JPS61243362A - Signal processing for determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To obtain a base sequence of nucleic acid handily and at a high accuracy, by passing a digital signal corresponding to an autoradiograph through a signal processing circuit for correcting the offset distortion. CONSTITUTION:In an autoradiograph 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 the one-dimensional direction, number is attached sequentially to lower bands of separate development trains and the correlationship between the number of the corresponding band and the separate development distance is obtained for each separate development train to determine the difference in the separate development distance between separate development trains from the correlationship. Then, the upper band is detected to define the difference in the separate development between trains as positional deviation and after the separate development position of the corresponding band is corrected, a number is attached thereto. About the bands involved, a correlationship is obtained anew between the number of band and the separate development distance for each separate development train. This operation is repeated and the sequence is attached to all bands based on the correction position to determine 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 nucleic acids.

[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 that carries specific genetic information
It has become essential to determine the base sequence of nucleic acids such as (or DNA fragments, the same shall apply hereinafter).

Maxim-Gilbert, who uses autoradiography, is a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
) law and Sanger-Courson (Sanger-C
Oulson) method is known. The former Maxam-Gilbert method first uses the D
3 at one end of a chain molecule of NA or DNA fragment.
2. By bonding a group containing a radioactive isotope such as p, the target substance is made into a radioactive labeling substance, and then the bonds between each constituent unit of the chain molecule are base-specifically made using chemical means. disconnect. 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. sequentially determine the bases in a certain positional relationship from
This allows the base sequence of the target ring to be determined.

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

With the aim of determining the base sequence of the above nucleic acids simply and with high precision, the present applicant has proposed a conventional radiographic method using a photosensitive material such as the above X-ray film in the autoradiograph measurement operation used therein. Instead, a patent application has already been filed for a method using a radiation image conversion method using a stimulable phosphor sheet (Japanese Patent Laid-Open No. 59-830).
57, No. 58-201231), here, the stimulable phosphor sheet is made of a stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, visible to By exciting it with electromagnetic waves (excitation light) in the infrared region, radiation energy can be emitted as fluorescence. According to this method, exposure time can be significantly shortened, chemical fog, etc., which has been a problem in the past, does not occur, and 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 in addition to images, it also contains symbols,
It is possible to display and record in any format such as numerical values.

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, 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. 1983)
-126527, JP-A-59-126278, Japanese Patent Application No. 59-89615, Japanese Patent Application No. 59-140908, etc.). When using a conventional radiographic film, the digital signal corresponding to the autoradiograph can be obtained by first making the autoradiograph a visible image on the film and then reading it photoelectrically using reflected or transmitted light. can get. In addition, 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 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 is caused by the fact that the shapes (sizes of depressions) of the many slots (sample injection ports) provided at the top of the medium are not completely the same and tend to differ individually. Distortion may also occur due to differences in the rate of penetration of the sample into the support medium, if the attachment positions shift from one another when attaching the sample to the medium, or if urea is not sufficiently washed out of the gel medium immediately before sample injection. It becomes.

FIG. 1 shows a specific example where offset distortion occurs in the separation development pattern due to the uneven shape of the Nuronto. Figure 1 (a) shows the upper end of the support medium used for separating and developing the sample, and (b) shows the same sample (1
) to (4), the putters obtained by injecting the mixture into each slot and then developing it in minutes are shown. As shown in Fig. 1(a), the third slot has a larger recess than the other slots, and as a result, only the separation deployment row of the third slot is shifted in the f direction as a whole, as shown in Fig. 1(b). , the misalignment (Δy
) is occurring. 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 provides 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 separation development pattern with offset distortion, 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 a plurality of separation and development arrays formed by separation and development of 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.

1) At least two bottom /< for each separation expansion column
2) obtaining a correlation between the band number and its separation development distance for each separation development column; 3) performing separation from the obtained correlation. 4) Detecting at least one upper band for each separation development column, and using the obtained difference in separation development distance as the positional shift between the columns, separating the bands. After correcting the deployed position, a step of assigning consecutive numbers to the band based on the position.

