JPS61243361A - Signal processing for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To automatically determine the base sequence of nucleic acid handily and at a high accuracy, by performing a signal processing a digital signal satisfactorily for an autoradiograph of even a separation development pattern suffering an offset distortion. CONSTITUTION:In an autoradiograph of a plurality of separate development trains in which a base specific DNA fragment or a mixture of this and a base specific RNA fragment with a radioactive label imparted to is separately developed on a support medium in one-dimensional direction, first,at least two lower bands of each separate development train are detected and number is attached to the lower end thereof sequentially to obtain the correlationship between the number of the band involved and the separate development distance for each of the separate development trains. then, the difference in the separate development distance is obtained between the separate development trains from the correlationship thus obtained and the separate development position is corrected for each train with the difference as positional deviation between the trains thereby determining the base sequence of nucleic acid.

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 becomes. In particular, DNA (which carries specific genetic information)
It has become essential to determine the base sequence of nucleic acids such as DNA fragments or DNA fragments (hereinafter the same).

Maxam Gilbert uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
) law and Sanger-Coursoy (Sanger-
Coulson) method is known. The former Maxam
In the Gilbert method, first, a group containing a radioactive isotope such as ff2p is attached to one end of a chain molecule of DNA or a DNA fragment whose base sequence is to be determined. After that, the bonds between each constituent unit of the chain molecule are cleaved base-specifically using chemical means. 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 separation development pattern is visualized on, for example, an X-ray film to obtain an autoradiograph thereof, 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, it is possible to determine the base sequence of the entire target object.

In addition, the latter Sanger φ Kuhn method involves base-specific synthesis of a DNA compound that is complementary to a chain molecule of DNA or DNA fragments and that has been given a radioactive label using chemical means. 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 radiation image conversion method 7 using a stimulable phosphor sheet (Japanese Patent Laid-Open No. 59-83
No. 057, Japanese Patent Application No. 58-201231). Here, the stimulable phosphor sheet is made of stimulable phosphor,
After radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, the radiation energy can be emitted as fluorescence by exciting it with electromagnetic waves (excitation light) in the visible to infrared region. According to this method,
Exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur.

In addition, autoradiographs of radiolabeled substances.

Since it is once stored in a phosphor sheet as radiation energy and then read out in time series as photostimulated light, it is possible to display and record it in any form such as symbols and numbers in addition to images.

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 separated development column (referred to as a specific fragment) and comparing them between each separation development row. Therefore, 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 Publication No. 128278-1982, Japanese Patent Application No. 89615-1987, Japanese Patent Application No. 140908-1980, etc.)
When using a conventional radiographic film, the digital signal corresponding to the autoradiograph is first visualized on the film and then read photoelectrically using reflected or transmitted light. It is obtained by 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 overall positional deviation between columns (so-called offset distortion) due to the difference in the starting position or start time of separation and development of the sample in each column. This occurs because the shapes (sizes of recesses) of the many slots (sample injection ports) provided at the upper end of the medium are not completely the same and tend to differ individually. In addition, when the sample is attached to the support medium, if the attachment positions are shifted from each other, or if urea is not sufficiently washed out of the gel medium immediately before sample injection, the rate of penetration of the sample into the support medium may be different, causing distortion. It is a contributing factor to the occurrence of

FIG. 1 shows a specific example where offset distortion occurs in the separation development pattern due to the irregular shapes of the slots. Figure 1 (a) shows the upper end of the support medium used for separation and development of the sample, and (b) shows the same sample (1
The patterns obtained by injecting into each slot of ) to (4) and then separating and developing are shown. As shown in Figure 1(a), as a result of the third slot having a larger recess than the other slots, (
As shown in b), only the separation and deployment row of the third slot is shifted downward as a whole, causing a shift (Δy) with the other rows. Such relative positional deviation between columns is called offset distortion.

Even when such distortion occurs, it is desired to efficiently process the digital signal corresponding to the autoradiograph and automatically determine the base sequence of the nucleic acid with high precision.

[Summary of the Invention] The present inventor has devised 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 separation pattern has offset distortion. By appropriately processing the signals, we have achieved automatic determination of the base sequence of nucleic acids with ease and high accuracy.

