JPH0570791B2 - - Google Patents

Description

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

[Background of the invention]

In the field of molecular biology, which has developed rapidly in recent years, it has become essential to clarify the genetic information of living organisms in order to elucidate their functions and replication mechanisms. . Above all,
It has become 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 uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
-Gilbert method and Sanger-Coulson method are known. The former Maxam-Gilbert method first attaches a group containing a radioactive isotope such as ^32P to one end of a chain molecule of DNA or DNA fragments whose base sequence is to be determined. After converting a substance into a radioactively labeled substance, chemical means are used to cleave the bonds between each constituent unit of the chain molecule in a base-specific manner. 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 is formed in which a large number of cleavage products are separated and developed (however, it is difficult to visually (cannot be seen). This separated development pattern is visualized on, for example, an X-ray film to obtain its autoradiograph,
Based on the obtained autoradiograph and each base-specific cleavage means, the bases located in a certain positional relationship from the end of the chain molecule to which the radioactive isotope is bound are sequentially determined. The sequence can be determined.

In addition, the latter Sanger-Coulson method is
A DNA compound that is complementary to a chain molecule of DNA or a DNA fragment and has been given a radioactive label is base-specifically synthesized using chemical means, and the base-specific DNA compound is synthesized using chemical means. This method uses a mixture and determines the base sequence from its autoradiograph in the same manner as above.

In order to easily and accurately determine the base sequence of the above nucleic acids, the present applicant has proposed a conventional radiographic method using photographic materials such as the above X-ray film in the autoradiograph measurement operation used therein. Instead, a patent application has already been filed for a method using a radiation image conversion method using a stimulable phosphor sheet (Japanese Patent Laid-Open No. 59-83057, Japanese Patent Application No. 58-201231 (Japanese Patent Laid-Open No. 60-93354). )). here,
The stimulable phosphor sheet is made of stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, it is excited with electromagnetic waves (excitation light) in the visible to infrared region. This allows radiation energy to be emitted as fluorescence. According to this method, the exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur.
Furthermore, 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. I can do it.

Conventionally, the base sequence of a nucleic acid is 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) for a visualized autoradiograph. This is determined by visually determining the separated development sites (bands) and comparing the positions of those 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. 126527, 1982, Patent Application No. 126527, Patent Application No. 1983-
No. 89615 (Japanese Unexamined Patent Publication No. 1983-23557), Patent Application No. 1983-
No. 226091 (Unexamined Japanese Patent Publication No. 85861, 1983), Patent Application No. 1983-
No. 226092 (Unexamined Japanese Patent Publication No. 62-85862), etc.). When using a conventional radiation film, the digital signal corresponding to the autoradiograph can be obtained by first making the autoradiograph into a visible image on the film and then photoelectrically reading it using reflected or transmitted light. can get. When a stimulable phosphor sheet is used, an autoradiograph can be obtained by directly reading out the phosphor sheet on which the stimulable phosphor sheet has been stored.

However, various distortions and noises tend to occur in separation and development patterns obtained by actually separating and developing a radiolabeled substance on a support medium by electrophoresis or the like. For example, when preparing a sample, the base-specific fragments of a nucleic acid may be insufficiently prepared or separated, or when a sample is injected into each slot of the support medium, samples from other slots may be mixed into the sample. Bands (referred to as ghost bands or extra bands) may appear in positions where they should not appear. Alternatively, noise may occur due to radioactive impurities being mixed into the sample or exposure to natural radiation during the exposure process.

Furthermore, in a region where the bands are dense near the separation/deployment start position, each band may not be sufficiently separated, and a plurality of bands may be fused.

As a result, since bands are comparatively identified including these extra bands, noise, and fusion bands, errors occur in base sequence determination and the accuracy of the information obtained decreases. Even when such noise or band fusion occurs, it is desirable 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]

In a method for automatically determining the base sequence of a nucleic acid using autoradiography, the present invention preferably uses a digital signal corresponding to an autoradiograph, even if the separation pattern includes noise or fusion of bands. By applying signal processing to the system, we were able to easily and automatically determine the base sequence of nucleic acids with high accuracy.

