JPS638558A - Signal processing for determination of base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To obtain a base sequence of nucleic acid at a high accuracy, by passing a digital signal corresponding to an autoradiograph in a separation/ development pattern of base specific fragments of nucleic acid through a processing circuit having a signal processing function for separation of fused bands. CONSTITUTION:A signal processing for the separation of fused bands is performed for a digital signal corresponding to an autoradiograph in a separation/development pattern as developed by separating four kinds of base specific DNA fragments in a 1-D manner. Then, at least two bands are detected continuously at the lower region of the separation/development pattern to obtain information on pitch Pij of the bands. The position in the direction of the separation and development of the subsequent band to be detected is controlled from the pitch information to detect another band. New pitch information is obtained from the positions of the band just detected and the bands already detected, while the position is forecast in the direction of the separation and development of a band to be detected next. These operations are repeated sequentially to detect all bands on the separation/development pattern.

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 important to clarify the genetic information of living organisms in order to elucidate the mechanisms of their survival and replication. It has become mandatory. Among other things.

It is essential to determine the base sequence of nucleic acids such as DNA (or DNA fragments, hereinafter the same) that carry specific genetic information.

Maxam Gilbert □lama layer - Gilbert uses autoradiography as a typical method to determine the base sequence of nucleic acids such as DNA and RNA.
) law and Sanger-C
Oulson) method is known. The former Maxam-Gilbert method first involves attaching a group containing a radioactive isotope such as 32p to one end of a chain molecule of DNA or DNA fragments whose base sequence is to be determined. After turning a substance into a radioactively labeled substance, the bonds between each constituent unit of a chain molecule are base-specifically cleaved using chemical means.Next, base-specific DNA cleavage and decomposition obtained by this operation is performed. A mixture of substances is separated and developed by gel electrophoresis, and a separated development pattern (which cannot be visually seen) is obtained by separating and developing a large number of cleaved and decomposed products. This separation development pattern is visualized on, for example, a x6 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 isotope is bound is determined. 11 in a certain positional relationship from! By sequentially determining the bases, it is possible to determine the base sequence of the entire target object.

The latter Sanger Φ Coulson 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. 1. 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 an M-laminated phosphor sheet (Japanese Patent Laid-Open No. 59-830).
57, Japanese Patent Application No. 58-201231), where the stimulable phosphor sheet is made of a stimulable phosphor, and the stimulable phosphor of the stimulable phosphor sheet absorbs radiation energy. By subsequently exciting it with electromagnetic waves (excitation light) in the visible to infrared region, the radiation energy can be emitted as fluorescence. According to this method,
Exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur.

Furthermore, since an autoradiograph of a radiolabeled substance is once stored in a phosphor sheet as radiation energy and then read out photoelectrically as photostimulated light, it is necessary to obtain it directly as a digital signal and then save it on a suitable recording medium. Can be done.

Conventionally, the base sequence of a nucleic acid has been expressed in a visualized autoradiograph using a base-specific cleavage degradation product or a base-specific composite of a radioactively labeled nucleic acid (hereinafter simply referred to as a base-specific fragment of a nucleic acid). It is determined by visually determining the separation development positions (bands) of and comparing the positions of the bands with each other. Therefore, analysis of autoradiographs is usually performed through human vision, which requires a great deal of time and effort.

Furthermore, since it relies on the human eye, there are limits to the accuracy of base sequence information, such as the base sequence of nucleic acids obtained by analyzing autoradiographs differing depending on the analyst.

Therefore, the applicant has already 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-
No. 126527, patent application No. 1989-89615, patent application No. 1983
0-226091, patent application No. 60-226092, etc.)
. When using a conventional radiographic film, the digital signal corresponding to the autoradiograph can be obtained by visualizing the ridge autoradiograph on the film and then reading it 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.

For example, a separation pattern typically has separation bands that are sparsely spaced in the lower regions (i.e., regions with large separation distances), while bands become more closely spaced as one approaches the start of the separation development at the top. It has become.

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, two or three bands tend to connect to form one wide band because the bands are not sufficiently separated in the upper region.

