JPS6293643A - Signal processing method for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To obtain the base sequence of nucleic acid with high accuracy, by calculating a migration speed on the basis of the temp. of a support medium in a separation/development process and correcting the strain of a migration distance due to a smiling phenomenon on the basis of the migration speed. CONSTITUTION:Constant interrelation is present between the temp. T of a support medium and the migration speed (v) of a specimen as shown by a drawing. The temp. of the gel support medium is measured by a microthermistor or an infrared thermometer. From the calibration curve of the drawing, the migration speed v(x') corresponding to each inputted measured temp. T(x') is immediately led out. Next, the position of a band is determined with respect to each band and the migration speed (v) at said position is determined. Subsequently, the position of the band is corrected on the basis of said migration speed. That is, a migration speed d(x') is calculated from the position of each band and the arithmetic operation of Formula is performed to convert said calculated distance d(x') to distance D(x') which will be considered to be inherently migrated if no smiling phenomenon is generated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Part 9f of the invention] The present invention provides 11 riU (1□-1 for sequencing) of nucleic acids.
This relates to a processing method.

[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 possessed by living organisms in order to elucidate their functions and replication mechanisms. It is said that In particular, D is responsible for specific genetic information.
It is essential to determine the base sequence of nucleic acids such as NA (or DNA fragments, etc.).

Maxam Gilbert uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
) law and Sanger-Coulson (Sanger-G.
Oulson) method is known. In the former Maxam-Gilbert method, first, a group containing a radioactive isotope such as 22p is attached to the - end of the chain molecule of the DNA or DNA fragment whose base sequence is to be determined. After converting the object into a radiolabeled substance, one chemical step is used to base-specifically cleave the bonds between each constituent center of the chain molecule. 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 S expansion pattern is visualized using, for example, an X-ray filter AL to obtain an autoradiograph, and from the obtained autoradiograph and each base-specific cutting means, the end of the chain molecule to which the radioactive element is bound is determined. By sequentially determining the bases in a certain positional relationship from the base, it is possible to determine the salt sequence of the entire target object.

In addition, the latter Sanger Tube-Luzon method uses a chemical f-stage to i ! This is a method for determining the 1u concentration from the autoradiograph in the same manner as described in 1-1 using a mixture of base-specific DNA compounds.

The applicant uses photographic materials such as X-ray film as described in 1- in an autoradiographic measurement operation used for the purpose of determining the base sequence of the nucleic acid described in 1- in a simple and highly accurate manner. Instead of traditional radiography? A patent application has been filed for a method using a radiation image conversion method using a B-transparent phosphor sheet (Japanese Unexamined Patent Publication No. 5
No. 9-83057, Japanese Patent Application No. 58-201231-).

Here, the stimulable phosphor sheet is made of stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, electromagnetic waves (excitation light) in the visible to infrared region are generated. ) can emit radiation energy as fluorescence. According to this method, the exposure time can be significantly shortened,
In addition, chemical fog, which has been a problem in the past, does not occur.Furthermore, autoradiographs of radiolabeled substances are stored in a phosphor sheet as radiation energy and then read out in chronological order as photostimulated light. Therefore, in addition to images, it is possible to display and record in any format such as symbols and numbers.

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

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

Therefore, the applicant has already filed a patent application for a method for automatically determining the base sequence of DNA by obtaining the above-mentioned autoradiograph as a digital signal and then subjecting this digital signal to appropriate signal processing. (Unexamined Japanese Patent Publication No. 59
-126527, Japanese Patent Application Laid-Open No. 59-126278, Japanese Patent Application No. 59-89615, Japanese Patent Application No. 59-140908, etc.), digital signals corresponding to autoradiographs cannot be obtained when using conventional radiation film. -H autoradiograph is visualized on the film and then read photoelectrically using reflected or transmitted light. 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 the electrophoretic pattern obtained when a radiolabeled substance is actually separated and developed on a support medium by electrophoresis. A typical example of this is the phenomenon (smiling phenomenon) in which the migration distance at both ends of the support medium is shorter than the migration distance at the center of the support medium. It is desired to automatically determine the base sequence of a nucleic acid with high accuracy by efficiently signal processing the corresponding digital signal.

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

That is, 1 the present invention is formed by spreading a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in one dimension on a support medium by electrophoresis. By performing signal processing on digital signals corresponding to autoradiographs of multiple electrophoresis columns.

In a method for determining the base sequence of a nucleic acid.

