JPH0529072B2 - - Google Patents

Description

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

[Background of the invention] In the field of molecular biology, which has developed rapidly in recent years, it is essential to clarify the genetic information of living organisms in order to elucidate their functions and replication mechanisms. It has become commonplace. Above all,
It has become essential to determine the base sequence of nucleic acids such as DNA (or 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 (however, visually (cannot be seen). This separated development pattern is visualized on, for example, an X-ray film to obtain its autoradiograph,
Based on the obtained autoradiograph and each base-specific 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 been filed for a method using a radiation image conversion method using a stimulable phosphor sheet (Japanese Patent Laid-Open No. 59-83057, Japanese Patent Application No. 58-201231). Here, the stimulable phosphor sheet is made of stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, electromagnetic waves (excitation light) in the visible to infrared region are emitted.
By exciting it with , radiation energy can be emitted as fluorescence. According to this method, the exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur. Furthermore, 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 these bands with each other. Therefore, analysis of autoradiographs is usually performed through human vision, which requires a great deal of time and effort.

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

Therefore, the applicant has already filed a patent application for a method for automatically determining the base sequence of DNA by obtaining the above-mentioned autoradiograph as a digital signal and then subjecting this digital signal to appropriate signal processing. (Unexamined Japanese Patent Publication No. 126527, 1982, Patent Application No. 126527, Patent Application No. 1983-
No. 89615, Patent Application No. 1983-226091, Special Application No. 1983-
226092). When using a conventional radiation film, the digital signal corresponding to the autoradiograph can be obtained by first making the autoradiograph into a visible image on the film and then photoelectrically reading it using reflected or transmitted light. can get. When a stimulable phosphor sheet is used, an autoradiograph can be obtained by directly reading out the phosphor sheet on which the stimulable phosphor sheet has been stored.

However, various distortions and noises tend to occur in separation and development patterns obtained by actually separating and developing radiolabeled substances on a support medium by electrophoresis or the like. For example, the separation and development column itself may become bent and meander due to insufficient development conditions during the separation and development process, or the superposition of the support medium and the photographic material or stimulable phosphor sheet may become misaligned during the exposure process. read (read)
During the process, the entire pattern may be tilted due to insufficient placement of the photosensitive material or phosphor sheet.

Furthermore, positions that should not appear may be caused by insufficient preparation and separation of base-specific fragments of nucleic acids during sample preparation, or by contamination of other samples into the slot when injecting the sample into the support medium. A band (called a ghost band or extra band) may 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.

Even if distortion occurs in the separation expansion sequence in this way, even if the obtained digital signal contains distorted information of the separation expansion sequence, and even if noise or band fusion occurs However, it is desirable to efficiently process the digital signals corresponding to the autoradiograph and automatically determine the base sequence of the nucleic acid with high precision.

[Summary of the Invention] The present inventor has proposed a method for automatically determining the base sequence of a nucleic acid using autoradiography,
By suitably processing the digital signal corresponding to the autoradiograph of a separation/development pattern in which the separation/development sequence is distorted, it is possible to automatically determine the base sequence of a nucleic acid easily and with high accuracy. did.

Furthermore, the present inventors have achieved automatic determination of the base sequence of a nucleic acid with ease and high accuracy even when noise or band fusion occurs in the separation and development pattern.

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) for each separation deployment column, detecting one band along the separation deployment direction x from the tracking start point of the column; (2) detecting the upper part of these bands closest to the separation deployment start position; After finding the maximum and minimum values of the region of band A in the direction y perpendicular to the x direction for the separation expansion array having band A, the average value ya of the maximum and minimum values is calculated as the average value ya of the next tracking start point. y coordinate, and the maximum value xa in the x direction of the points having ya in the band area is set as the x coordinate of the next tracking start point. (3) For the remaining separation expansion columns, (a) If the region in the x direction overlaps the region of band B of the remaining separation expansion column, xa
is the x-coordinate of the next tracking start point, and the average value yb of the maximum and minimum values in the y direction of the band B area is
(b) If the area of band A in the x direction and the area of band B of the remaining separation deployment row do not overlap,
Let xa be the x-coordinate of the next tracking start point, and add the difference between ya and the y-coordinate of the tracking start point of the column with band A to the y-coordinate of the tracking start point of the column with band B to the next tracking. A method for determining the base sequence of a nucleic acid, comprising the following steps: determining the y-coordinate of the starting point; and (4) following the sequence for each separation development sequence by sequentially repeating the first to third steps above. The present invention provides a signal processing method for