5) Adding the band newly detected in the fourth step to the already detected bands, and newly obtaining the correlation between the band number and its separation distance for each separation expansion column for these bands; and 6) By sequentially repeating steps 3 to 5,
The present invention provides a signal processing method for determining the base sequence of a nucleic acid, which comprises the step of ranking all bands on each separation and development array based on their complementary IE 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, the digital image corresponding to the autoradiograph can be By passing the signal through an appropriate signal processing circuit having a signal processing function for correcting offset distortion, the base sequence of a 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 separation and development columns and the band positions are shifted, the order of the bands can be relatively easily determined by comparing the band positions of each column in the lower region. It is possible to decide.

However, since the bands are closely spaced in the upper region, positional deviations between columns make it difficult to determine the order of the bands, causing errors in the base sequence of the resulting nucleic acid.

The present inventor focused on the fact that the interval between 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, and based on the offset We have found a method to correct distortion appropriately and easily. In other words, since the order of the bands in the lower region is easily determined and a certain correlation is obtained between the serial number of the band and its separation development distance, based on this relationship, the order of the bands in the upper region is determined more easily. It is possible to partially determine the positional deviation of
This allows offset distortion to be successively corrected.

In particular, since the correlation between the serial number of a band and its separation distance can be expressed locally by a straight line, it is possible to approximate it by a straight line if the number of bands used to obtain the correlation is not very large. This correlation can be easily obtained.

Therefore, the present invention is a method for sequentially correcting positional deviations between columns, and even when one positional deviation differs locally in the separation and development direction, positional correction can be performed with high accuracy.

Furthermore, the base sequence of a nucleic acid can be determined simply and with high precision based on the digital signal that has been corrected for offset distortion.

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

Examples include mixtures of 48 acid base-specific fragments 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 mixture, which is a type of base-specific DNA fragment mixture, is a mixture of DNA fragments that have been radiolabeled according to the Maxam-Gilbert method described above.
It is obtained by base-specific cleavage and decomposition of A.

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

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

The radioactive label is added to these substances using a method suitable for 32p.
, +4 (: , 31+ 3.3 H,"I Su,
! ') It is given by retaining a radioactive isotope.

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

Next, the support medium on which the radiolabeled substance has been separated and developed is subjected to radiography using a conventional photographic material.
Alternatively, an autoradiograph 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 -
After exposing the radiographic film for a long time (several tens of hours) at 70°C, the autoradiograph of the radiolabeled substance is made into a visible image on the radiographic film by development6.
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 the 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 sheet (for several tens of minutes) and accumulating the radiation energy emitted from the radiolabeled substance on the phosphor sheet. The body sheet is, 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. 28527 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. is possible.

Furthermore, in any of the above methods, reading (or reading out) the autoradiograph need not be performed over the entire surface of the radiation film (or stimulable phosphor sheet), but can of course be performed on only one 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 at a scanning line density that allows the light to pass through, the reading time can be shortened and necessary information can be efficiently obtained.

Note that in the present invention, the digital signal corresponding to an autoradiograph includes the digital signal obtained in this manner.

The obtained digital signal I)
). 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 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 CG) 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 a DNA fragment of various lengths that has been cleaved, degraded, or synthesized in a base-specific manner, that is, has the same terminal base. consists of things.

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

A digital signal corresponding to this autoradiograph is stored in a -H memory (buffer memory or nonvolatile memory such as a magnetic disk) in the 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 position (y) of the extracted signal and the level (Z) of that signal is created for each lane. .

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

The right side of each -dimensional waveform in Figure 2 (region 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 electrophoresis pattern, as is clear from the one-dimensional waveform in Figure 82, 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 with the band number (n) on the horizontal axis and the migration distance (y') on the vertical axis,
Regression lines as shown in lines 1 to 4 in FIG. 4 are obtained. Note that FIG. 4 shows the regression line 1 consisting of the band number (n) and migration distance gl (y') for each lane.
~4 corresponds to the slot number.