1) determining the relationship between a position in a direction perpendicular to the migration direction and the migration speed at that position based on information about the position on the support medium and the electrophoresis temperature at that position, and 2) for each band, A method for determining the base sequence of a nucleic acid, comprising the steps of determining the migration speed at the band position from the relationship between the position and the migration speed, and correcting the migration distance of the band based on the obtained migration speed. A signal processing method is provided.

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

The smiling phenomenon in the separation and development process of a sample by electrophoresis is considered to be caused by the following heat dissipation effect (edge effect) on the -shell.

Electrophoresis is carried out by applying a voltage to both ends of a gel support medium and separating and developing the sample due to the potential difference between the two ends.During the separation and development process, a current uniformly flows through the support medium, which generates heat. However, since heat is dissipated from both ends of the support medium, the temperature at both ends is lower than that at the center of the support medium. The migration speed of the sample is affected by the temperature of this support medium and is slower at both ends than at the center. As a result, in the resulting migration pattern, the migration distance at both ends of the support medium is shorter than the migration distance at the center (occurrence of the smiling phenomenon).

Note that the smiling phenomenon generally appears symmetrically in the width direction of the support medium.

As a result of research, the present inventor found that there is a certain correlation between the temperature of the support medium and the migration speed of test key 1 of 1 m. As a result, we found that the migration speed can be determined based on the temperature of the support medium during the DC opening process, and that distortions in the migration distance due to the ring phenomenon can be easily corrected based on this migration speed. , unreleased 1. ! It has reached lj.

Therefore, in the present invention, there is no need to detect distortion in migration distance using digital image data L, and it is not necessary to perform complex signal processing for this purpose. 1. Manually input the data into the signal processing circuit. By obtaining information about the actual migration temperature during signal processing, it is possible to easily calculate the deviation (correction (11'j)) in the migration distance based on the temperature difference.
The position of each band can be corrected based on this correction value σl.

By further performing appropriate signal processing on the digital signal that has been corrected for the smiling phenomenon, the base sequence of a nucleic acid can be determined easily and with high precision.

[Structure of the invention] Examples of samples used in the present invention include:

Examples include mixtures of base-specific fragments of nucleic acids such as DNA and RNA tagged with radioactive labels. here,
A fragment of a nucleic acid means a portion 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 prepared by adding a radiolabeled D
It is obtained by base-specific cleavage and decomposition of NA.

Also, a! The group-specific DNA synthesis mixture can be obtained by synthesizing the mixture using DNA as a template, a deoxynucleotide triphosphate labeled with a radioactive label, and a DNA synthase, etc., according to the Sanger φ Coulson 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. In addition, DNA consists of four types of bases as its constituent units: azonide, guanine, thymine, and cytosine, while RNA consists of four types of bases: adenine, guanine, uracil, and cytosine.

Radioactive labeling can be done in a manner appropriate for these substances: 12p,
It can be investigated by retaining radioactive diagonal elements such as IaC, 36S, 3H, and +j'.

The sample (radioactively labeled or tagged nucleic acid salt), (a mixture of specific fragments) is separated and developed on a support medium by electrophoresis using a gel support medium such as agarose gel or polyacrylamide gel. 5.

Note that these supporting media for electrophoresis may be provided with supporting aids such as glass plates, plastic sheets, and the like.

Next, an autoradiograph of the support medium on which the radiolabeled substance has been separated and developed is obtained by radiography using a conventional photosensitive material F (or by a radiation image conversion method using a strawberry-plantable phosphor sheet). A digital signal corresponding to the autoradiograph of the radiolabeled substance is then obtained via a suitable readout system.

When using the former radiographic method, first the support medium and the photographic material such as X-ray film are heated at a low temperature (-90 to -
The radiographic film is exposed to light by overlapping at 70℃ for a long time (several tens of hours), and then developed to create a visible image of the autoradiograph of the radiolabeled substance on the radiographic film.
The autoradiograph visualized on the radiographic film is then read using an image reading device. For example, an autoradiograph is obtained as an electrical signal by irradiating a radiation film with a light beam and photoelectrically detecting the transmitted or reflected light. Furthermore, by A/D converting this electrical signal, a digital signal corresponding to an autoradiograph can be obtained.

When using the latter radiation image conversion method, first, the support medium and stimulable phosphor sheet are heated at room temperature for a short period of time (several seconds to
The autoradiograph is recorded as a kind of latent image on the phosphor sheet by overlapping the phosphor sheets (for several minutes) to accumulate the radiation energy emitted from the radiolabeled substance on the phosphor sheet. Here, the stimulable phosphor sheet includes a support made of, for example, a plastic film, divalent europium activated barium fluoride bromide (BaFBr:Eu2°
) 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 accumulates the radiation energy, and is then excited by light in the visible to infrared region. Then, it has the property of emitting the accumulated 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 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.