Furthermore, there is an overall positional deviation between the columns (so-called offset distortion) due to the fact that the start position or start time of separation and development of the sample is different in each column. Offset distortion is caused, for example, by the fact that the shapes (distortion magnitudes) of the numerous slots (sample injection ports) provided at the top of the support medium, such as gel media, are not exactly the same and tend to vary individually. occurs. 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

Furthermore, the preparation and separation of base-specific fragments of nucleic acids may be insufficient during sample preparation, or samples from other slots may be mixed in when the sample is injected into each slot of the support medium. , a band (this is called a coast band or extra band) may appear in a position other than the place where it appears. Alternatively, noise may occur due to radioactive impurities being mixed into the sample or exposure to natural radiation during the exposure process.

Even when such band concatenation (fusion) occurs, as well as offset distortion and noise, the digital signal corresponding to the autoradiograph can be efficiently processed to determine the base sequence of the nucleic acid with high precision. It is desirable that the decision be made automatically.

[Summary of the Invention] In a method for automatically determining the base sequence of a nucleic acid using autoradiography, the present inventor has proposed a method for automatically determining the base sequence of a nucleic acid using autoradiography. By suitably processing digital signals, we have achieved automatic determination of the base sequence of nucleic acids with ease and high accuracy.

Furthermore, the present invention has achieved automatic determination of the base sequence of a nucleic acid with ease and high accuracy even when band fusion and offset distortion occur in the separation and expansion pattern.

That is, the present invention provides a radioactively labeled salt-specific DNA fragment or if! Signal processing is performed on a digital signal corresponding to an autoradiograph of a separation pattern consisting of a plurality of minute # development columns formed by separation and development of a mixture of group-specific RNA fragments in a one-dimensional direction on a support medium. In a method for determining the base sequence of a nucleic acid.

1) The step of continuously detecting at least two bands in the lower region of the separation development pattern, 2) Obtaining information about the pitch of the band from the position of the detected band, and determining the next band to be detected from this pitch information. 3) detecting one band on the separation development pattern based on the predicted position; 4) detecting the position of the already detected band and newly detected in the third step above; a step of newly obtaining information about the pitch of the band from the position of the band that has been detected, and predicting the position of the band to be detected next in the separation development direction from this pitch information; and 5) the third and fourth steps above. The present invention provides a signal processing method for determining the base sequence of a nucleic acid, comprising the steps of: detecting all bands on a separation development pattern by sequentially repeating the step of detecting all the bands on the separation development pattern.

According to the present invention, even if in a separation and development pattern obtained by separating and developing a mixture of base-specific fragments of nucleic acids on a support medium, the band separation is partially insufficient and a fused band occurs. By passing the digital signal corresponding to the autoradiograph through a processing circuit having a signal processing function for separating the fused bands, the base sequence of the nucleic acid can be obtained easily and with high precision. In addition, since it is possible to separate fusion bands, which was extremely difficult in the past, the amount of base sequence information obtained in -1 autoradiographic measurements increases, which also increases the amount of base sequence information obtained from -1 autoradiographic measurements. This facilitates base sequencing.

Furthermore, even if an offset distortion occurs in the separation development pattern at the same time as band fusion, the bands can be accurately separated and detected. Further, even if noise such as an extra band occurs, only the true band can be detected without misidentifying the noise as a band.

In other words, the interval between the bands is different on the upper and lower parts of the separation development pattern, and each band is sufficiently separated in the lower region, so even if the entire 5-minute MU open row is misaligned, the order from the lower end is different. It is possible to detect each band one by one without making any mistakes. Then, the interval between consecutively detected bands within the same column or between different columns (this is called the pitch) is determined, and based on this, the position where the next band should exist is predicted individually for each column. be able to. Thereby, even if a plurality of bands are fused, it is possible to properly and easily separate them into individual bands.

Furthermore, by predicting the position of the next band across two columns for each column, even if there is a positional shift between columns, the position can be predicted in consideration of the misalignment.

By being able to detect bands while separating fusion bands in this manner, base sequencing of nucleic acids can be easily and highly accurately determined.