That is, the present invention provides base-specific DNA fragments or base-specific RNAs to which a radioactive label has been added.
Determining the base sequence of a nucleic acid by performing signal processing on digital signals corresponding to autoradiographs of multiple separation and development columns formed by separating and developing a mixture of fragments in one-dimensional direction on a support medium. In the method, 1) On a straight line along the separation development direction x for each separation development column, based on the signal level ls at the search starting point and the signal level threshold ln (where n is 0 or a positive integer) 2) finding the band closest to the search starting point, and 2) determining the area of the band based on the threshold ln, and based on the size of this area: a) determining that the band is a true band; or b) determining that the band is not a true band and starting over from the first step, thereby detecting one band for each separation expansion column. The present invention provides a signal processing method for determining the base sequence of a nucleic acid.

According to the present invention, with respect to a digital signal corresponding to an autoradiograph of a separation pattern obtained by separating and developing a mixture of base-specific fragments of a nucleic acid on a support medium, noise and/or fusion in the separation pattern can be obtained. Even if a band occurs, a signal processing circuit that has a function that can effectively detect only the true band without determining noise as a band and separating the fused band into individual bands. By passing it through, the base sequence of a nucleic acid can be automatically obtained easily and with high precision.

That is, a signal level threshold and a certain range of band sizes are set in advance, and if a signal exceeding the threshold is found on the search line along the separation development direction, it is assumed that a band exists, and then this band is By determining that it is a genuine band when the size is within a certain range, the band can be accurately determined without mistaking noise such as extra bands as a band, or misreading a fused band as one band. can be detected.
In particular, if the size of the band exceeds the preset range, change the threshold to a larger value and repeat the above operation to eliminate the influence of noise and accurately separate the fused band. You can confirm the band.

In addition, since the separation and development conditions of the sample are easily influenced by the development conditions, the separation and development column itself may curve or meander, or when the support medium and the photographic light-sensitive material or stimulable phosphor sheet are overlapped and exposed. In many cases, the entire pattern is tilted due to misalignment of the overlapping patterns. In order to automatically determine the base sequence of a nucleic acid with high precision, it is generally desirable to create a one-dimensional waveform (profile) that precisely follows the lane for each separation/development column (lane). Alternatively, if the pattern is tilted, it is necessary to track each lane when creating the pattern. According to the present invention, since the true band and its shape can be determined, it is possible to suitably perform lane tracking using this result.

Then, by performing more suitable signal processing on the detected authentic band and the one-dimensional profile based thereon, the base sequence of the nucleic acid can be automatically determined simply and with high precision.

[Structure of the invention]

Examples of samples used in the present invention include:
Examples include mixtures of base-specific fragments of nucleic acids such as DNA and RNA that have been given radioactive labels. Here, the nucleic acid fragment means a part of a long chain molecule. For example, a base-specific DNA cleavage mixture, which is a type of base-specific DNA fragment mixture, is produced by base-specific cleavage and decomposition of radiolabeled DNA according to the Maxam-Gilbert method described above. can get.

In addition, the base-specific DNA compound mixture is prepared using the Sanger-Coulson method as described above, using DNA as a template and adding deoxynucleoside triphosphate to which a radioactive label has been added.
It can be obtained by synthesis using DNA synthase.

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

Radioactive labels can be applied to these substances in an appropriate manner.
It is imparted by retaining radioactive isotopes such as ³² P, ¹⁴ C, ³⁶ S, ³ H, ¹²⁵ I, etc.