That is, the present invention provides a plurality of separated spread arrays formed by separating and spreading a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in a one-dimensional direction on a support medium. A method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to an autoradiograph of The process of sequentially numbering the bands.

2) obtaining a correlation between the band number and its separation expansion distance for each separation expansion column; and 3) obtaining a difference in separation expansion distance between separation expansion columns from the obtained correlation;
The present invention provides a signal processing method for determining the base sequence of a nucleic acid, which comprises the following steps: correcting the separation and expansion position for each column by using this difference as a positional shift between columns.

The present invention also provides a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to the autoradiograph, comprising: 1) detecting at least two bands at the bottom of each separation development column; Step of assigning serial numbers to the bands in order from the bottom end; 2) For each separation development row, the number of the band and its corresponding number l! !
! The process of obtaining a correlation with the deployment distance.

3) Obtaining the difference in separation development distance between the separation development columns from the obtained correlation, and correcting the separation development position for each column by using this difference as a positional deviation between the columns, and 4) All of the separation development positions for each separation development position. The present invention also provides a signal processing method for determining the base sequence of a nucleic acid, which comprises the steps of: detecting the bands of the nucleotide sequence, and ranking the bands based on their positions.

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

Generally, in the separation development pattern, the spacing between the separation development bands is sparse in the lower part (ie, the region where the separation development distance is large), while the spacing between the bands becomes closer as the separation development start position in the upper part is approached. Here, the term "lower part" generally refers to the area below the vicinity of the center of the support medium, and the term "upper part" generally refers to the area above the vicinity of the center. Therefore, even if offset distortion occurs between the one-separation development rows and the band positions are shifted, the order can be relatively easily determined by comparing the band positions of each row in the lower region. It is possible to decide.

However, in the upper region, the spacing between the /hends is close, and thus the positional deviation between the columns makes it difficult to determine the order of the bands, causing errors in the base sequence of the obtained nucleic acid.

The inventor noticed that the interval between the /< bands is different between the upper and lower parts of the separated development pattern, and that it is easy to determine the order of the bands even when offset distortion occurs in the lower region. , we have discovered a method for appropriately and easily correcting offset distortion. In other words, in the lower region, not only is the order of the bands easily determined, but also the correlation between the serial number of the band and its separation development distance for each column is linear, so it is possible to easily correct the positional deviation between the columns. This allows offset distortion to be corrected all at once.

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

[Configuration 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, to which radioactive labels have been added. 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 is synthesized using DNA as a template and a radioactively labeled deoxynucleoside triphosphate and a DNA synthesizing enzyme according to the aforementioned Sanga Ichiban-Luzon method. It can be obtained by

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

- The radioactive label is added to these substances in a manner appropriate to co2P,
IAC, 3S3, KoH, L2! It is given by retaining a radioactive isotope such as 'I.

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

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

When using the former radiographic method, first the support medium and 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 tens of minutes) and accumulating the radiation energy emitted from the radiolabeled substance in the phosphor sheet. Here, the stimulable phosphor sheet includes, for example, a support made of a plastic film, divalent europium-activated barium fluoride bromide (BaFBr:Eu")
A phosphor layer made of a stimulable phosphor such as phosphor and a transparent protective film are laminated in this order. When the stimulable phosphor contained in the stimulable phosphor sheet is irradiated with radiation such as X-rays, it absorbs the radiation energy and accumulates it.
It has the characteristic that when excited with light in the visible to infrared region, the accumulated radiation energy is released as photostimulated light.

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

For details of the above-mentioned autoradiograph measurement operation and method of obtaining a digital signal corresponding to the autoradiograph, see the above-mentioned JP-A-59-83057 and JP-A-59-1.
It is described in various publications such as No. 26527 and Japanese Unexamined Patent Publication No. 59-126278.

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

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 C or the 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 in the reading operation, a scanning line is scanned on each band. By performing scanning with a light beam so that it passes through, the reading time can be shortened and necessary information can be efficiently obtained. In addition,
In the present invention, the digital signal corresponding to an autoradiograph includes the digital signal obtained in this manner.

The obtained digital signal (a) is the coordinate (x, y) expressed in the coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (Z) at that coordinate.
The level of the signal represents the image density at that coordinate, that is, 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 base sequence will be determined.