J: For details of the above-mentioned autoradiograph measurement operation and method of obtaining a digital signal corresponding to the autoradiograph, please refer to the above-mentioned JP-A-59-83057 and JP-A-59-83057.
It is described in various publications such as No. 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 any digital signal obtained by any other method as long as it has a correspondence with the autoradiograph of the radiolabeled substance. 6 Also, it is possible to
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 sheet), but it is of course possible to read out the autoradiograph over only the image area.

Furthermore, in the present invention, readout conditions are set by inputting information about the position of each electrophoresis column and band width in advance, and in the readout operation, one or more of each band hill is set. By performing scanning with a light beam at a scanning line density such that the scanning lines pass through the image, 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 I)
), and the signal level represents the image density at that coordinate, ie, the amount of radiolabeled substance. Therefore, = the series of digital signals (ie, digital image data) has two-dimensional positional information of the radiolabeled substance.

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

An embodiment of the signal processing method of the present invention will be described with reference to a case where the electrophoresis array is formed by a combination of base-specific DNA fragments labeled with the following four types of radioactive labels.

l) Guanine (G) specific DNA fragment 2) Adenine (
A) Specific DNA fragment 3) Thymine (T) specific DNA
Fragment 4) Cytosine CC) Specific DNA fragment Here, each base-specific DNA fragment is a DNA fragment of various lengths that has been cut, degraded, or synthesized in a base-specific manner, that is, has the same terminal base. Consisting of

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

The digital signal corresponding to this autoradiograph is processed in the signal processing circuit.
or non-volatile memory such as a magnetic disk).

The signal processing circuit also contains information about the relationship between the temperature of the support medium and the migration speed. The relationship between temperature and migration speed (calibration curve) for this support medium is
By performing measurements under rotating electrophoresis conditions using a support medium similar to that in which the sample was actually separated and developed, 1! It can be found feasibly.

The temperature of the gel support medium may be measured, for example, by attaching a microthermistor to the gates plate for holding the support medium, or may be measured using a non-contact type infrared source JRI meter. A non-contact type infrared temperature sensor is preferable because it can measure the temperature of the support medium itself. Since the temperature of the support medium is usually non-uniform along the direction perpendicular to the migration direction (width direction of the support medium) due to the heat dissipation effect at both ends of the support medium, temperature measurement is performed at multiple points along this orthogonal direction. Do it in position.

For example, in the case of a gel support medium with a width of 30 cm, the temperature is measured at 1 cm intervals at the center, and narrows as it approaches both ends, and at 3 mm intervals at both ends (measurement points =
In addition, in order to increase the accuracy of the measured temperature, it is possible to measure at multiple positions in the electrophoresis direction and take the average, or to measure over time at each measurement position and calculate proportional distribution or integration. May take a value.

The electrophoresis speed can be suitably measured by separating and developing a colored reagent having a molecular weight similar to that of the base-specific DNA fragment serving as a sample on a support medium, and measuring the electrophoresis distance per time. Bromophenol blue (BPB) or xylene cyantool (XC) as a coloring reagent
Dyes such as can be used. Alternatively, it can be preferably determined by measuring an autoradiograph of a base-specific DNA fragment obtained by separation and development under the same electrophoresis conditions using a support medium similar to that in which the actual sample is separated and developed. can.

The calibration curve representing the relationship between the temperature of the support medium and the migration speed is
For example, it is represented by a straight line as shown in FIG. In FIG. 2, the horizontal axis represents the temperature T of the gel support medium, and the vertical axis represents the migration speed V of the substance. Since the temperature of the support medium is high at the center and low at both ends, the higher the temperature T in FIG. 2, the closer it is to the center of the support medium. usually,
Is the temperature non-uniformity due to heat radiation effect in the center of the support medium?
Since it is symmetrical in the width direction with respect to the center, it is possible to measure only one region of the support medium when creating a calibration curve.

In addition, as shown in Figure 2, the temperature T is in a very low range (
In other words, in the area (close to the spacer at both ends), the temperature changes greatly due to large heat dissipation, and it is also susceptible to D"W due to other factors such as air movement, making it difficult to detect lines. It tends to deviate from a straight line.Therefore, it is preferable to use a linear range as the test j-line.

Note that the calibration curve does not necessarily have to be a straight line, but can also be represented by a curve approximated by an appropriate higher-order function.

