JPS63191959A - Signal processing method for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To detect only the true band by determining whether the peak detected in the direction perpendicular to a sepn. development direction is the peak to indicate the true band or noise on the many rasters formed with one sepn. development array. CONSTITUTION:Two or more one-dimensional waveforms (rasters) consisting of the position along a migration direction and the level of the signal are formed with one sepn. development array. The peaks are then detected on all the rasters. Whether the peak exists or not within the specified range centering at the peak of one peak on one raster on the adjacent rasters is then retrieved with respect to said one peak. the retrieval of the peak on the adjacent rasters is thereafter successively repeated as far as the peak is found out. Determination is made that the band exists in the positions of a series of the peaks, if the peaks are detected on plural pieces of the rasters adjacent to each other. The base sequence of nucleic acid is thus easily and automatically determined with high accuracy.

Description

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

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

Maxim Gilbe uses autoradiography as a typical method to determine the base sequence of nucleic acids such as DNA and RNA.
rt) method and Sanger-Kuhlen (Sanger
-C (Oulson) method is 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 turning a substance into a radioactively labeled substance, the bonds between each constituent unit of a chain molecule are base-specifically cleaved using chemical means.Next, base-specific DNA cleavage and decomposition obtained by this operation is performed. A mixture of substances is separated and developed by gel electrophoresis, and a separated development pattern (however, it cannot be seen visually) is obtained by separating and developing a large number of cleavage products. This separation development pattern is visualized on, for example, an X-ray film to obtain its autoradiograph, and from the obtained autoradiograph and each base-specific cutting means, the end of the chain molecule to which the radioactive isotope is bonded is determined. By sequentially determining the bases in a certain positional relationship from the base, it is possible to determine the base sequence of the entire target object.

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

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

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. ) of the separation expansion position δ(
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 patented a method for automatically determining the base sequence of DNA by performing continuous digital signal processing on the digital image data obtained from the above-mentioned autoradiograph as digital image data. Applications have been filed (Japanese Unexamined Patent Publication No. 126527/1982, Japanese Patent Application No. 8/1983
No. 9615, patent application No. 1983-226091, patent application No. 1983
-226092, etc.), digital image data corresponding to autoradiographs is obtained by first visualizing the autoradiograph on the film when using conventional radiation film, and then using reflected light or transmitted light. It can be obtained by reading it photoelectrically. Furthermore, 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, the sample may be contaminated with radioactive impurities, or it may be exposed to natural radiation during exposure. Noise may appear on the autoradiograph. As a result, since bands are comparatively identified including 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 perform signal processing on digital image data including the autoradiograph to automatically determine the base sequence of a nucleic acid with high precision.

[Summary of the Invention] Even when noise occurs in an autoradiograph of a separated development pattern consisting of a radioactively labeled substance, the present inventors have demonstrated the ability to detect nucleic acids by applying suitable digital signal processing to the digital image data. We have achieved automatic determination of base sequences with ease and high accuracy.

That is, the present invention provides automated separation and development patterns formed by spreading a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments on a support medium in a one-dimensional direction. A method for determining the base sequence of a nucleic acid by performing digital signal processing on digital image data including a radiograph,
1) Obtaining at least two one-dimensional waveforms consisting of positions along the separation development direction and signal levels for one separation development sequence, 2) Detecting peaks on all one-dimensional waveforms, 3) A step of searching for one peak on one one-dimensional waveform to see if a peak exists within a certain range centered on the position of the peak on an adjacent one-dimensional waveform, 4) In the third step If a peak exists, repeating the third step on adjacent one-dimensional waveforms in sequence; 5) In the third and fourth steps, peaks are detected on a plurality of mutually adjacent one-dimensional waveforms; If so, it is determined that a band exists at the position of a series of peaks, and in other cases, it is determined that a band does not exist at the position of the peak, and 6) repeating the above third to fifth steps in sequence. Accordingly, the present invention provides a signal processing method for determining the base sequence of a nucleic acid, which comprises the step of detecting all bands on a separation and development array.

According to the present invention, separated U13t! obtained by separating and developing a mixture of base-specific fragments of nucleic acids on a support medium! In digital image data including pattern autoradiographs, nucleic acid can be extracted by passing it through a processing circuit with a signal processing function that can eliminate the noise and detect only the true band, even if noise occurs in the separated and expanded pattern. The base sequence of can be obtained easily and with high accuracy.

Simply detecting a peak on a one-dimensional waveform (raster) consisting of the position and signal level along the separation development direction of a separation development sequence tends to misidentify noise as a band in addition to the true band. Therefore, accurate base sequence information cannot be obtained. Typically, the band has a width in a direction perpendicular to the direction of separation development depending on the size of the slot.