[Structure of the Invention] Examples of samples used in the present invention include a mixture of base-specific fragments of nucleic acids such as DNA and RNA labeled with radioactive labels. Here, the nucleic acid fragment means a part of a long chain molecule. for example,
A base-specific DNA cleavage product mixture, which is a type of base-specific DNA fragment mixture, is obtained by base-specific cleavage and decomposition of radiolabeled DNA according to the aforementioned Maxam-Gilbert method.

In addition, the base-specific DNA compound mixture is synthesized according to the Sanger-Cournon method described above, using DNA as a template (type 9) and using a radioactively labeled deoxynucleoside triphosphate and a DNA synthase. can get.

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. Furthermore, DNA has four types of constituent units: adenine, guanine, thymine, and cytosine. On the other hand, RNA consists of four types of bases: adenine, guanine, uracil, and cytosine.

Radioactive labels can be added to co2P, I by a method appropriate to these substances.
It is given by retaining radioactive isotopes such as A, C1, S, H, and I.

The sample, a mixture of IiX group-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 by various separation and development methods such as photography.

Next, an autoradiograph of the support medium in which molecules a of the radiolabeled substance have been 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 autoradiogram of the radiolabeled substance is obtained via a suitable readout system.

When using the former radiographic method, first the supporting medium and the photographic light-sensitive material such as X-ray film are heated to a low temperature.

After exposing the radiographic film to light by overlapping it at room temperature or room temperature for a long time (several hours to several tens of hours), it is developed and an autoradiograph of the radiolabeled substance is made into a visible image on the radiographic film.Next, an image reading device is used. Read the autoradiograph visualized on the radiographic film using a radiograph. 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 brought together at room temperature for a short period of time (several seconds to several tens of minutes) to allow the phosphor sheet to accumulate the radiation energy emitted from the radiolabeled substance. The graph is recorded as a kind of latent image on a phosphor sheet. Here, the stimulable phosphor sheet has a support made of, for example, a plastic film,
Divalent europium activated barium fluoride bromide (BaFBr:
A phosphor layer consisting of a stimulable phosphor such as Eu2°) and a transparent protective film are layered in this order. *The stimulable phosphor contained in the phosphor sheet is: When irradiated with radiation such as X-rays, it absorbs and accumulates the radiation energy, and when excited with light in the visible to infrared region, it emits the accumulated radiation energy as photostimulated light.

Next, the autoradiograph accumulated and recorded on the stimulable phosphor sheet is read out using a reading device.
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 addition, in the above, a method using conventional radiography and radiographic image conversion methods has been described as a method for obtaining a digital signal corresponding to an autoradiograph of a radiolabeled substance separated and developed on a support medium bead. 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 go there.

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 perform it only on the image area. .

Furthermore, readout conditions are set by inputting information about the position of each separation expansion column, band width, etc. in advance, and in the readout operation, one or more scanning lines are By performing scanning with a light beam at a scanning density m such that , 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 nxy consists of the coordinates (x, y) expressed in the coordinate system fixed to the radiation film (or phosphor sheet) and the signal level (Z) at those coordinates.
One signal corresponds to one pixel.

The level of the signal represents the image density at that coordinate, ie, the amount of radiolabeled material. 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 on the support medium obtained in this way is subjected to signal processing by the method of the present invention as described below to obtain the base sequence of the target nucleic acid. A decision is made.

An embodiment of the signal processing method of the present invention will be described with reference to a case where it consists of a migration column (separation and development column) formed by a combination of base-specific DNA fragments to which four types of primary radiolabels have been added.

■) Guanine CG) Specific DNA fragment 2) Adenine (
A) Specific DNA fragment 3) Thymine (T) specific DNA
Fragment 4) Cytosine (C) specific DNAAllIr fragment, where:
Each m group-specific DNA fragment is base-specifically cleaved, decomposed, or synthesized, that is, it consists of DNA fragments of various lengths having the same terminal base.

FIG. 1 shows an autoradiograph of the electrophoresis pattern obtained by electrophoresing the above-mentioned four types of base-specific DNA fragments into four slots, respectively. The electrophoresis direction is the direction of the arrow (↓), and the electrophoresis pattern is such that the band spacing is sparse in the lower region, but the spacing becomes denser toward the north.

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

First of all, two or more bands are sequentially detected in the lower region of the migration pattern.