Since the migration speed depends on the length and number of bases of the DNA fragment, if the absolute value of the migration speed is plotted on the vertical axis in FIG. 2, multiple calibration curves will be obtained.

On the other hand, if we plot the migration speed ratio on the vertical axis, which is obtained by normalizing the migration speed at a certain temperature, for example, the temperature at the center of the support medium in the width direction (generally the highest temperature in the width direction). Even for groups of DNA fragments that differ somewhat in length and number of bases, the calibration curves almost match. For bands with significantly different DNA fragment lengths (number of bases), it is preferable to separately obtain a calibration curve as necessary.

During the electrophoresis process of the sample L, the temperature of the gel support medium can be measured in the same manner as in the preparation of the calibration curve. At this time, it is necessary to clarify the measurement position of the support medium 1. For example, measurements are taken at positions corresponding to the positions of each slot.

In digital signal processing, first, information about the temperature of the support medium during the migration process of the sample and the position indicating the temperature [T (x');x' represents the position along the direction perpendicular to the migration direction of the support medium. Squirrel] is input to the signal processing circuit. Information can be entered manually or automatically, since there must be a pairwise correspondence between the measurement position and the coordinate system of the digital signal.
The position of the support medium must be specified by the digital signal 5 obtained by attaching a marker with an appropriate radioactive label to the support medium or by exposing the support medium to light while fixing its position. Alternatively, a measurement position may be determined in advance and this measurement position may be incorporated into the signal processing circuit.

From the test line 11 regarding the temperature T and the migration speed V, the migration speed v corresponding to each input measurement temperature T (x')
(x') is immediately derived. This allows for position 2 along the direction perpendicular to the electrophoresis direction. x' and migration speed v
(x') can be found. Here, the influence of the heat dissipation effect is usually small near the center of the support medium, and the temperature difference occurs symmetrically in the width direction of the support medium, so the position The center is the reference position (x' = O), and the position is positive (+
), and it is preferable to set the left direction as negative (-).

Next, for each band, the band position is determined, and the migration speed V at that position is determined.

The position of the band can be determined, for example, by scanning along the electrophoresis direction at a scanning line density such that each band has a plurality of scanning lines as described above. ), then detect the position (peak position) where the signal level is maximum on each -dimensional waveform, and determine the position where the signal level at the peak position is maximum among multiple one-dimensional waveforms. the position of the band (
x, y). Alternatively, the peak position of the one-dimensional waveform corresponding to the center position of each electrophoresis column can be used as the band position.

For details on the method of determining the band position by digital signal processing, please refer to Japanese Patent Application No. 181432, filed on August 19, 1985 by the applicant,
This is stated in each specification of Japanese Patent Application No. 1983 filed on October 11th.

The migration speed at the determined band position (X) can be immediately derived from the relationship between the already determined position 2iX' and the velocity v (x').

Next, the position of the band is corrected based on this migration speed.

For example, the migration speed v at the reference position 2t (x'=o)
(0), and the position of each band can be corrected based on the ratio of the migration speed at the reference position and the migration speed at the band position. That is, the migration distance d (x') is calculated from the position (y) of each band, and for this migration pressure gld (X'), D (x')=d (x')xv (0)/v
By performing the calculation consisting of (x'), it can be converted into the migration distance at the reference position. Obtained D
(x') represents the distance that would have migrated if the smiling phenomenon had not occurred.

By converting the migration distance for all bands in the migration pattern to the migration distance at the reference position, the band position can be expressed by the corrected coordinates [x', D (x')], and in this way, the smiling phenomenon It is possible to correct the distortion of migration distance caused by

If each lane is straight and parallel to the electrophoresis direction, the electrophoresis distance of the band can be corrected for each lane at once. Furthermore, when temperature measurement is performed at each slot position, the migration speed of each lane can be derived directly based on the L-diameter m-line, which further simplifies signal processing for smile correction. can.

Note that in the present invention, if offset distortion and/or band fusion occur in the electrophoretic pattern, these corrections may be made before or after subjecting the digital signal to the above-described signal processing.

Here, the offset distortion refers to an overall positional shift between lanes, and is caused by the fact that the starting position of sample electrophoresis differs in each slot due to differences in the shape of the slots. Furthermore, fusion of bands means that two or three bands are connected to form a wide punt because the electrophoresis is not 1 minute. - Generally tends to occur in the region near the migration start position of the L portion of the pattern.