Here, the migration distance 111 (y') is the distance II from the reference origin (yo) to the position of each band (y' = y-Vo)
The reference origin, which represents , can 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, and the regression line for each lane is expressed by the general formula: Y'=an+l). Here, a and b are each constants.

Usually, in the lower region of the electrophoresis pattern, there is a local linear relationship between the band number and the electrophoresis distance, which can be approximated by a regression line as shown in straight lines 1 to 4 in FIG. Here, if offset distortion does not occur,
The detected/hendo should have been 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 electrophoresis positions is not limited to the above regression line; for example, it can be approximated with an appropriate higher-order curve to form a regression curve to achieve even higher precision. Correlations can also be determined.

Next, the difference in migration distance between the lanes is determined from the obtained correlation, and this difference is taken as the positional shift between the columns in the band region to be detected next.

The difference in migration distance between lanes can be obtained, for example, by first extrapolating each regression line in the direction in which n increases, and then determining the migration distance gly' at an arbitrary point on the horizontal axis. Then, this difference in migration distance is taken as the positional shift between the columns in the vicinity (that is, in the band region to be detected next). Specifically, the difference in migration distance between the lanes of the first slot and the second slot is as follows:
(in the direction where n becomes larger) to any direction (n =
It is obtained as the difference in migration distance y゛, 2 at n a). This difference corresponds to a positional shift between lanes due to offset distortion.

One or more bands are detected in the region above the lower region where bands have already been detected, and the migration positions of these bands are corrected based on the positional shift between the lanes determined above, and then the order is determined. wear.

Band detection can be performed by finding the maximum value of the one-dimensional waveform of each lane in the same manner as described above.

To correct the electrophoresis position, the position 11(y) of the newly detected band is shifted toward the L part or the r part by the difference in the obtained migration distance.For example, for the band in the second slot,
The electrophoresis position can be corrected by subtracting the value y′,2 with respect to y. In this way, positional deviation between lanes can be corrected for each lane, and offset distortion of the electrophoretic pattern can be corrected.

Each band is compared with each other based on this corrected position and numbered sequentially from the bottom. At this time, since the combination of the four types of base-specific DNA fragments is an exclusive combination, The order of bands can be easily determined by taking advantage of the fact that one band is never present in two or more lanes at the same time.

Furthermore, for each lane, this newly detected band is added to the already detected bands, and the band with the lowest band number is excluded instead, and again the correlation between the band number and its migration distance ( regression line or regression curve)
seek. For example, a new regression line as shown in the straight line l''~4° in Figure 4 can be obtained.In this case, since the amount of band data used to find the correlation is always constant, the obtained correlation The relationship can be expressed by a nearly constant relational expression such as a straight line without changing depending on the data.

Based on this corrected regression line, calculate the positional shift between lanes in the band region to be detected in the first order again,
The migration position of the newly detected band in the region is corrected, and the bands are ranked.

In this way, by dividing each lane into predetermined areas and repeating the above-described operation for each division, it is possible to correct positional deviations due to offset distortion for all bands on each lane.

Since the positional deviation between lanes is not necessarily uniform throughout the lanes, offset distortion can be corrected with high precision by correcting the positional deviation piecewise.

The number of bands detected and position corrected in one operation is preferably less than half the number of bands used to obtain the correlation, for example about 3 to 5 per lane.

Alternatively, the correlation between the serial number of a band and its migration distance may be calculated for all already detected bands by adding newly detected bands to the bands already detected. As the above operations are repeated, the correlation becomes not a straight line, but a regression curve (fifth
(see figure).

In FIG. 5, curves 1 to 4 are regression curves consisting of band numbers (n) and migration distances (y') for all bands detected in each lane.

Alternatively, the above operation may be repeated for each band. In other words, each time one band is detected, the correlation between the serial number of the band and its migration distance is corrected, and the correlation between the lanes in the band region to be detected next is adjusted. The positional deviation may also be determined, and thereby the migration position can be corrected with higher accuracy. For example, as shown in FIG.
After calculating regression curves 1 to 4 for each lane based on the data of the nbth band, by extrapolating in the direction where n becomes larger, the first and third slots for the next nb+1th band are calculated. The difference in migration distance between lanes is Y'
+ko is determined.