Here, "continuously detecting one band" means detecting one band and then detecting the closest band in terms of the position in the electrophoresis direction, regardless of the difference in the electrophoresis row.

For example, after extracting digital signals within a certain area along the migration direction of each migration column (lane), a one-dimensional waveform consisting of the position of the extracted signal and the level of the signal for each lane is created. Note that the detection of digital signals is
When scanning is performed in the electrophoresis direction at a scanning line density such that each band is covered with a scanning line as described above,
A one-dimensional waveform can be created for each lane directly from the obtained digital signal.

FIG. 2 partially shows a -dimensional waveform consisting of signal position (X) and signal level (2) for each lane. Here, the X direction is the opposite direction to the electrophoresis direction.

In a one-dimensional waveform, 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.The maximum value is the same in the -dimensional waveform. Regardless of whether the direction of migration is f! It is necessary to search for 1(X) in at least two consecutive locations. The position of this maximum value is defined as the band position. Thereby, at least one band spacing (pitch) can be obtained. The number of bands to be detected varies depending on the total number of bands with the electrophoresis pattern and the state of the pattern, but for example, if the total number of bands is 15
When the number is in the range of 0 to 200, it is preferable to detect several to several dozen bands in total, and it is preferable to detect the band at the bottom of the electrophoresis pattern.

Each hunt is ranked based on the order m(x) of the detected band. As shown in FIG. 1, in the lower region of the electrophoresis pattern, the intervals between the bands are sparse and each band is sufficiently separated, so that the order of the bands can be immediately determined. In Figure 1, the left X of the six bands in the lower region
The number 1 represents the order of the band.

Second, information about the distance # (pitch) between these bands is obtained, and from this pitch information, the position of the next band to be detected in the migration direction is predicted.

band pitch P1'' (where i and j are positive integers in the range of 1≦i≦4 and 1≦j≦4, respectively, and P
l, is the pitch of the bands from the i lane to the j lane) can be determined as the difference in the position of each band in the X direction. For example, by detecting the −·th band and the second band, the hunt pitch PI2 between the first lane and the second lane is obtained. In this way, pitch P12 of the six bunts shown in FIG. P21. Pla, Psv
, P'J3 is obtained.

In addition, the pitch Pii of the band in the i-lane is determined when the band is detected continuously in the same lane (i=j
), the same lane.

When two bands are detected, the pitch can be calculated as the average value. For example, the Pth band can be determined by detecting the third band.

In other words, since the first band has already been detected on the same first lane and the distance between the two bands corresponds to the distance between two bands, the distance between these bands (difference in position in the X direction) is calculated as the pitch number 2. The average value obtained by dividing by is taken as pH.

The band pitch gradually decreases as it approaches the electrophoresis start position t of the pattern L portion, but if it is locally up to about 10 pifts, even the average value can be used to predict the band position with sufficiently high accuracy. can.

In this way, information about the pitch of the band is stored. The obtained band pitch information is, for example, a matrix: (where i and J are 1≦i≦n and l≦, respectively)
j is an integer in the range of n, where n is an integer greater than or equal to 2 and equal to the number of separation and expansion columns, pH is the pitch of the band in the i column, and Pit is the pitch of the band from the i column to the j column. ).

1. By detecting the six bands, the matrix: Incidentally, a blank portion in row 9 means that the pitch Pij is undefined.

Based on this pitch information, the position of the band to be detected first is predicted. Specifically, the value obtained by adding Pij to the position x1 of the last detected band on lane i is the predicted position of the next band to be detected on lane j [X
j].

[Xjl=Xi +pij For example, if a band is next detected in the third lane, the value obtained by adding P33 to the position of the sixth band detected last is its predicted position 2ff[x3]. For lanes other than the third lane, the band pitch P3i is not obtained, so the band positions cannot be predicted.

In this way, the position of the next band to be detected can be predicted for each lane individually and in consideration of the positional relationship between lanes. Therefore, it is possible to predict the band position for each lane by taking into account the positional deviation of the entire lane such as offset distortion, so that the fused band can be separated more accurately, the extra band can be abstracted, and only the true band can be isolated. can be detected.