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 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. 2 shows an autoradiograph of the electrophoresis pattern formed by electrophoresing the above-mentioned four types of base-specific DNA fragments into four slots. As shown in FIG. 2, offset distortion occurs in the obtained autoradiograph.

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

First, two or more bands are detected for each electrophoresis column (lane) and their order is determined.

For example, after extracting a digital signal within a certain area along the electrophoresis direction of each lane, a -dimensional waveform consisting of the extracted signal position N (y) and the signal level (Z) for each lane is created. do.

If the digital signal is detected by scanning in the electrophoresis direction at a scanning line density that covers each band as described above, a one-dimensional waveform is directly created for each lane. be able to.

FIG. 3 shows a -dimensional waveform consisting of a signal position (y) and a signal level (Z) for each lane. In addition, the third
In the figure, the position of the vertical axis (y=yo) indicates the reference origin on the digital image data.

The right side of each -dimensional waveform in Figure 3 (area where y is large)
For example, by finding the point where the sign of the signal level difference value is reversed (changes from + to -), the position where the signal level is maximum is found, and the position where this maximum value is taken is the band position. . The number of bands to be detected varies depending on the total number of bands on the electrophoresis pattern and the state of the pattern, but for example, if the total number of bands is 150 to
200, it is preferable to detect about 10 bands for each lane.

All of the obtained bands are assigned serial numbers (n) in order of the migration position (y) farthest from the reference origin.

In the lower region of the migration pattern, as is clear from the one-dimensional waveform in Figure 3, the band spacing is sparse;
Even if offset distortion occurs, the band positions will not be reversed between lanes, and the order of the bands can be easily determined.

Next, for each lane, the correlation between the band number and its migration distance is determined.

For example, by creating a graph in which the horizontal axis represents the band number (n) and the vertical axis represents the migration distance (yo), a regression line is obtained as shown in FIG. 4. Note that FIG. 4 shows a regression line consisting of the band number (n) and migration distance (yo) for each lane. Lines 1 to 4 correspond to slot numbers.

Here, the migration distance (yo) represents the distance (y'=y-yo) from the reference origin (yo) to the position of each band. The reference origin may be, for example, the position of the slot.

Therefore, when there is a positional shift between the lanes, the distance y' from the common reference origin is not necessarily the true migration distance. Further, the regression line of each lane is expressed by the general formula: V'=an+b. Here, a and b are each constants, and b represents the y' intercept.

Usually, there is a local linear relationship between the band number and the migration distance in the lower region of the migration pattern, which can be approximated by a regression line as shown in FIG. Therefore, if offset distortion had not occurred, the detected band would have existed on one regression line. In other words, the four regression lines obtained for each lane should have overlapped into one.

Note that the method of expressing the correlation between band numbers and their migration positions is not limited to the regression line described above; for example, the correlation can be expressed with higher precision by approximating an appropriate higher-order curve to create a regression curve. Relationships can also be determined.

Next, the difference in migration distance between the lanes is determined from the obtained correlation, and the migration position of each lane is corrected based on this difference.

In FIG. 4, the difference in migration distance between lanes appears as the difference in the y' intercept when the migration distance y' is determined at any point on the horizontal axis. For example, the difference in migration distance between the first slot and the second slot is b12. This difference in y° intercept corresponds to a positional shift between lanes due to offset distortion.

If the slopes of the regression lines (a value in the above general formula) are different, the average value of the difference in yo at any point on the horizontal axis.

Alternatively, the difference in yo at a suitable point on the horizontal axis can be taken as the difference in migration distance. Further, even when the correlation is represented by a regression curve, the difference in migration distance, that is, the positional shift between lanes, can be determined in the same manner as described above.

Based on the difference in migration distance obtained, the position (y) of the signal on each lane is shifted upward or downward, for example,
For the lane of the second slot, by moving the entire one-dimensional waveform (2) shown in Figure 4 in the direction of the origin on the y-axis and shifting it by 2 (subtracting the value of 1 + 2 with respect to y),
The electrophoresis position can be corrected all at once. In this way, positional deviations between lanes can be collectively corrected for each lane, and offset distortion of the electrophoresis pattern can be corrected.

Regarding the one-dimensional waveform of each lane with the electrophoresis position 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 detected.