According to the present invention, with respect to a digital signal corresponding to an autoradiograph of a separation and development pattern obtained by separating and developing a mixture of base-specific fragments of nucleic acids on a support medium, separation and development columns (lanes) are distorted. Even when the base sequence of a nucleic acid is passed through a signal processing circuit that has a function of accurately tracking lanes, the base sequence of the nucleic acid can be automatically obtained easily and with high precision.

Further, according to the present invention, even if noise and/or fusion bands occur in the separation development pattern, the lane can be separated into individual fusion bands without picking up noise during the tracking process. However, only genuine bands can be detected effectively. By detecting this effective band, lane tracking can be performed with even higher accuracy.

That is, first, one band is detected in each lane, and for the lane with the band closest to the separation/deployment start position, the x and y coordinates of the next tracking start point are effectively determined from the band area, and this x coordinate is This is the x-coordinate of the tracking start point that is common to each lane. Regarding the y-coordinate of the next tracking start point in another lane, if the area of this band and the band of another lane overlaps even slightly, the y-coordinate is obtained from the band area in the same manner as above, and If the band areas of the lanes do not overlap at all, the y-coordinate is determined by adding the changed value of the y-coordinate of the lane. In this way, lane tracking can be performed with high precision by correcting the tracking start point of each lane every time one band is detected from the top to the bottom area of the pattern.

In particular, when detecting a band, a signal level threshold and a certain range of band size are set in advance, and if a signal exceeding the threshold is found on the tracking line along the separation deployment direction, it is determined that there is a band. Then, if the size of this band is within a certain range, it is determined as a genuine band, so that noise such as an extra band is not mistaken as a band, and the fused band is not mistaken as one band. Nor. 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 confirm the band while accurately separating the fused band. can do.

Based on this accurate lane tracking information, it is possible to obtain a one-dimensional waveform (a profile that matches the pattern) along each lane, and perform more suitable signal processing on this one-dimensional profile. By doing so, the base sequence of a nucleic acid can be automatically determined simply and with high accuracy.

[Structure of the Invention] In the present invention, examples of samples used include a mixture 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, base-specific DNA
A base-specific DNA cleavage and decomposition product mixture, which is a type of 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 fragment mixture is prepared using the above-mentioned Sanger-Coulson method, 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 DNA fragments can also be obtained as a mixture of cleavage and decomposition products or as a mixture of synthetic products by the same method as above. Furthermore, DNA consists of four types of bases as its constituent units: adenine, guanine, thymine, and cytosine, while RNA consists of adenine, guanine, uracil,
Consists of four types of bases: cytosine.

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

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

Next, an autoradiograph of the support medium on which the radiolabeled substance has been separated and developed is obtained by radiography using a conventional photographic light-sensitive material or by a radiation image conversion method using a stimulable phosphor sheet. A digital signal corresponding to the autoradiograph of the autoradiographic material 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 _D It corresponds to one pixel. 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 on the support medium thus obtained is subjected to signal processing according to the method of the present invention as described below, and the base sequence of the target nucleic acid is determined. 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 is formed by a combination of fragments (separation and development column).

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

FIG. 1 shows 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, one band is detected for each electrophoresis column (lane) on a straight line (tracking line) along the electrophoresis direction x starting from the tracking start point.

Band detection can be performed, for example, by the following operation.

First, a band is determined for each lane on a straight line along the migration direction x based on the signal level ls at the tracking start point and the initial signal threshold lo.