To correct offset distortion, in general, the spacing between bands is sparse in the dot region of an electrophoresis pattern, so first detect multiple bands in the dot region for each lane, and then sequentially number the bands from the dot end. After that, a correlation (for example, a regression line) between the band number and its migration distance is obtained. From this correlation, calculate the difference in migration distance between lanes,
The positional deviation between lanes can be corrected all at once by shifting the migration position of each lane as a whole.

Alternatively, offset distortion can be corrected piecewise for each lane.

To correct the fusion band, first, a plurality of bands are continuously detected in the F region of each lane, and serial numbers are assigned to the bands in order from the very edge, and then a correlation between the distance between the bands and the band number is obtained. Based on this correlation, the position of the undetected band is determined by Y full, and a new band is detected based on this Y=am position, thereby making it possible to separate the band into one individual band. Prediction of band positions may be performed all at once, or may be performed piecewise.

For details of these corrections by signal processing, see Japanese Patent Application No. 60-85275 by the present applicant.

Patent Application No. 1986-85276, Patent Application No. 111186/1983
and Japanese Patent Application No. 60-111187.

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

In this way, the base sequence of one chain molecule of DNA can be determined. Note that the information about the DNA base sequence is not limited to the display format shown in L. For example, if desired, the information about the intensity of each band (2
') can also be expressed as the relative amount of radiolabeled substance.
There are seven abilities. Furthermore, it is also possible to display the base sequence of both edges of a single chain molecule of DNA.

Alternatively, it is also possible to display the image as an image based on a digital signal that has been subjected to the smile correction described in L.

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

Note that in the embodiment described above, a mixture of base-specific DNA fragments as a sample (G.

Although the case where an exclusive combination of (A, T, C) is used has been described, the signal processing method of the present invention is not limited to this M1 combination; for example, (G, G+A, T+C1C
) can be applied to various combinations. Similarly, a mixture of base-specific RNA fragments (for example, G,
The signal processing method of the present invention can also be applied to combinations of A, U, and C). Furthermore, the correction of the smiling phenomenon is not limited to the separation and development array of base-specific fragments of nucleic acids of the set, but can be performed for all separation and development arrays that are simultaneously separated and developed on the support medium. It is.

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

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

Recording devices include, for example, those that optically record by scanning a photosensitive material with a single laser beam, those that record the symbol φ value 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 heat-sensitive recording materials.

[Brief explanation of drawings]

FIG. 1 is a partial diagram showing an example of a separation development pattern in which a smiling phenomenon occurs. FIG. 2 is a graph showing the relationship between the temperature T of the support medium and the migration speed V. Figure 3 shows the coordinate system [X'
, d (X')].

Claims (1)

[Claims] 1. A mixture of base-specific DNA fragments or base-specific RNA fragments to which a radioactive label has been added is separated and developed in one-dimensional direction on a support medium by electrophoresis. In a method for determining the base sequence of a nucleic acid by performing signal processing on digital signals corresponding to autoradiographs of a plurality of electrophoresis columns, (1) the position on the support medium and the electrophoresis temperature at that position are determined. (2) determining the relationship between the position in the direction perpendicular to the electrophoresis direction and the electrophoresis speed at that position based on the information; and (2) calculating the electrophoresis speed at the band position from the relationship between the position and the electrophoresis speed for each band. 1. A signal processing method for determining the base sequence of a nucleic acid, the method comprising: correcting the migration distance of a band based on the obtained migration speed. 2. In the first step, the relationship between a position in a direction perpendicular to the electrophoresis direction and the electrophoresis speed at that position is determined based on the known relationship between the electrophoresis temperature and the electrophoresis speed. A signal processing method for determining the base sequence of a nucleic acid according to item 1. 3. In the second step, the center of the support medium is used as a reference position, the ratio of the migration speed at this reference position and the migration speed at each band position is determined, and the migration distance of the band is calculated based on the obtained ratio. 2. A signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that the signal processing method is corrected. 4. In the second step, the electrophoresis speed at that position is determined for each electrophoresis column, and the electrophoresis distance of the bands is collectively corrected based on the obtained electrophoresis speed. or the signal processing method for determining the base sequence of a nucleic acid according to item 3. 5. A mixture of the above base-specific DNA fragments. It consists of four types: (1) guanine-specific DNA fragments, (2) adenine-specific DNA fragments, (3) thymine-specific DNA fragments, and (4) cytosine-specific DNA fragments. The nucleic acid base sequencing method according to claim 1 is characterized in that it consists of four electrophoretic rows formed by separating and developing these four types of base-specific DNA fragments on a support medium, respectively. signal processing methods for. 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 .