. The first search start point in the second step is set as the position with the largest separation and development distance, and the sixth step is performed in a direction opposite to the direction of separation and development. Signal processing method for base sequencing of nucleic acids. 3. Signal processing for determining the base sequence of a nucleic acid as set forth in claim 1, characterized in that in step 5a, the position of the peak closest to the search start point is set as the next search start point. Method. 4. Claims characterized in that the fixed interval and fixed value in the second step, the threshold value in the third step, and the fixed distance in the fifth b step are each set based on the position on the one-dimensional waveform. 2. A signal processing method for determining the base sequence of a nucleic acid according to item 1. 5. 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 separation 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. 6. 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, characterized in that 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 a nucleic acid as described in Section 3. 7. 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 .