Here, the direction of electrophoresis does not mean the direction of electrophoresis that completely matches the actual pattern when meandering lanes and tilted patterns occur, but rather the direction in which the sample was intended to be electrophoresed. It usually means the longitudinal direction (x direction) of the support medium (ie, the superimposed photographic material or stimulable phosphor sheet).

In addition, it is usually preferable to select the initial tracking start point near the migration start point (the left end in Figure 1), where the lane position is not too far off, and the tracking should be performed from the top to the bottom of the migration. do. The initial threshold lo is the initial value of the signal level threshold (reference value) ln (where n is 0 or a positive integer) for determining the presence or absence of a band, and is set to a relatively low value. If the size of the band is above a certain range, the setting may 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, 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.

If the cases are divided based on the relationship between the signal level ls at the tracking start point and the initial threshold value lo, five cases as shown in FIG. 3 may occur. Note that FIG. 3 is a diagram partially showing the one-dimensional waveform of FIG. 2, with white dots representing tracking start points and black dots representing band detection points. That is, (1) when the starting level ls is less than the initial threshold lo (ls≦lo) and the signal level tends to increase [Fig. 3 1], (2) when the starting level ls is less than the initial threshold 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 [Figure 3], (4) 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)
If [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].

Incidentally, case 5 above occurs when the bands are close to each other and the distance between them is not sufficient, and represents so-called band fusion.

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

In case 3 above (ls>lo), the start 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 start level ls is determined as the band detection point.

In case 4 above (ls>lo), the starting point is still in the detected band region, so the band detection point is defined as the point where the signal level once falls below the threshold lo and then becomes equal to or higher than the threshold lo for the first time.

In case 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,
A band search can be performed without detecting an already determined band again or failing to search for a band to be detected next.

Next, the area of the detected band is determined based on the threshold value lo, and then it is determined whether the band is a true band based on the size of the area, and it should be detected only if it is a true band. Band.

Assuming that the band is a set of points connected to the band detection point, a point is determined 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.

The size of the band area can be estimated, for example, by the maximum distance (difference between the maximum value and the minimum value) xo of the area in the x direction and the maximum distance yo in the y direction perpendicular to the x direction.

FIG. 4 shows an example of the 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) The distances xo and yo are both within the range of the above minimum width and maximum width (xmin≦xo≦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 considered to be an extra band and not a true band. After the determination, 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,
If at least one of distance 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 lo is changed to l ₁ and tracking is started. Start the band search again from point.

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

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

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 is detected for each lane (until case a occurs).

Second, among the bands detected for each lane, the next tracking start point is determined for the lane that has the band closest to the migration start position, that is, the band located at the top of the pattern (this is referred to as band A). .

The y-coordinate of the next tracking start point is the average value ya of the maximum and minimum values in the y-direction in region 3 of band A, as shown in FIG. Further, the x coordinate is the x direction and the maximum value xa among the points having the coordinate ya within the region of band A. This allows accurate tracking along the lane without failing to detect the band. In Figure 4, the coordinates (xa,
ya) 4 is the next tracking start point, and tracking continues in the direction of the arrow.

Third, the next tracking start point is determined for each of the remaining three lanes.

5 and 6 show the relationship between the band area and the next tracking start point.

First, the x coordinate of the next tracking start point in each lane is assumed to be the same coordinate xa as that of the band A lane. As a result, lane tracking is performed by correcting the tracking start point each time a band is detected in any one lane, so tracking can always be performed along the lane without missing a band detection.

Next, (a) As shown in Fig. 5, the band of the other lane (this is called band B) and the x of area 5 and band A.
If the directions and regions overlap even slightly, the average value yb of the maximum and minimum values in the y direction in region 5 of band B is set as the y coordinate of the next tracking start.
Tracking continues in the direction of the arrow from the next tracking start point 6. Band B is the coordinate of the next tracking start point
Since it is located close to xa, the y coordinate can be suitably determined based on the band B area.