On the other hand, noise that appears on an autoradiograph due to incomplete introduction of radioactive isotopes during the sample preparation process or contamination of natural radiation during the exposure process may be in the form of spots or even bands. Its width is narrower than a true band.

According to the present invention, a large number of rasters are created for one separation development column, and detected peaks are determined based on whether peaks exist continuously on these rasters in the direction perpendicular to the separation development direction. By determining whether this is a peak indicating a true band or noise, it is possible to exclude noise and detect only the true band.

By comparing the bands between the separation and development columns based on the position of the detected band, 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.

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

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

The radioactive label can be added to 32p, + by a method appropriate to these substances.
It is provided by retaining radioactive isotopes such as 4q, 418S, 3H, and 12sI.

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. The mixture is developed on a support medium by various separation and development methods such as photography.

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 is obtained by a radiation image conversion method using a stimulable phosphor sheet, and then digital image data including an autoradiograph of the radiolabeled substance is obtained via an appropriate readout system.

When using the former radiographic method, first, a support medium and a photographic material such as X&lit film are superimposed at low temperature or room temperature for a period of time (several hours to several tens of hours), and the radiation film is then exposed. The autoradiograph of the radiolabeled substance is developed into a visible image on the radiographic film. The autoradiograph visualized on the radiographic film is then read using an image reading device. For example, an autoradiograph is obtained as an electrical signal by irradiating a radiation film with a light beam and photoelectrically detecting the transmitted or reflected light. Further, by A/D converting this electrical signal, digital image data including 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, a phosphor layer made of a stimulable phosphor such as divalent europium-activated barium fluoride bromide (BaFBr: Eu2), and a transparent protective layer. The films are stacked 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. in particular,
For example, by scanning a phosphor sheet with a laser beam to emit radiation energy as photostimulated light, and detecting this photostimulated light photoelectrically, radiotradiographs of radiolabeled substances can be obtained without creating a visible image. Obtained directly as an electrical signal. Further, by A/D converting this electrical signal, digital image data including an autoradiograph can be obtained.

For details of the above-mentioned autoradiograph measurement operation and method of obtaining digital image data including the autoradiograph, please refer to the above-mentioned JP-A-59-83057 and JP-A-Sho.
It is described in various publications such as No. 9-126527 and Japanese Unexamined Patent Publication No. 126278-1982.

In addition, in the above, a method using conventional radiography and radiographic image conversion methods was described as a method for obtaining digital image data including 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 image data obtained by any other method as long as it includes an autoradiograph of a radiolabeled substance. It is possible to run one race.

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

Furthermore, read conditions are set by inputting information about the position of each separation expansion column, band width, etc. in advance, and in the read operation, two or more scanning lines are passed on each band. Reading (reading) is performed by scanning with a light beam at a scanning line density such that the
You can save time and efficiently obtain the necessary information. Note that in the present invention, digital image data including autoradiographs also includes digital signals obtained in this manner.

The obtained digital image data is a digital signal oxy consisting of coordinates (x, y) expressed in a coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (2) at the coordinates. 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 digital image data has two-dimensional positional information of the radiolabeled substance.

The digital image data including the autoradiograph of the radiolabeled substance on the support medium thus obtained is subjected to signal processing according to the method of the present invention as described below to determine the base sequence of the target nucleic acid. 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.

1) 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 DNA fragment of various lengths that has been cleaved, degraded, or synthesized in a base-specific manner, that is, has the same terminal base. consists of things.

FIG. 1 shows an example of an autoradiograph of the electrophoresis pattern in which the four types of base-specific DNA fragments are electrophoresed into four slots, respectively, and the electrophoresis direction is the X direction.

Digital image data including this autoradiograph is temporarily stored in a memory (buffer memory or nonvolatile memory such as a magnetic disk) in a signal processing circuit.

Normally, digital image data obtained by reading a radiation film or a stimulable phosphor sheet contains image information not only about the migration pattern but also about the entire surface of the film or phosphor sheet, and includes areas other than the image area (pattern area). Contains extra digital data. Therefore, prior to the signal processing for base sequencing of the present invention, in order to improve the quality of the processing and improve the processing efficiency, it is necessary to extract only the image data corresponding to the image region from the obtained digital image data. It is desirable to do this (so-called "cutting out of image area J").

For example, image data corresponding to a migration pattern can be extracted by the following signal processing. First, the obtained image data is transferred in the direction perpendicular to the migration direction (X direction).
By dividing the image data into two or more blocks along the , and then adding the image data for each block along the separation development direction (X direction), a one-dimensional waveform (projection) consisting of the position in the X direction and the signal level is created. Next, by detecting the boundaries of the electrophoresis pattern area and furthermore the boundaries of each electrophoresis column (lane) from the projection of each block, and interpolating these boundaries between blocks, the The boundaries of the entire migration pattern area and lanes can be determined. Similarly, boundaries in the length direction (X direction) can also be determined.

