JPH0467918B2 - - Google Patents

Description

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

[Background of the invention] In the field of molecular biology, which has developed rapidly in recent years, it is essential to clarify the genetic information of living organisms in order to elucidate their functions and replication mechanisms. It has become commonplace. Above all,
It has become essential to determine the base sequence of nucleic acids such as DNA (or RNA fragments, hereinafter the same) that carry specific genetic information.

Maxam Gilbert uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
-Gilbert method and Sanger-Coulson method are known. The former Maxam-Gilbert method first attaches a group containing a radioactive isotope such as ^32P to one end of a chain molecule of DNA or DNA fragments whose base sequence is to be determined. After making a substance into a radioactive labeling substance, chemical means are used to base-specifically cleave the bonds between each constituent unit 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 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 cutting means, bases located in a certain positional relationship from the end of the chain molecule to which the radioactive element is bound are sequentially determined, thereby determining the base sequence of all the target objects. can be determined.

In addition, the latter Sanger-Coulson method is
A radioactively labeled DNA compound that is complementary to a DNA or RNA fragment chain molecule is synthesized in a base-specific manner 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 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 emitted.
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, an autoradiograph of a radiolabeled substance is once stored as radiation energy in a phosphor sheet and then read out in chronological order as photostimulated light, so it can be displayed and recorded in any format such as symbols and numbers in addition to images. Is possible.

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

Additionally, because it relies on the human eye, there are limits to the accuracy of the information that can be obtained, such as the fact that the base sequence of a nucleic acid determined by analyzing an autoradiograph differs depending on the analyst.

Therefore, the applicant has already filed a patent application for a method for automatically determining the base sequence of DNA by obtaining the above-mentioned autoradiograph as a digital signal and then subjecting this digital signal to appropriate signal processing. (Unexamined Japanese Patent Application No. 126527, No. 126527, Unexamined Japanese Patent Publication No. 59-
No. 126278, Patent Application No. 89615, Patent Application No. 1987-
140908 etc.). When using a conventional radiation film, the digital signal corresponding to the autoradiograph can be obtained by first making the autoradiograph into a visible image on the film and then photoelectrically reading it using reflected or transmitted light. can get. When a stimulable phosphor sheet is used, an autoradiograph can be obtained by directly reading out the phosphor sheet on which the stimulable phosphor sheet has been stored.

However, various distortions and noises tend to occur in separation and development patterns obtained by actually separating and developing radiolabeled substances on a support medium by electrophoresis or the like. A typical example of this is the phenomenon in which the separation distance at both ends of the support medium is shorter than that at the center (smiling phenomenon).
There is. The smiling phenomenon occurs due to heat dissipation (so-called edge effect) during the separation and expansion process. Even if such distortion 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 of a separated development pattern in which the smiling phenomenon occurs, it has been possible to automatically determine the base sequence of a nucleic acid easily and with high precision.

That is, the present invention provides base-specific DNA fragments or base-specific RNAs to which a radioactive label has been added.
Determining the base sequence of a nucleic acid by performing signal processing on digital signals corresponding to autoradiographs of multiple separation and development columns formed by separating and developing a mixture of fragments in one-dimensional direction on a support medium. The method includes: (1) detecting the position and slope of at least one band with respect to the separation and expansion direction for each separation and expansion column; (2) from the position and slope of the band, formula (1) { η(x) = e ^-a a=∫ ^x ₀ tanθ(x)/D(x)dx (1) (where x is the distance from the center point in the width direction of the support medium, and D(x) is the band at position x , tanθ(x) is the slope of the band at position x, and η(x) is the separation expansion speed correction coefficient at position x). and (3) in each separation expansion column, based on the separation expansion speed correction coefficient, formula (2): D ₀ (x)=D(x)/η(x) (2 ) (where D ₀ (x) is the smiley-corrected separation distance at position x); correcting the separation distance of the column by The present invention provides a signal processing method.

