JPH0529070B2 - - 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 multiple mechanisms. It has become commonplace. Above all,
It has become essential to determine the base sequence of nucleic acids such as DNA (or DNA fragments, hereinafter the same) that carry specific genetic information.

Maxam Gilbert uses autoradiography as a typical method to determine 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 converting a substance into a radioactively labeled substance, chemical means are used to cleave the bonds between each constituent unit of the chain molecule in a base-specific manner. Next, the mixture of base-specific DNA cleavage products obtained by this operation is separated and developed by gel electrophoresis, and a separated development pattern (however, visually (cannot be seen). This separated development pattern is visualized on, for example, an X-ray film to obtain its autoradiograph,
Based on the obtained autoradiograph and each base-specific cleavage means, the bases located in a certain positional relationship from the end of the chain molecule to which the radioactive isotope is bound are sequentially determined. The sequence can be determined.

In addition, the latter Sanger-Coulson method is
A DNA compound that is complementary to a chain molecule of DNA or a DNA fragment and has been given a radioactive label is base-specifically synthesized using chemical means, and the base-specific DNA compound is synthesized using chemical means. This method uses a mixture 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 transferred to the stimulable phosphor of the phosphor sheet, electromagnetic waves (excitation light) in the visible to infrared region are applied.
By exciting it with , radiation energy can be emitted 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 an autoradiograph of a radiolabeled substance is once stored in a phosphor sheet as radiation energy and then read out photoelectrically as photostimulated light, it is necessary to obtain it directly as a digital signal and then save it on a suitable recording medium. I can do it.

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. ) is determined by visually determining the separation development position (band) and comparing the positions of these 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. 126527, 1982, Patent Application No. 126527, Patent Application No. 1983-
No. 89615, Patent Application No. 1983-226091, Special Application No. 1983-
226092 etc.). When using a conventional radiation film, the digital signal corresponding to the autoradiograph can be obtained by first making the autoradiograph a visible image on the film and then reading it photoelectrically 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.

For example, the preparation and separation of base-specific fragments of nucleic acids 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. , bands (referred to as ghost bands or extra bands) may appear at positions where they should not originally 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 if such noise occurs,
It is desired to efficiently process digital signals corresponding to the autoradiograph to automatically determine the base sequence of a 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 even in the case of a noisy separation/development pattern, it has been possible to automatically determine the base sequence of a nucleic acid with ease and high accuracy.

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) obtaining a one-dimensional waveform consisting of a position along the separation development direction and a signal level for each separation expansion column; (2) obtaining a one-dimensional waveform consisting of a position along the separation expansion direction and a signal level; (3) determining a threshold based on the average level; (4) a peak having a signal level equal to or higher than the threshold exists within the section; (5a) If a peak exists in the fourth step, determining that a band exists at the position of the peak, and setting the position of the peak as the next search reference point; (5b) If there is no peak in the fourth step, a step of setting the next search reference point at a position a certain distance ahead of the search reference point, and (6) repeating the second to fifth steps above in sequence. The present invention provides a signal processing method for determining the base sequence of a nucleic acid, which comprises the step of determining the positions of all bands on a separation and development array.

According to the present invention, in a digital signal corresponding to an autoradiograph of a separation pattern obtained by separating and developing a mixture of base-specific fragments of nucleic acids on a support medium, noise is generated in the separation 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 that appear on an autoradiograph due to sample contamination, natural radiation, etc. is lower than that of the true band. However, in methods such as the Sanger-Coulson method, as the sample base-specific fragment increases in molecular weight, it contains more radioactive isotopes.
Due to the increased radioactivity intensity, a concentration gradient (change in signal level) occurs in the autoradiograph, where the image density decreases as the separation development distance increases. Therefore, when determining whether a genuine band exists or whether a band is a genuine band, a certain threshold value is set and the peak of the signal level appearing on the separated development pattern is higher than this threshold value. If bands are uniformly selected based on whether or not they are present, extra bands tend to be detected as genuine bands, or conversely, genuine bands tend to be mistakenly excluded, 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 value is set based on this for the true band and noise. By setting a suitable value that can separate the true bands from each other, 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. By comparing the positions of the bands between the separation and development arrays based on the positions of the detected bands, 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:
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 compound mixture is prepared using the Sanger-Coulson method as described above, using DNA as a template and adding deoxynucleoside triphosphate to which a radioactive label has been added.
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 light-sensitive material such as an X-ray film are overlapped for a long time (several hours to several tens of hours) at low temperature or room temperature, and then the radiographic film is exposed. , and developed to produce a visible image of the autoradiograph of the radiolabeled substance on 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 transmitted or reflected light. Furthermore, by A/D converting this electrical signal,
A digital signal corresponding to the autoradiograph can be obtained.