Third, one band is detected on the migration pattern based on the predicted position.

a) First, if the signal level reaches a maximum near the predicted position in the one-dimensional waveform of any lane, it is determined that a band exists at the position where the maximum occurs.
Detect one band. In addition, the above th! ! group-specific D
Since the combinations of NA fragments (G, A, T, C) are mutually exclusive, if a band is detected in one lane, it is assumed that no band exists at the same position in other lanes. can be determined.

b) If the signal level is maximum near the predicted position and there is a lane whose position cannot be predicted, there is no band in the lane whose position is predicted and there is a band in other unpredictable lanes. If there is a band, the band is detected by finding the position l at which the signal level first becomes maximum on another lane above the position of the last detected band, and determining this position as the band position.

C) Or, if the signal level does not reach a maximum near the predicted position and is predicted to be around the predicted position in all lanes,
A band exists in the lane with the highest signal level near the predicted position, and the band is detected by determining the predicted position for each band position.

For example, as shown in FIG. 2, for the last detected band of the second lane (position a), the pitch is P21 to P2
4, the predicted positions 71 b - e of the bands on the one-dimensional waveform of each lane are determined.

Since the signal level does not reach a maximum near any of the predicted positions, it is determined that the band exists in the fourth lane (position e) where the signal level is maximum.

Thereby, even if the peak of a band is uncertain due to the fusion of two or more bands or the generation of noise, the bands can be suitably separated and detected.

Fourth, the pitch information of the band is updated from the position of the newly detected band and the position of the already detected band, and the position of the next band to be detected is predicted again based on this pitch information. do.

First, the pitch of l(nd) from the i lane where a band was last detected to the j lane where a new band was detected can be directly obtained.
Let it be j.

Additionally, if a band is detected on a lane different from the lane where the band was last detected (i≠j), and a band has already been detected on lane j, the band and the newly detected band The average value obtained by dividing the distance between the two by the corresponding number of pitches is calculated, and this value is set as Pjj in the matrix.

Furthermore, P kj (where k is 1 other than i and j
≦ is an integer in the range of ≦4), it is also a rotation to obtain the average value by using the already determined Pki and calculating P kJ = (P k1 + P 1'') / 2.

Alternatively, for example, when the pitch of the band from the first lane to the second lane is updated from PI3 to P'12, the change in pitch is ΔF +2=P'12- P12. The relative position of each lane remains unchanged (i.e.
If there is no local positional shift or variation between lanes, then this change in pitch can be said to be purely a change in pitch depending on the migration distance. It can be assumed that the pitch of a band changes similarly if it is near the position of the band.

ΔP12=ΔP 32=P 42 Therefore, P'32=P]2+ΔP32 P'42=P42+ΔP42 can be updated for each band.

The latter two update means are effective when the positional deviation between lanes is not so large, and has the advantage that pitch can be finely adjusted over a wide range by detecting one band.

Next, based on this partially updated pitch information, the predicted position of the next band is determined in the same manner as above.

In this way, every time one band is detected based on the predicted position, the pitch information is updated and the operation of predicting the 6th order of the first band is repeated until the upper end of the electrophoresis pattern. bands can be detected one by one.

Since the pitch information is updated every time one band is detected, the position of the next band can always be predicted with high accuracy based on the latest pitch information.

In addition, if the electrophoresis pattern contains various distortions such as smiling phenomenon and offset distortion, or noise such as extra bands, in addition to band fusion, please check these before or during the above operation. Signal processing for correction may be performed.

Here, the smiling phenomenon is a phenomenon in which the migration distance of the slots at both ends of the support medium is shorter than the migration distance of the slots at the center of the support medium, and is caused by the heat dissipation effect (so-called edge effect) during the migration process. It is something that occurs. For details of these corrections by signal processing, see Japanese Patent Application No. 60-74899 and Japanese Patent Application No. 60-7 by the applicant.
No. 4900, Patent Application No. 85275, Patent Application No. 1988-
No. 85276, Patent Application No. 1988-226091, 1982
It is described in the specifications of Japanese Patent Application No. 61-71881 filed on March 29, 1988 and Japanese Patent Application No. 61-90607 filed on April 19, 1988.