Based on the position of the detected band, all bands are ranked in order from the bottom of the electrophoresis pattern. At this time, the combination of the above four types of base-specific DNA fragments is an exclusive combination. Therefore, by changing lanes and taking advantage of the fact that two or more bands are not detected at the same position, the ranking can be easily determined. (1) to (4) above
The slots are CG), (A), (T), and (C), respectively.
Since it has information about the terminal base consisting of
-A -G-...) can be obtained.

In the present invention, if a smiling phenomenon occurs in the electrophoretic pattern, correction of the smiling may be performed before the digital signal is corrected for the offset distortion described in E.

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. be.

Smiling correction can be performed, for example, as follows.

In the electrophoresis pattern where the smiling phenomenon occurs, the band (long strip in the width direction) is usually not perpendicular to the electrophoresis direction but at an angle depending on the degree of the smiling effect. Detect the slope of at least one band for each lane. For example, the slope can be calculated by scanning the digital image data so that at least two scanning lines span each / It can be determined from a regression line obtained by connecting the positions. Alternatively, the digital signal as described above may be detected in advance in the autoradiograph reading process.

Next, one band (reference band) on one lane (reference lane) with the smallest degree of smiling effect is determined, and from the slope of this band and the slope of the nearest band on the other lanes, the standard band is determined. Extrapolate to the other lane and determine the relative position of the reference band in the other lane. Then, from this relative position and the position on the reference lane,
Determine the migration distance ratio for each lane. The obtained ratio represents the degree of the smiling effect of each lane, and the migration distance of each lane is collectively expanded or contracted based on this ratio. In this way, smiling can be corrected for all lanes.

Alternatively, the migration distance can be corrected for each band individually by detecting the slope of all bands and extrapolating each band on a lane other than the reference lane to the reference lane based on its slope. can.

For details on the smiling phenomenon caused by digital signal processing, please refer to the applicant's Japanese Patent Application No. 1986 [filed April 9, 1985 (1) Ko] and Japanese Patent Application No. 1988-
No. 1 [filed on April 9, 1985 (2)].

By the method described above, the base sequence of one chain molecule of DNA can be determined. Furthermore, DNA
The information regarding the one-base sequence is not limited to the above display format; for example, if desired, the intensity (2°) 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 two stranded molecules of DNA.

Alternatively, the DNA base sequence information can be displayed as an image based on a digital signal that has been corrected for offset distortion (and smile correction) as described above. At the same time, the original autoradiograph may be visualized and displayed. In this case, it is possible for the analyst himself to determine the final base sequence based on this displayed image.

In addition, although the case where an exclusive combination of (G, A, T, C) is used as a mixture of base-specific DNA fragments as a sample has been described above, the signal processing method of the present invention applies to this combination. For example, C
It can be applied to various combinations such as G, G+A, T+'C, 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, offset distortion correction is not limited to the molecule a of the base-specific fragments of the set of nucleic acids, and is not limited to the development array, but should be performed for all the molecule a development arrays that are simultaneously separated and developed on the support medium. is possible.

In addition to using the base sequence information obtained in this way, it is also possible to perform genetic linguistic information processing, such as comparing it with the base sequences of other nucleic acids that have already been recorded. .

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 either 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 laser beam, etc., those that record the code number displayed on a CRT etc. on a video printer, etc., and those that record using heat rays. Recording devices based on various principles can be used, such as those that record on material.

[Brief explanation of drawings]

FIG. 1(a) is a partial view showing the slot shape provided at the upper end of the support medium, and FIG. 1(b) is a partial view. FIG. 3 is a diagram showing an example of a separated development pattern in which offset distortion has occurred. FIG. 2 is a diagram showing an example of a migration pattern in which offset distortion has occurred. FIG. 3 is a diagram showing a -dimensional waveform consisting of a signal position (y) and a signal level (2) for each lane. FIG. 4 is a diagram showing a regression line consisting of band number (n) and electrophoresis pressure Ill (y') for each lane. Straight lines 1 to 4 correspond to slots (1) to (4), respectively. Patent applicant Fuji Photo Film Co., Ltd. Representative
Person Patent Attorney Yasuo Yanagawa Figure 2 Figure 4 o 1 Li 15 Procedural Amendment June 10, 1985 Patent Application No. 85275 2, Title of Invention Signal processing for determining the base sequence of nucleic acids Method 3: Relationship with the I-correction case Patent applicant name (520) Fuji Photo Film Co., Ltd. 4, agent address 8, Miya Yotsuya Building, 2-14 Yotsuya, Shinjuku-ku, Tokyo
Floor 6. Number of inventions increased by amendment None. 7. Target of amendment: "Detailed description of the invention" column 8 of the description. Contents of the amendment. We will make supplements as described in the above. (1) No. 33, line 6, by digital signal processing
1 Correct J by the method described above. (2) 1st patent application from the 7th stroke of page 33 to the 8th stroke of the same page 1986-
No. 1 is amended to read "R Japanese Patent Application No. 1983-74899". (2) P. 33, line 8, 1986-1
Patent Application No. 60-74900]. Below 1;