When using the latter radiation image conversion direction,
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, 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 _D A 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 on the support medium obtained in this way is subjected to signal processing by the method of the present invention as described below to obtain the base sequence of the target nucleic acid. A decision is made.

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 compound (2) Adenine (A)-specific DNA compound (3) Thymine (T)-specific DNA compound (4) Cytosine (C)-specific DNA compound Here , each base-specific DNA compound is synthesized in a base-specific manner by the Sanger-Coulson method, that is, it consists of DNA compounds of various lengths having the same terminal base.

FIG. 1 shows an autoradiograph of the electrophoresis pattern obtained by electrophoresing the four types of base-specific DNA compounds described above into four slots. The autoradiograph shows the electrophoresis direction (direction of arrow →)
A concentration gradient occurs along the .

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, 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. In addition, the right part is the one-dimensional waveform of the part far from the migration start position (the migration distance is large), and the peak B
is the true band from comparison with other peaks on the right. However, between peak A and peak B, peak A
It can be seen that the signal level is higher in the case of , and therefore 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, search reference point x _i of the band
An average level value Z _i of the signal level Z within a certain period L is calculated from .

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

It is usually preferable to select the initial search reference point 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 of the run to the top. It is preferable to proceed in order. The fixed section L 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 that it changes depending on the search position. In this case, the number of peaks detected within one section can be made almost constant regardless of the search position. Further, as shown in FIG. 4, the section L may be taken in the search direction (arrow ←) with the reference point x _i as the starting point, or it may be taken 1/2L on each side with the reference point as the center point.

The average level value Z _i is obtained by calculating the average value of the signal levels for the regions where the signal level is equal to or higher than the constant value Z _a , that is, the sections L ₁ , L ₂ and L ₃ . Specifically, the average value of signals having a level equal to or higher than Z _a is determined. This makes it possible to remove comparatively small extra bands and noise (background noise) appearing in the entire migration pattern.

A threshold value T _i is determined based on this average level value Z _i . The threshold value T _i is obtained, for example, by substituting Z _i into a function whose variable is a preset average value [T _i =f(Z _i )]. Specifically, it can be set as T _i =α·Z _i (however, the coefficient α is a positive number other than 0). Thereby, the threshold value T _i can be set to a suitable value that can separate the extra band and the true band. For example, the coefficient α
By setting is a constant larger than 1, even when the peak of the extra band is relatively large or when there are many extra bands, it can be suitably excluded. The function f(Z _i ) 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, the section L starting from the search reference point x _i
Search for a peak within which the signal level of the peak is greater than or equal to the threshold T _i . A peak is a point on a one-dimensional waveform where the signal level is maximum. At this time, the search time can be shortened by searching for peaks not for the entire section L but only for the sections L ₁ to _{L 3} having a constant value Z _a or higher.

Then, a peak whose signal level is equal to or higher than the threshold T _i is determined to be a true band, and a peak smaller than T _i is determined to be an extra band or noise and ignored. As a result, in Fig. 4, the two peaks D and E in the section L ₁ to _{L 3} have signal levels that are equal to the threshold value.
Only the peak E that exceeds T _i is determined to be the true band, and the extra band, peak D, is excluded.