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

Normally, in a separation and development pattern where a smiling phenomenon occurs, the separation and development speed of the sample is slower as it approaches the edge in the width direction of the support medium, and therefore the separation and development distance is shorter. Each band on the separation development row (band-shaped separation development region long in the width direction) is not perpendicular (that is, horizontal) to the separation development direction, but has an inclination depending on the degree of the smiling effect. The present inventor focused on the slope of this band and theoretically found that there are factors specific to the support medium (i.e., physical quantities that do not depend on the separation and development time and the number of bases in base-specific fragments of nucleic acids). This has been confirmed experimentally, and a method has been found to suitably and easily correct the smiling effect based on this factor (correction coefficient). Then, by further performing appropriate signal processing on the smiling-corrected digital signal, the base sequence of a nucleic acid can be determined easily and with high precision.

[Structure of the Invention] Correction of the smiling effect in the present invention is based on the theory described below.

First, it is assumed that there is an inherent factor in the support medium itself that causes the smiling effect, and this factor depends on the separation development position of the sample, that is, the distance (x) from the center point in the width direction of the support medium. can be expressed as a physical quantity (η(x)) that does not depend on the separation/development time or the number of bases contained in the sample. Further, as a second assumption, the slope of the band on the support medium (θ(x)) is caused by the smiling effect and has a correlation with this factor.

Therefore, this physical quantity η(x) can be expressed as a separation/deployment speed correction coefficient in relation to the separation/deployment speed v(x) as follows.

v ₀ (x)=v(x)/η(x) (3) (where, v(x) is the separation and development velocity of an arbitrary band at position x in the width direction of the support medium, and v ₀ (x ) is the smiling-corrected separation deployment velocity at the position.) In equation (3), the corrected separation deployment velocity v ₀ (x)
represents the hypothetical rate of separation development that the band at position x would have exhibited had the smiling effect not occurred.

The smiling effect generally refers to a phenomenon in which the distance of separation and development becomes shorter as it approaches both ends in the width direction compared to the center of the support medium. The separation and development speed is maximum in the center of the support medium without being affected by the edge effect, and is minimum at both ends. Therefore, the corrected separation and development velocity vo(x) in equation (3) can be considered to be equal to the actual separation and development velocity v(0) at the central position of the support medium (x=0).
Further, the separation and deployment speed correction coefficient η(x) satisfies the following conditions: η(0)=1, η≦1.

Therefore, the separation development distance D(x) is D(x)=v(x)×t=v ₀ (x)×η(x)・t...(4) (However, D(x) is the position is the separation distance of the bands at x, and t is the separation time. Also, if the smiling-corrected separation development distance corresponding to this D(x) is D ₀ (x), then
D ₀ (x) is expressed as follows.

D ₀ (x)=v ₀ (x)×t=D(x)/η(x) (2) Similarly, separation development distance D (x+Δx) at position x+Δx, which is moved from position x by Δx in the width direction
is expressed as follows.

D(x+Δx)v(x+Δx)・t =v ₀ (x)・η(x+Δx)・t (5) As shown in Figure 2, if the positions x and x+Δx are placed so that they span one band, these The angle θ between the line segment connecting the peak points of the signal level of the band at the position and the perpendicular line in the direction of separation development.
can be expressed as tanθ(x)={D(x)−D(x+Δx)}/Δx (6). By substituting equations (4) and (5) into equation (6), tanθ(x)/v ₀ (x)・t = −{η(x+Δx)−η(x)}/Δx (7) can get.