By comparing the positions of the detected authentic bands with each other between lanes, the bands can be immediately ranked. At this time, since the combination of the above four types of base-specific DNA fragments is an exclusive combination, two or more bands cannot exist at the same position on different lanes. The ranking can be determined. the above(
Slots 1) to (4) are (G), (A), (
Since it has information about the terminal bases consisting of
-TA-A-G-...) can be obtained.

In this way, 1 for one chain molecule of DNA
′! ! The base sequence can be determined. Note that information about the DNA base sequence is not limited to the above display format; for example, it is also possible to simultaneously display the intensity (Z゛) of each band as the relative amount of the radiolabeled substance, if desired. 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 subjected to the above signal processing. That is, the corrected position of each/hand can be visualized and displayed together with the original autoradiograph. In this case, it is almost possible for the analyst to make the final base sequence determination based on this displayed image.

In addition, in the above, a case was explained in which an exclusive combination of a mixture of base-specific DNA fragments as a sample and L4 (G, A, T, C) was used, but the signal processing method of the present invention uses this combination. The present invention is not limited to, and can be applied to various combinations such as (G, G+A, T+C, C), for example. 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).

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

The information about the base sequence of the nucleic acid determined by the signal processing described above is output from the signal processing circuit and then stored in a recording device 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 symbols @ numerical values 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 recording material.

[Brief explanation of drawings]

FIG. 1 is a diagram showing an example of an electrophoresis pattern in which bands are fused. FIG. 2 is a partial diagram showing a -dimensional waveform consisting of a signal position (X) and a signal level (Z) for each lane.

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. 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 separation pattern consisting of columns, comprising: 1) consecutively detecting at least two bands in the lower region of the separation pattern; 2) obtaining information about the pitch of the band from the position of the detected band, and predicting the position of the next band to be detected in the separation development direction from this pitch information; 3) based on the predicted position. 4) Obtaining new information about the pitch of the band from the position of the already detected band and the position of the newly detected band in the third step. , a step of predicting the position of the next band to be detected in the separation development direction from this pitch information, and 5) a step of detecting all bands on the separation development pattern by sequentially repeating the third and fourth steps above. A signal processing method for determining the base sequence of a nucleic acid, comprising: 2. In the first and third steps above, after obtaining a one-dimensional waveform consisting of the position along the separation development direction and the signal level for each column, the position where the signal level is maximum is determined on this one-dimensional waveform. 2. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein the band is detected by determining the base sequence of a nucleic acid. 3. In the second and fourth steps above, the information about the band pitch is expressed as a matrix: P=(P_1_1P_1_2...P_1_nP_2_1
P_2_2...P_2_n :P_i_iP_i_j: :: P_n_1...P_n_n) (However, i and j are 1≦i≦n and 1≦, respectively.
j is an integer in the range of n, where n is an integer greater than or equal to 2 and equal to the number of separation and expansion columns, P_i_i is the pitch of the band in column i, and P_i_j is the pitch of the band from column i to column j. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, characterized in that the signal processing method is obtained as follows. 4. In the second and fourth steps, the position x_j of the band to be detected next in column j is predicted by calculating the value obtained by adding P_i_j to the position x_i of the last detected band. A signal processing method for determining the base sequence of a nucleic acid according to claim 3. 5. In the third step above, a) If the signal level becomes maximum near the predicted position,
It is determined that a band exists at the position where this maximum occurs. b) If there is a column where the signal level does not reach a maximum near the predicted position and the position cannot be predicted, the position of the last detected band is determined. Find the position where the signal level first reaches a maximum on the column above the column and determine that a band exists at this position, or c) The signal level does not reach a maximum near the predicted position and the position is the same in all columns. If predicted, the band is detected by determining that the band exists in a column with the highest signal level near the predicted position. Signal processing methods for decisions. 6. In the fourth step, a) the pitch of the band from column i where a band was detected last to column j where a band was newly detected is expressed as P in the matrix.
_i_j, and b) If a band has already been detected on column j, the average value obtained by dividing the distance between this band and the newly detected band by the number of band pitches is set as P_j_j in the matrix. 4. The signal processing method for determining the base sequence of a nucleic acid according to claim 3, wherein information about the pitch of the band is newly obtained. 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 separation 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 .