When the determination of the band within the interval L is completed in this way, the position of the peak E (true band) which is equal to or higher than the threshold value T _i in the interval is determined as the next search reference point _{x i +1} , and this The above operation is repeated for a certain section L from the search reference point x _i+1 . If there are two or more peaks that are equal to or higher than the threshold T _i , by selecting the peak closest to the search reference point in the search direction,
By shifting the sections little by little and 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 reference 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.

Peak greater than threshold T _i within section L (genuine band)
If the search reference point x _i does not exist at all, the above operation is repeated with a position obtained by advancing the search reference point x i by a certain distance dL in the search direction as the next search reference point x _{i +1} . 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 reference point.

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

If the electrophoresis pattern contains noise such as extra bands, or various other distortions or noises such as smiling phenomenon, offset distortion, or band fusion, perform the above-mentioned band determination. Signal processing for these corrections may be performed during the process.

Here, the smiling phenomenon is a phenomenon in which the migration distance in the slots at both ends of the support medium becomes shorter than the migration distance in the slots in 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, and is caused by the fact that the start position and start time of sample electrophoresis differ for each slot due to differences in the shape of the slots. Furthermore, band fusion refers to three or three bands joining together to form one wide band due to insufficient migration. Generally, this tends to occur in the area near the migration start position at the top of the pattern.

For details of these corrections by signal processing, please refer to Japanese Patent Application No. 60-74899 and Japanese Patent Application No.
No. 60-74900, Special Application No. 1983-85275, Special Application No. 1983-
No. 85276, Special Application No. 111186, Special Application No. 111186, Special Application No. 1983-
It is described in each specification of No. 111185.

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 above four types of base-specific DNA compounds 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 above
The slots (1) to (4) 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. The DNA base sequence (e.g. A-G-
C-T-A-AG-...) can be obtained.

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

Alternatively, DNA base sequence information can also 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, in the above, the sample base-specific
As a mixture of DNA compounds (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). Furthermore, the method of the present invention is not limited to base-specific DNA composites obtained by the Sanger-Coulson method, and can be applied to base-specific DNA fragments prepared by any method. I can do it.

Similarly, the method of the present invention can also be applied to mixtures of base-specific RNA fragments (for example, a combination of G, A, U, and C). moreover,
Band determination 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.

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. 1 is a diagram showing an example of an autoradiograph of a migration pattern in which a concentration gradient occurs. Second
The figure 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. Figure 4 shows the second
FIG. 3 is a partially enlarged view of a one-dimensional waveform in the figure, and is a diagram illustrating a processing method of the present invention. x _i : search reference point, L: constant interval, Z _a : constant value,
T _i :Threshold value, Peaks C, E: True band, Peak D: Extra band.

[Claims]

1 Compatible with autoradiographs of multiple separation agents formed by separating and developing 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) obtaining a one-dimensional waveform consisting of a position along the separation development direction and a signal level for each separation development column; (2) a step of dividing a one-dimensional waveform into at least two sections; and (3) a step of smoothing the signal level by a smoothing means having different characteristics for each section. Signal processing method for base sequencing. 2. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein in the third step, the smoothing means is a moving average filter. 3. The signal processing method for determining the base sequence of a nucleic acid according to claim 2, wherein in the third step, the mask size of the moving average filter is changed for each section.

Claims (1)

(6) A signal processing method for determining the base sequence of a nucleic acid, comprising the step of determining the positions of all bands on the separation and development array by sequentially repeating the second to fifth steps. 2. The method according to claim 1, characterized in that the first search reference point in the second step is the position with the greatest separation and development distance, and the sixth step is performed in a direction opposite to the direction of separation and development. A signal processing method for determining the base sequence of nucleic acids. 3. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that in step 5a, the position of the peak closest to the search reference point is set as the next search reference point. . 4 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. 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 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. 6 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 on the phosphor sheet. Claim 1, characterized in that the autoradiograph is obtained by accumulating and recording the phosphor sheet, irradiating the phosphor sheet with excitation light, and photoelectrically reading out the autoradiograph as photostimulated light. A signal processing method for base sequencing of the described nucleic acid. 7. A digital signal corresponding to the above 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. 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.