Here, the right side of equation (7) is, when Δx→0, −lim{η(x+Δx)−η(x)}/Δx = −dη(x)/dx=−η′(x) (8) It can be expressed as Moreover, since v ₀ (x)·t=D(x)/η(x) from equation (4), equation (7) can be expressed as follows.

tanθ(x)/D(x)=−η′(x)/η(x) ……(9) As is clear from equation (9), the value obtained by dividing the slope of the band by its separation expansion (left side ) has nothing to do with the number of bases in the base-specific fragment of the sample nucleic acid or its separation distance, but simply the position x in the width direction.
The value determined by

Generally, since η′/η=(log _e η)′, equation (9) can be expressed as −(log _e (x))′=tanθ(x)/D(x) ……(10) I can do it.

tanθ(x) and D(x) on the right side of (10) are values that can be obtained by actual measurement from the separation and development pattern of the actual sample. For example, if the actual measured values are expressed in a graph, the graph will be as shown in FIG.

FIG. 3 is a graph in which the horizontal axis represents the position x in the width direction, and the vertical axis represents tanθ(x)/D(x).

In equation (10), since log _e η(0), −log _e η(x)=∫ ^x ₀ tanθ(x)/D(x)dx ...(11) That is, η(x)=e ^-a a=∫ ^x ₀ tanθ(x)/D(x)dx (1). Therefore, in the graph of Figure 3, 0 to x
By calculating the accumulation of tan θ(x)/D(x) (area of the shaded portion), the separation and expansion speed correction coefficient η(x) can be determined.

For example, if the cumulative value for the separation development row of the i-th slot (the distance from the center point of the support medium is x _i ) is: mg ^xi ₀ tanθ(x)/D(x)dx = a _i , The separation deployment speed correction coefficient and the corrected separation deployment distance are expressed as η(xi)=e ^-ai D ₀ (xi)=D(xi)/η(xi) from equations (1) and (2) above. be able to. Therefore, the smiling effect can be corrected by extending the separation distance of the i-th slot separation expansion column by 1/η(xi).

The signal processing method of the present invention will be explained below.

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 DNA as a template and a radioactively labeled deoxynucleotide triphosphate according to the Sanger-Coulson method described above.
It can be obtained by synthesis using DNA synthase.

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

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

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

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

When using the former radiographic method, first the support medium and a photographic material such as X-ray film are superimposed at a low temperature (-90 to -70°C) for a long time (several tens of hours) to expose the radiation film. After development, an autoradiograph of the radiolabeled substance is visualized on the radiographic film. Next, the autoradiograph visualized on the radiographic film is read using an image reading device. For example, an autoradiograph is obtained as an electrical signal by irradiating a radiation film with a light beam and photoelectrically detecting the transmitted or reflected light. moreover,
By A/D converting this electrical signal, a digital signal corresponding to the autoradiograph can be obtained.

When using the latter radiation image conversion method,
First, the support medium and the stimulable phosphor sheet are overlapped for a short time (several seconds to several tens of minutes) at room temperature, and the radiation energy emitted from the radiolabeled substance is accumulated in the phosphor sheet. is recorded on the phosphor sheet as a kind of latent image. Here, the stimulable phosphor sheet is made of a support made of, for example, a plastic film, divalent europium-activated barium fluoride bromide (BaFBr: Eu ²⁺ )
A phosphor layer made of a stimulable phosphor such as phosphor and a transparent protective film are laminated in this order. The stimulable phosphor contained in the stimulable phosphor sheet absorbs and accumulates radiation energy when it is irradiated with radiation such as X-rays, and then accumulates when excited with light in the visible to infrared region. It has the property of emitting radiation energy as photostimulated light.

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

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

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

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

Furthermore, in the present invention, reading conditions are set by inputting information about the position of each separation expansion column, band width, etc. in advance, and two lines are read on each band in the reading operation. Reading is performed by scanning with a light beam at a scanning line density that allows the scanning lines above to pass through.
You can save time and efficiently obtain the necessary information. 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 Dxy consists of coordinates (x, y) expressed in a coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (z) at the coordinates. The level of the signal represents the image density at that coordinate, ie, the amount of radiolabeled substance. Therefore, the series of digital signals (ie, digital image data) has two-dimensional positional information of the radiolabeled substance.

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

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

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

FIG. 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. In FIG. 1, a smiling phenomenon occurs in the migration pattern.