The serial number of the band thus obtained means the base sequence of the DNA of interest. ): Note (1)
~(4) slots are (G), (A), and (T), respectively.
, (C), by replacing the base with the base corresponding to the slot to which each band belongs, the DNA base sequence (for example, A-G-C-
T-A-AG-...) can be obtained.

In the present invention, if a smiling phenomenon occurs in the migration pattern, the smiling phenomenon may be corrected before the digital signal is corrected for the above-described offset distortion.

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 determined by scanning digital image data so that each band has at least two scanning lines, extracting a digital signal, and then creating a one-dimensional waveform for each scanning line.
It can be determined from the regression line obtained by connecting the positions of the maximum values. Alternatively, reading the autoradiograph (
The digital signal as described above may be detected in advance in the readout 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. In this case, all bands are detected in advance, and smile correction is performed on a band-by-band basis.

For details on the smiling phenomenon caused by digital signal processing, please refer to Japanese Patent Application No. 1986 [filed on April 9, 1985 (1)] 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 about the base sequence is not limited to the previous display format; for example, if desired, it is also possible to simultaneously display the intensity (Z'') of each band as the relative amount of the radiolabeled substance.Also, at the same time, It is also possible to display it as an image together with the visible image of the autoradiograph.Furthermore, it is also possible to display the base sequences of both two stranded molecules of DNA.

In addition, in the above, a case was explained in which an exclusive combination of (G, A, T, C) was used as a mixture of base-specific DNA fragments as a sample, but the signal processing method of the present invention uses this combination. For example, it is not limited to (
It can be applied to various combinations such as G, G+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 offset distortion is not limited to the separation and development columns of base-specific fragments of nucleic acids of the set, but can be performed for all separation and development columns 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 sent to a recording device directly or, if necessary, via a storage means such as a magnetic disk or magnetic tape. transmitted.

Recording devices include, for example, those that optically record by scanning a photosensitive material with a laser beam, etc., those that record symbols and numbers 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 shape of the slot provided at the upper end of the support medium, and FIG. 1(b) is a view showing an example of a separated developed 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 migration ratio fll (y') for each lane. Straight lines 1 to 4 and straight lines 1' to 4'' are (1) to 4, respectively.
This corresponds to slot (4). Figure 5 shows the band number (n) and migration ratio for each lane! 0 curve 1 which is a diagram showing a regression curve consisting of (y')
~4 correspond to the slots (19~(4)), respectively. Patent applicant Fuji Photo Film Co., Ltd. Agent
Person Patent Attorney Yasushi Yanagawa 11111〉X Figure 2: Figure 4 n We will amend the "Detailed Description of the Invention" column of the specification as follows. procedure? 10th official book June 10, 1985

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; 3) calculating the difference in separation distance between the separation expansion columns from the obtained correlation; 4) Detecting at least one upper band for each separation and development column, correcting the separation and development position of the band by using the difference in the obtained separation and development distance as a positional shift between the columns, and then determining the position of the band. 5) Adding the newly detected bands in the fourth step to the already detected bands, and assigning the band number and its number for each separation and expansion column for these bands. By sequentially repeating the step of newly obtaining the correlation with the separation development distance, and 6) the above third to fifth steps,
1. A signal processing method for determining the base sequence of a nucleic acid, comprising the step of assigning a ranking to all bands on each separation and development array based on their corrected positions. 2. 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 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 development distance between the separation development rows is obtained as the difference in separation development distance when the regression line or regression curve is extrapolated in a direction where the band number increases. A signal processing method for determining the base sequence of a nucleic acid according to claim 3. 5. In the fifth step above, add the band newly detected in the fourth step to the already detected bands, exclude the bands with small numbers among the already detected bands, and separate these bands. 2. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein a correlation between a band number and its separation distance is newly obtained for each development column. 6. 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. 7. 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. 8. 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. 9. 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 .