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

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

When using the former radiographic method, first the support medium and a photographic light-sensitive material such as an X-ray film are overlapped for a long time (several hours to several tens of hours) at low temperature or room temperature, and then the radiographic film is exposed. , and developed to produce a visible image of the autoradiograph of the radiolabeled substance on radiographic film. Next, the autoradiograph visualized on the radiographic film is read using an image reading device. For example, an autoradiograph is obtained as an electrical signal by irradiating a radiation film with a light beam and photoelectrically detecting the transmitted or reflected light. 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 the stimulable phosphor sheet are overlapped for a short time (several seconds to several tens of minutes) at room temperature, and the radiation energy emitted from the radiolabeled substance is accumulated in the phosphor sheet. is recorded on the phosphor sheet as a kind of latent image. Here, the stimulable phosphor sheet is made of a support made of, for example, a plastic film, divalent europium-activated barium fluoride bromide (BaFBr: Eu ²⁺ )
A phosphor layer made of a stimulable phosphor such as phosphor and a transparent protective film are laminated in this order. The stimulable phosphor contained in the stimulable phosphor sheet absorbs and accumulates radiation energy when it is irradiated with radiation such as X-rays, and then accumulates when excited with light in the visible to infrared region. It has the property of emitting radiation energy as photostimulated light.

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

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

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

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

The obtained digital signal Dxy consists of coordinates x, y expressed in a coordinate system fixed to the radiation film (or phosphor sheet) and the signal level z at those coordinates, and one signal corresponds to one pixel. It corresponds to The level of the signal represents the image density at that coordinate, ie, the amount of radiolabeled substance. Therefore, a series of digital signals (i.e.
(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.

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

1) Guanine (G) specific DNA fragment 2) Adenine (A) specific DNA fragment 3) Thymine (T) specific DNA fragment 4) Cytosine (C) specific DNA fragment Here, each base specific DNA fragments are DNA fragments of various lengths that have been cleaved or synthesized in a base-specific manner, that is, have the same base at the end.
Consists of fragments.

FIG. 1 shows an autoradiograph of the electrophoresis pattern obtained by electrophoresing the four types of base-specific DNA compounds described above into four slots. The migration direction is the x direction.

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

First of all, a straight line (search line) along the electrophoresis direction x starting from the search start point for each electrophoresis column (lane)
In the above, a band is searched based on the signal level ls at the search starting point and the initial threshold value lo of the signal.

Here, the direction of electrophoresis does not mean the direction of electrophoresis that completely matches the actual pattern in the case of meandering lanes and inclination of the pattern, but rather the direction in which the sample is intended to be electrophoresed. means the long axis direction (x direction) of the support medium (ie, the superimposed photographic material or stimulable phosphor sheet).

Furthermore, any point in the migration direction can be selected as the search starting point for each lane. Search for bands in the migration direction (from the top of the pattern to the bottom)
It is preferable to do so. The initial threshold lo is the signal level threshold (reference value) for determining the presence or absence of a band.
This is the initial value of ln (where n is 0 or a positive integer) and is set to a relatively low value. If the size of the band is above a certain range, the setting can be sequentially changed to higher values as will be described later.

Specifically, bands are detected in the following manner, for example.

FIG. 2 1 is a partial autoradiograph for one lane, and is a diagram in which band 1 is displayed as a two-dimensional grayscale image. The band search is performed on a straight line 2 along the migration direction x.

FIG. 2 shows a one-dimensional waveform of the straight line 2 consisting of the position in the x direction and the signal level. That is, the band search is performed based on this one-dimensional waveform, but since the digital signal corresponds to each readout (readout) pixel, the one-dimensional waveform is actually a set of discontinuous points.

When cases are divided based on the relationship between the signal level ls at the search starting point and the initial threshold lo, five cases as shown in FIG. 3 can occur. Note that FIG. 3 is a diagram partially showing the one-dimensional waveform of FIG. 2, with white dots representing search start points and black dots representing band detection points. That is, (1) If the starting level ls is less than the initial threshold lo (ls≦lo) and the signal level tends to increase (see Figure 3 1), (2) If the starting level ls is less than the initial threshold lo (ls≦lo) (ls≦lo) and the signal level tends to decrease [Figure 3 2], (3) The starting level ls is higher than the initial threshold lo (ls>
lo), and the signal level tends to increase [Fig. 3 3], (4) If the starting level ls is higher than the initial threshold lo (ls>
lo), and the signal level tends to decrease (and the signal level once falls below the threshold lo)
[Fig. 3 4], and (5) the starting level ls is higher than the initial threshold lo (ls>
lo), and the signal level tends to decrease but does not fall below the threshold lo [Fig. 3, 5].