The details of the signal processing method for cutting out the image area are described in the specification of Japanese Patent Application No. 1983 filed on December 27, 1988 by the present applicant.

Alternatively, after electrically displaying an autoradiograph on a screen such as a CRT based on the obtained digital image data, the migration pattern area can be determined by operating a cursor, mouse, etc. on the screen, or by operating a keyboard. It can be entered as information.

Note that since the approximate position of the boundary in the length direction is known based on the injection position (slot) of the sample, electrophoresis conditions, etc., it may be set in advance as fixed coordinates.

First of all, two or more one-dimensional waveforms (raster) consisting of positions along the migration direction and signal levels are created for each migration pattern 1 (lane) based on image data corresponding to the migration pattern area. .

If the digital image data was read by scanning along the migration direction at a line density such that each band has at least two scan lines, as described above, each band can be directly scanned. About the scanning line! (y)
and the signal level (2). If the entire autoradiograph has been read, a raster is created after signals are extracted along each lane by scanning the image data in the same way as above.

The number of rasters per lane depends on the slot shape (width)
Although it varies depending on the amount of sample and the number of pixels, it is generally in the range of 5 to 40 from the viewpoint of band detection accuracy. - As an example, the slot width is 5 mm and the pixel spacing is 0.
．． 2mm, and the raster interval is also 0.2mm (rasters are created in pixel units), the number of rasters per lane is 25.
It is individual.

FIG. 2 partially shows a number of rasters created for the first lane, in other words the rasters
This corresponds to a concentration cross-sectional diagram when cut at each position in the direction.

Second, detect peaks on all rusks.

The peak can be detected, for example, by determining the point at which the sign of the difference in signal levels is reversed.

In FIG. 2, arrows (↓) each indicate a peak.

Third, focusing on one peak on one rask, it is searched whether or not a peak exists within a certain range centered on the position of the peak on an adjacent rask. Fourth, as long as a peak is found, the peak search is repeated on successively adjacent rusks.

Here, the fixed range centered on the peak position is usually a range of Ya±σmm centered on the peak position Va, and σ is determined depending on the raster interval. - As an example, when the raster spacing is 0.2 mm, σ=0.4. By limiting the range of peak search, a band is determined only when a peak without a large shift in the electrophoresis direction exists in a plurality of rasters, and the accuracy of band detection can be improved.

Further, the peak search may be performed on one adjacent raster, or may be performed on two or more rasters at the same time. For example, it is possible to search on 2 to 5 rasters at the same time. This simultaneous search for a plurality of rasters is a suitable method when there is a break in the peak, which will be described later.

FIG. 3 shows the presence of peaks searched for each raster in FIG. The X mark represents the peak position.

Fifth, the continuity of the searched peaks is determined, and based on this determination, it is determined whether a series of peaks within a certain range (ya±σ) is a band.

Peak continuity can be determined by band width (slot width)
Generally speaking, if beat is continuously detected in N or more rasters, it can be determined that a band exists at the position of a series of peaks, although this varies depending on the shape of the pattern, raster spacing, etc. Here, N is when the slot width is 5 mm and the raster spacing is 0.2 mm (
The number of rasks per lane: 25) is usually 13≦N≦, considering that the band width expands and contracts during electrophoresis.
is an integer in the range of 37.

Preferably, it is determined that a band exists when peaks are consecutively detected in N or more and M or less rasters. ”
- In the case of the above conditions, for example, N=15 and M=3
It can be set to 0.

Furthermore, if the peaks are dull or the signal level is locally low, a series of peaks may not necessarily be detected consecutively between rusks. Therefore,
In a series of rasters for which a peak search has been performed, if peak discontinuities (existence of rasters in which no peak is detected) are within a certain range, it is desirable to determine that this is also a band. For example, if the peak breaks are at L points or less,
If the number of rasters in which no peak is detected at each interrupted location is equal to or less than 3, it is determined that a band exists at the series of peak positions. Here, K and L are determined depending on the raster interval, for example, when the raster interval is 0.2 mm, 1≦≦3 and 1
It is an integer in the range of ≦L≦3.

In cases other than the above, it is determined that no band exists at the peak position.

FIG. 4 is another example showing the existence of peaks searched for a plurality of rasters (x mark: peak position).

In FIG. 4, it is determined that the series of peaks A and E are each a band, and the remaining peaks B, C and D are not bands but noise.

The above operation is performed for all peaks to detect all bands on the electrophoresis pattern. In this way, bands can be detected with high accuracy even when noise occurs in the electrophoresis pattern.

Note that, although the case where the image area is cut out in units of migration patterns has been described above, it is also possible to first cut out the image area in units of lanes and repeat the above-mentioned operation for each cut out lane.