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, the position of at least one band on each electrophoresis column (lane) and the inclination with respect to the electrophoresis direction are detected.

If the digital signal is detected as described above by scanning along each lane at a scanning line density such that at least one scanning line is applied to each band (see Figure 1, 1: Migration band, 2: scan line), position for each scan line directly
A one-dimensional waveform consisting of signal level (y) and signal level (z) can be created. When the autoradiograph is read over the entire surface, a one-dimensional waveform is created after signals are extracted along each lane by scanning the digital image data in the same manner as described above.

The band position is determined by detecting the position (peak position) where the signal level is maximum on each one-dimensional waveform, and determining the position where the signal level at the peak position is maximum among multiple linear waveforms. D(x)]. Alternatively, the peak position on the one-dimensional waveform corresponding to the center position of the electrophoresis column may be set as the band position. Here, x is the distance from the center point in the width direction of the support medium, and D
(x) is the distance from the migration start position (migration distance).

For details on how to determine band positions by digital signal processing, please refer to the applicant's patent application
It is described in the specifications of Japanese Patent Application No. 60-181432 and Japanese Patent Application No. 60-226091 filed on October 11, 1985.

To determine the band slope, first find the position where the signal level is maximum on two one-dimensional waveforms. For example, as shown in Figure 2, after finding the peak position of the signal level at the center and end of the band ([x, D(x)] and [x+Δx,
+Δx)]) into the above equation (6), the band slope tanθ(x) can be obtained.

From the position and slope of this band, tanθ(x)/D(x)
Calculate.

By performing the above operations for each lane, tan θ(x)/D(x) is obtained for each lane, and from this result, tan(x)/D(x) is expressed as an approximate expression as a function of x. For example, if the obtained results are summarized in a graph, the position x in the width direction of the support medium and
A graph similar to the graph representing the relationship with tanθ(x)/D(x) is obtained.

Next, for each lane, the cumulative value (integral value) of tanθ(x)/D(x) from the center point (x=0) to the lane position (xi): ∫ ^xi ₀ tanθ(x)/D( x)dx is calculated using the above approximate value or approximate graph. By substituting the obtained cumulative value into Equation (1) above, the migration speed correction coefficient η(xi) for each lane is obtained: η(xi)=e ^-ai a _i =∫ ^xi ₀ tanθ(x)/D( x)dx can be obtained.

By substituting this correction coefficient η(xi) into the above equation (2), D ₀ (xi)=D(xi)/η(xi) can be obtained.

The obtained D ₀ (xi) is the smiling-corrected migration distance of the i-th lane.

From the viewpoint of improving accuracy, it is generally preferable to detect the band inclination for a lower band with a large slope (region where the migration distance is relatively long). Alternatively, the lane may be divided into a plurality of sections in the migration direction, and the above-described operation may be performed for each section to perform the smiling correction.

In addition, since the smiling effect normally appears symmetrically in the width direction of the support medium, the above tanθ(x)/D
The approximate expression (x) may be obtained only for one side of the support medium.

The migration distance obtained by the correction indicates the position where the migration would have occurred if the smiling effect had not occurred.

In this way, even if a smiling phenomenon occurs in the migration pattern, the smiling effect can be corrected by performing signal processing to correct the migration distance for each slot.

Furthermore, for the one-dimensional waveform of the scanning line whose migration distance has been corrected, for example, by detecting all positions where the level of the signal appearing on the waveform is maximum, the positions of all bands on each lane can be determined. can be detected. The band position may be the position of the maximum value of a scanning line passing approximately at the center of the lane, or may be the average of the positions of the maximum value of each scanning line of the lane.

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

Here, offset distortion refers to the overall positional deviation 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, band fusion refers to two 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.