Note that the case (5) above occurs when the bands are close to each other and the interval between the bands is not sufficient, and represents so-called fusion of the bands.

Cases (1) and (2) above (ls≦lo) are the simplest cases, and the next point on the one-dimensional waveform where the signal level becomes equal to or higher than the threshold lo for the first time is defined as a band detection point.

In the case of (3) above (ls>lo), the starting point is already in the region of the band to be detected, so the point where the signal level next becomes equal to or higher than the starting level ls is set as the band detection point.

In case (4) above (ls > lo), the starting point is still in the detected band area, so the band detection point is the point where the signal level once falls below the threshold lo and then exceeds the threshold lo for the first time. do.

In the case of (5) above (ls>lo), since the signal level between bands does not become less than the threshold value lo, the next point at which the signal level becomes equal to or higher than the starting level ls for the first time is set as the band detection point.

By determining the band detection point in this way,
Band detection can be performed without detecting an already determined band again or failing to find the next band to be detected.

Second, the area of the found band is determined based on the threshold lo, and then it is determined from the size of the area whether or not the band is a true band,
A band should be detected only if it is a true band.

First, assuming that a band is a set of points connected to the band detection point, a point is found that is continuous from the band detection point either vertically, horizontally, or diagonally, and has a signal level equal to or higher than the threshold lo. A continuous area including the obtained band detection points is a band area.

Next, the size of the band area is determined. The size of the area is, for example, the maximum distance in the x direction of the area x ₀
and the maximum distance y ₀ in the direction y perpendicular to the x direction.

FIG. 4 shows an example of the searched band area (hatched area: 3).

The minimum width xmin, ymin in the x direction and y direction, and the maximum width xmax, ymax in the x direction and y direction, which are acceptable as one band, are set in advance. In other words, xmin and ymin are the set values to distinguish between true bands and noise,
xmax and ymax are setting values for distinguishing between a single band and a band in which multiple bands are combined.

a) Distances x ₀ and y ₀ are both within the above minimum width and maximum width (lmin ≦x ₀ ≦xmax and
ymin≦yo≦ymax), the obtained band is determined to be a true band, and this band is set as the band to be detected.

b ₁ ) If at least one of distance xo and distance yo is smaller than the above minimum width (xo < xmin or yo < ymin), the obtained band is determined to be an extra band and not a true band. After that, a band search is performed for an area further below the band detection point.

b ₂ ) In cases other than a and b ₁ above, the distance
At least one of xo and distance yo is larger than the above maximum width (xo > xmax or yo
>ymax), it is assumed that the band separation is not sufficient, and the threshold value l ₀ is changed to l ₁ and the band search is performed again from the search starting point.

Here, the new threshold l ₁ is the value obtained by adding a constant increment Δl to l ₀ (l ₁ = l ₀ + Δl), and by increasing the threshold and re-searching for bands, the fused bands can be removed one by one. The true band can be determined by separating the signal into two, or the extra band with a relatively low signal level can be suitably removed.

In general, if the band area is too large, the threshold value ln can be changed to a higher value l _o+1 (l _o+1 = l _o +Δl; where n+1 represents the number of times the threshold value has been changed) as many times as necessary. , the band search can be repeated from the search starting point. In this case, the maximum value of the threshold value
lmax is set, and if the changed threshold l _o+1 is less than the maximum value (l _o+1 ≦ lmax), this threshold l _o+1
The band search is redone from the search starting point based on , but the threshold l _o+1 becomes larger than the maximum value (l _o+1 >
lmax), it is preferable from the viewpoint of processing efficiency and accuracy to determine that the band is not a true band and perform a band search in the region below the band detection point, since it is impossible to separate the band.

Note that the determination of the size of a band is not limited to the above-mentioned means, and can also be determined based on, for example, the number of signals included in a band region.

In this way, the band search is repeated until one true band for each lane is detected (until case a above occurs).