In addition to noise, if various distortions such as smiling phenomenon, offset distortion, or band fusion occur in the electrophoresis pattern, perform signal processing to correct these after detecting the above bands. It's okay.

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 occurs when two or three bands connect to form one wide band due to insufficient migration, and generally occurs in the area near the migration start position at the top of the pattern. It's easy to do.

For details of these corrections by signal processing, please refer to Japanese Patent Application Nos. 60-74899 and 1986-74 filed by the applicant.
No. 900, Japanese Patent Application No. 85275/1983, Japanese Patent Application No. 85/1983
No. 5276, patent application No. 111186, patent application No. 1983
-111187.

The DNA base sequence can be immediately determined by comparing the positions of the detected bands with each other and ranking the bands. At this time, the above four types of base-specific DN
Since the combination of A fragments is an exclusive combination, the order can be easily determined by taking advantage of the fact that two or more bands cannot exist at the same position on different lanes. Each of the slots (1) to (4) above is (G)
, (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 of the two chain molecules of I) NA.

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

In addition, in the above, a case was explained in which an exclusive combination of 8 complexes of base-specific DNA fragments as a sample and 1, te (G, A, T, C) was used, but the signal processing of the present invention The method is not limited to this combination, but can be applied to various combinations such as (G, G+A, T+C, C). 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, band detection 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 single laser beam, those that record symbols and numbers displayed on a CRT, etc., on a video fist printer, and those that use heat rays. Recording devices based on various principles can be used, such as those that record on heat-sensitive recording materials.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of an autoradiograph of an electrophoresis pattern in which noise occurs. FIG. 2 is a partial diagram showing a plurality of rasters for the first lane. FIG. 3 is a partial diagram showing the presence of peaks searched for the raster shown in FIG. FIG. 4 is a partial diagram showing the presence of peaks searched for different rasters. Arrow (↓): Beak, × mark: Beak position, patent applicant
Representative of Fuji Photo Film Co., Ltd. Patent attorney
Yasushi Yanagawa Figure 1 (+) (2) (3) (4) omitted, =,
・e Figure 3 ABC:DE

Claims (1)

[Scope of Claims] 1. A separation pattern formed by separating and developing a mixture of base-specific DNA fragments or base-specific RNA fragments on a support medium in a one-dimensional direction on a support medium. In a method for determining the base sequence of a nucleic acid by performing digital signal processing on digital image data including an autoradiograph, 1) With respect to one separation development column, from the position and signal level along the separation development direction. 2) Detecting peaks on all the one-dimensional waveforms; 3) For one peak on one one-dimensional waveform, detecting the peak on the adjacent one-dimensional waveform. 4) If a peak exists in the third step, repeat the third step on adjacent one-dimensional waveforms in sequence. Step 5) If peaks are detected on multiple one-dimensional waveforms adjacent to each other in the third and fourth steps, it is determined that a band exists at the position of the series of peaks; is a step of determining that no band exists at the position of the peak, and 6) by sequentially repeating the third to fifth steps above,
A signal processing method for determining the base sequence of a nucleic acid, comprising the step of detecting all bands on a separation and development column. 2. Before the first step, the image data corresponding to the separated development pattern region is extracted from the digital image data, and the first step to the sixth step are performed on the image data corresponding to the pattern region. A signal processing method for determining the base sequence of a nucleic acid according to claim 1. 3. Before the above first step, by extracting image data corresponding to each separation expansion column from the digital image data,
The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that the first to sixth steps are repeatedly performed on image data corresponding to the separation and development column for each separation and development column. . 4. The mixture of the above base-specific DNA fragments includes (1) guanine-specific DNA fragment, (2) adenine-specific DNA fragment, (3) thymine-specific DNA fragment, and (4) cytosine-specific DNA. These four types of base-specific DNA fragments are separated and developed on a support medium, and the separation and development pattern consists of four separate and development rows formed by separating and developing these four types of base-specific DNA fragments, respectively. A signal processing method according to claim 1. 5. The digital image data including the autoradiograph is obtained by superimposing a support medium and a stimulable phosphor sheet containing a stimulable phosphor to generate an autoradiograph of the radiolabeled substance on the support medium. The autoradiograph is obtained by accumulating and recording on a phosphor sheet and then photoelectrically reading out the autoradiograph as photostimulated light by irradiating the phosphor sheet with excitation light. The signal processing method according to item 1. 6. The digital image data including the autoradiograph is obtained by superimposing a support medium and a photographic light-sensitive material, photosensitively recording an autoradiograph of the radiolabeled substance on the support medium on the photosensitive material, and then transferring the digital image data to the photosensitive material. Claim 1 is obtained by photoelectrically reading the autoradiograph visualized above.
Signal processing method described in section.