To compensate for offset distortion, since the spacing between bands is generally sparse in the lower region of the electrophoresis pattern, first detect multiple bands in the lower region for each lane, number the bands sequentially from the bottom, and then identify the bands. Obtain a correlation (eg, regression line) between the number and its migration distance. The difference in migration distance between lanes is determined from this correlation, and the migration position of each lane can be collectively corrected by shifting the migration position of each lane as a positional deviation between lanes.
Alternatively, offset distortion can be corrected piecewise for each lane.

To correct the fusion band, first, a plurality of bands are detected in a connected manner in the lower region of each lane, serial numbers are assigned to the bands in order from the lower end, and then a correlation between the distance between the bands and the band number is obtained. By predicting the position of an undetected band from this correlation and newly detecting a band based on this predicted position, it is possible to separate the bands into individual bands. 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 and Japanese Patent Application No. 85275 filed by the applicant.
No. 60-85276, Japanese Patent Application No. 60-111186 and Japanese Patent Application No.
It is described in each specification of No. 60-111187.

Based on the corrected band positions, each band is ranked in order from the bottom of the electrophoresis pattern. At this time, the above four types of base-specific DNA
Since the combination of fragments is an exclusive combination, the order can be easily determined by taking advantage of the fact that only one band can exist at the same position along the migration direction. The slots (1) to (4) above each have information about the terminal bases consisting of (G), (A), (T), and (C), so replace them with the base corresponding to the slot to which each band belongs. By this,
DNA base sequence (e.g. A-G-C-T-A-
A-G-...) can be obtained.

In 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, it is also possible to display the image as an image based on the digital signal that has been subjected to the smile correction described above. At the same time, it is also possible to display the original autoradiograph as a visible image. In this case, the final base sequence can be determined by the analyst himself based on this displayed image.

In the above embodiment, as a mixture of base-specific DNA fragments (G, A,
Although the case has been described in which an exclusive combination of (G, G+
It can be applied to various combinations such as A, T+C, and C). Similarly, the signal processing method of the present invention can be applied to a mixture of base-specific RNA fragments (for example, a combination of G, A, U, and C). Furthermore, the correction of the smiling effect is not limited to the separation and development array of a set of base-specific fragments of nucleic acids, but can be performed for all separation and development arrays that are simultaneously separated and developed on the support medium. be.

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

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

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 partial diagram showing an example of an electrophoretic pattern in which a smiling phenomenon occurs. FIG. 2 is an enlarged view of the slanted band. FIG. 3 is a graph showing the relationship between the position x in the width direction of the support medium and tanθ(x)/D(x). 1: electrophoresis band, 2: scanning line.

Claims (1)

[Scope of Claims] 1. A plurality of separated and developed arrays formed by separating and developing a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in a one-dimensional direction on a support medium. In a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to an autoradiograph, the method includes: (1) detecting the position of at least one band and its inclination with respect to the separation development direction for each separation development column; (2) From the position and slope of the band, equation (1): η(x)=e ^-a a=∫ ^x ₀ tanθ(x)/D(x)dx (1) (where x is the distance from the center point in the width direction of the support medium, D(x) is the separation distance of the band at position x, tanθ(x) is the slope of the band at position x, and η(x ) is the separation deployment speed correction coefficient at position Then, by equation (2): D ₀ (x)=D(x)/η(x) (2) (where D ₀ (x) is the smiling-corrected separation expansion distance at position x), the corresponding 1. A signal processing method for determining a base sequence of a nucleic acid, comprising: correcting a separation distance of a column. 2 Obtain the above digital signal by scanning the autoradiograph so that each band has at least two scanning lines, and in the first step, find the position where the signal level in each scanning line is maximum. , the signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that the slope is detected based on this position. 3. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that in the first step, the position and slope of the lower band of each separation and expansion column are detected. 4. Signal processing for determining the base sequence of a nucleic acid as set forth in claim 1, wherein the separation and development array is divided in the separation and development direction, and the first to third steps are performed for each division. Method. 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, and then photoelectrically reading out the autoradiograph as photostimulated light by irradiating the phosphor sheet with excitation light. A signal processing method for base sequencing of the described nucleic acid. 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.