Using the authentic bands detected in this way, the base sequence of DNA can be automatically determined. For example, each lane is appropriately tracked using the obtained true band region to obtain an accurate one-dimensional waveform (profile lane) consisting of the position in the x direction and the signal level, and then The base sequence can be determined automatically by performing appropriate signal processing.

For example, lane tracking can be performed as follows.

First, among the bands detected in each lane, for the lane that has the band closest to the electrophoresis start position, that is, the band located at the top of the pattern (this is referred to as band A), select the next tracking start point (simultaneously with the next band). (which is also the starting point of the search).

The y-coordinate of the next tracking start point is the midpoint of the maximum distance in the y-direction in region 3 of band A, as shown in FIG. Furthermore, the x-coordinate is the x-coordinate of the point farthest from the tracking start point among the points having this y-coordinate within the region of band A. In FIG. 4, coordinates xa and ya are the next tracking start point, and lane tracking (and band search) is performed in the direction of the arrow.

Next, for each of the remaining three lanes,
Determine the next tracking start point.

Band of other lane (this will be called band B)
If the region of band A and the region of band A in the x direction overlap even slightly, the coordinate xa is set as the x coordinate of the next tracking start point, and the midpoint of the maximum distance in the y direction in the region of band B is set as the next tracking start point. Let be the y coordinate of the point.

In addition, the band B area of other lanes and the band A area
If the area in the x direction of
The value obtained by adding the difference between the coordinates ya of the next tracking start point in the lane and the first tracking start point (search start point) ys to the first tracking start point ys' of the band B lane: ys' + (va −ys) is the y-coordinate of the next tracking start point.

Then, a band search is performed again according to the method described above on a straight line along the x direction from the tracking start point newly set for each lane toward the bottom of the pattern.

At this time, the threshold value ln always starts from the initial value _l0 .
It is preferable that the threshold value ln is set to be less than or equal to lmax.

The band detection operation and the lane tracking operation according to the present invention are sequentially repeated until no bands are detected. Thereby, lane tracking can be completed from the upper end of the lane to the lower end.

For details on lane tracking mentioned above,
Patent application filed on April 19, 1988 by the applicant
-90607 (Japanese Unexamined Patent Publication No. 62-247225).

Based on the lane tracking information obtained in this way, for example, a constant width signal along the lane is extracted to obtain a one-dimensional profile, and then the position where the signal level is maximum is determined for the profile. Determine the position of the band by By comparing the positions of authentic bands determined between lanes, it is possible to immediately rank the bands. At this time, since the combination of the above four types of base-specific DNA compounds is an exclusive combination, two or more bands cannot exist at the same migration position on different lanes. The order can be determined. Above (1)
The slots in ~(4) each have information about the terminal bases consisting of (G), (A), (T), and (C), so by replacing the bases with the bases corresponding to the slots to which each band belongs, the DNA base sequence (e.g. A-G-C
-TA-A-G-...) can be obtained.

In this way, the base sequence of one chain molecule of DNA can be determined. In addition,
Information about the DNA base sequence is not limited to the above display format; for example, if desired, the intensity z' of each band can be displayed simultaneously as the relative amount of the radiolabeled substance. moreover,
It is also possible to display the base sequences of both DNA strands.

Alternatively, DNA base sequence information can also 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 band can be visualized and displayed together with the original autoradiograph. In this case, it is possible for the analyst himself to determine the final base sequence based on this displayed image.

In addition, in the above, the sample base-specific
We have explained the case where an exclusive combination of G, A, T, and C is used as a mixture of DNA compounds.
The signal processing method of the present invention is not limited to this combination, and can be applied to various combinations such as G, G+A, T+C, and C, for example. At the same time, the method of the present invention can also be applied to a mixture of base-specific RNA fragments (for example, a combination of G, A, U, and C). Furthermore, band detection is not limited to the separation and development column of a set of base-specific fragments of nucleic acids, but can be performed for all separation and development columns that are simultaneously separated and developed on the support medium. .

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.

Examples of recording devices include those that optically record by scanning a photosensitive material with a laser beam or the like;
Recording devices can be used based on various principles, such as those that record symbols and numbers displayed on a CRT or the like on a video printer or the like, and those that record on a heat-sensitive recording material using heat rays.