Claims (1)

[Claims] 1. A plurality of separations formed by separating and spreading a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in one-dimensional direction on a support medium. In a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to an autoradiograph of a column, 1) detecting at least two bands at the bottom of each separation development column, and sequentially dividing the bands from the bottom end; 2) obtaining a correlation between the band number and its separation distance for each separation expansion column; and 3) determining the difference in separation distance between the separation expansion columns from the obtained correlation. obtained,
A signal processing method for determining the base sequence of a nucleic acid, comprising the step of correcting the separation development position for each column by using this difference as a positional shift between columns. 2. In the first step, after extracting the digital signal along the separation development direction of each column, the band is detected by finding the position where the level of the extracted signal in each column is maximum. A signal processing method for determining the base sequence of a nucleic acid according to claim 1. 3. In the second step, the correlation between the band number and its separation distance is obtained as a regression line or a regression curve, for determining the base sequence of a nucleic acid according to claim 1. Signal processing method. 4. In the third step, the difference in separation and development distance between the separation and development columns is obtained as a difference in the intercept of a regression line or a regression curve. signal processing methods for. 5. Before the first step, by detecting the inclination of at least one band with respect to the separation and development direction for each separation and development row, and then determining the relative position of the band in other separation and development rows based on this inclination. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that the separation distance of each band is corrected. 6. The mixture of the base-specific DNA fragments includes (1) a guanine-specific DNA fragment, (2) an adenine-specific DNA fragment, (3) a thymine-specific DNA fragment, and (4) a cytosine-specific DNA. The method is characterized in that the separation and development column consists of four separate and development columns formed by separating and developing these four types of base-specific DNA fragments on a support medium, respectively. A signal processing method for determining the base sequence of a nucleic acid according to claim 1. 7. 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. 8. 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 . 9. An autoradiograph of multiple separation and development columns formed by separation and development of a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in one-dimensional direction on a support medium. A method for determining the base sequence of a nucleic acid by performing signal processing on a corresponding digital signal, comprising the steps of: 1) detecting at least two bands at the bottom of each separation and development column and serially numbering the bands from the bottom; 2) Obtaining the correlation between the band number and its separation expansion distance for each separation expansion column, 3) Obtaining the difference in separation expansion distance between the separation expansion columns from the obtained correlation, and converting this difference into a column. 4) detecting all the bands on each separation development column and ranking the bands based on the positions; and 4) detecting all bands on each separation development column. A signal processing method for determining the base sequence of a nucleic acid. 10. In the first and fourth steps above, after extracting the digital signal along the separation development direction of each column, detect the band by finding the position where the level of the extracted signal in each column is maximum. A signal processing method for determining the base sequence of a nucleic acid according to claim 9. 11. For determining the base sequence of a nucleic acid according to claim 9, wherein in the second step, the correlation between the band number and its separation distance is obtained as a regression line or a regression curve. Signal processing method. 12. Base sequencing of nucleic acids according to claim 11, characterized in that in the third step, the difference in separation and development distances between separation and development columns is obtained as a difference in intercepts of regression lines or regression curves. signal processing method for. 13. Before the first step, by detecting the inclination of at least one band with respect to the separation and development direction for each separation and development row, and then determining the relative position of the band in other separation and development rows based on this inclination. 10. The signal processing method for determining the base sequence of a nucleic acid according to claim 9, characterized in that the separation distance of each band is corrected. 14. 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 9. 15. 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 9, 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. 16. 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. 10. The signal processing method for determining the base sequence of a nucleic acid according to claim 9, wherein the signal processing method is obtained by photoelectrically reading an autoradiograph visualized in .