[Brief explanation of the drawing]

FIG. 1 is a partial diagram showing an example of an autoradiograph of a migration pattern. 1 in FIG. 2 is a partial diagram showing an example of an autoradiograph (gradation image) for one lane, and 2 shows a one-dimensional waveform consisting of the position in the x direction and the signal level for the straight line 2. In FIG. FIGS. 3 1-5 are partially enlarged views of the one-dimensional waveforms of FIG. 2. FIG. 4 is a diagram showing an example of a detected band area. 1: band, 2: straight line (search line), 3: band area, white dot: tracking start point, black dot: band detection point.

Claims (1)

[Scope of Claims] 1. A plurality of separated and developed arrays formed by separating and developing a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments in a one-dimensional direction on a support medium. In a method for determining the base sequence of a nucleic acid by performing signal processing on a digital signal corresponding to an autoradiograph of 2) finding one band closest to the search starting point based on the level ls and the signal level threshold ln (where n is 0 or a positive integer); and 2) determining the region of the band based on the threshold ln. Then, based on the size of this region, a) the band is determined to be a true band and the band is detected; or b) the band is determined not to be a true band and the above first step is performed. 1. A signal processing method for determining a base sequence of a nucleic acid, comprising the steps of: starting over from scratch, and detecting one band for each separation and expansion column. 2 In the above first step, regarding the signal level ls at the search starting point and the signal level threshold ln, a) When ls≦ln, the signal level is next to the threshold
The point where the signal level is equal to or higher than ln is the band detection point, or b) When ls>ln, the signal level is next to the threshold value.
Claim 1, characterized in that the band detection point is defined as a point where the signal level becomes equal to or higher than the threshold value ln after falling below the threshold value ln, or a point where the signal level next becomes equal to or higher than the starting level ls without falling below the threshold value ln. A signal processing method for determining the base sequence of a nucleic acid as described in Section 3. 3 In the second step above, the signal level reaches the threshold ln
After finding a continuous area including the above band detection points, regarding the maximum distance xn in the x direction and the maximum distance yn in the y direction of this area, a) xmin≦xn≦xmax, and ymin≦yn
When ≦ymax, the band is detected by determining the area separated by distances xn and yn as the true band, b ₁ ) When xn<xmin or yn<ymin,
After determining that the area separated by distances xn and yn is not a true band, the process starts over from the first step in the search direction from the band detection point. or b ₂ ) When xn>xmax or yn>ymax, the area separated by the distances xn and yn is determined not to be a true band, and then the threshold is set as ln+Δl=
l _o+1 and start over from the first step in the search direction from the search starting point. (However, xmin, xmax, ymin, and ymax are constants, respectively, , the minimum width in the y direction, and the maximum width in the y direction; Δl is a constant increment of the threshold value ln). Signal processing method. 4 In the second step _b2 above, _b21 ) When l _o+1 ≦lmax, set the threshold to l _o+1 and start over from the first step in the search direction from the search starting point, or _b22 ) l _o+1 >lmax, the distances xn and yn
After determining that the area delimited by A signal processing method for determining the base sequence of a nucleic acid according to claim 3. 5. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein in the first step, a band is searched in the direction of separation and development. 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 fragment. A patent claim characterized in that the separation and development column consists of four separation and development columns formed by separating and developing each of these four types of base-specific DNA fragments on a support medium. A signal processing method for base sequencing of cough acid according to item 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 on the phosphor sheet. Claim 1, characterized in that the autoradiograph is obtained by accumulating and recording the phosphor sheet, irradiating the phosphor sheet with excitation light, and photoelectrically reading out the autoradiograph as photostimulated light. A signal processing method for base sequencing of the described nucleic acid. 8 A digital signal corresponding to the above-mentioned autoradiograph is transferred onto the photosensitive material after superimposing the support medium and the photosensitive material to photosensitively record the autoradiograph of the radiolabeled substance on the support medium on the photosensitive material. 2. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein the signal processing method is obtained by photoelectrically reading a visualized autoradiograph.