(b) Band B of other lanes as shown in Figure 6.
If the x-direction area of band A in area 7 does not overlap at all, the coordinates ya of the next tracking start point in the lane of band A and the first tracking start point
Let the value obtained by adding the difference from ys to the beginning of the band B lane and the tracking start point ys': yb'=ys'+(ya-ys) be the y-coordinate of the next tracking start point. Tracking continues in the direction of the arrow from the next tracking start point 8. In general, lane meandering often appears in a similar tendency throughout the pattern, so if there is no band near the coordinate xa of the next tracking start point of another lane, tracking in lane band A will start. By assuming that the corrected value of the y-coordinate of the point is the same and changing the y-coordinate of the lane by that amount, tracking can be carried out further along the lane.

Fourth, a band search is performed again in the same manner as 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 threshold value lo. It is preferable that the threshold value ln is set to be less than or equal to lmax.

Repeat the above operations until no bands are detected. Thereby, lane tracking can be completed from the upper end of the lane to the lower end of the lane.

FIG. 7 partially shows another example of a migration pattern in which lane tracking has been completed. The polygonal line on the lane represents the tracking trajectory, and the rectangle represents the size of the band area.

Based on the lane tracking information obtained in this way, the DNA base sequence can be automatically set with high accuracy.

For example, a signal with a constant width along the lane is extracted based on lane tracking information, a one-dimensional waveform (profile) consisting of the position in the x direction and the signal level is obtained with high accuracy, and this one-dimensional profile is By further subjecting the isle to appropriate signal processing, the base sequence can be determined.

Furthermore, if various distortions such as a smiling phenomenon or offset distortion occur in the migration pattern, signal processing may be performed to correct these distortions before determining the band position for the profile. .

Here, the smiling phenomenon is a phenomenon in which the migration distance in the slots at both ends of the support medium is shorter than the migration distance in the slot in the center of the support medium, and is caused by the heat dissipation effect (so-called edge effect) during the migration process. arise. Offset distortion refers to the overall positional deviation between lanes, and is caused by the fact that the start position and start time of sample electrophoresis differ for each slot due to differences in the shape of the slots.

For details of signal processing for these corrections, see Japanese Patent Application No. 60-74899 and Japanese Patent Application No. 74899 filed by the applicant.
No. 60-74900, Special Application No. 1983-85275, Special Application No. 1983-
It is described in each specification of No. 85276.

Next, by determining the position of the profile where the signal level is maximum, for example, the position of the band can be determined accurately. 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 ranking can be easily determined. The slots 1 to 4 above 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-T-A-AG-...) can be obtained.

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

Alternatively, 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
As a mixture of DNA compounds (G, A, T, C)
Although the case where exclusive combinations of (G, G+A, T+
It can be applied to various combinations such as C and C). Similarly, the method of the present invention can also be applied to a mixture of base-specific DNA fragments (for example, a combination of G, A, U, and C). Furthermore, lane tracking 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 recording 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 based on various principles can be used, such as those that record symbols and numbers displayed on a CRT or the like using a video printer, or 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 (shaded 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 the detected band area and the next tracking start point. FIG. 5 and FIG. 6 are diagrams showing other examples of the detected band area and the next tracking start point, respectively. FIG. 7 is a partial diagram showing an example of a migration pattern for which lane tracking has been completed. 1: Band, 2: Straight line (tracking line), 3, 5,
7: band area, 4, 6, 8: next tracking start point, white dot: tracking start point, black dot: band detection point.

[Claims]

1. Two sound collecting means that are provided so as to aim upward with their central axes tilted symmetrically from the vertical line, and have directivity for collecting flight sounds emitted from flying objects and outputting collected sound signals; a first calculation means that adds and subtracts the sound collection signals of both the sound collection means to form a sum signal and a difference signal; and a second calculation means that derives a signal of a level difference between the sum signal and the difference signal. and a time period during which the sum signal, the difference signal, and the level difference signal exceed different threshold levels, and a time period during which the level difference signal exceeds the respective threshold levels. The present invention is characterized by comprising processing means for detecting the intrusion of the flying object into the detection area and identifying the intruding flying object only when the time difference until the threshold level is exceeded satisfies a predetermined condition. A flying object detection device.

Claims (1)

(b) If the area of band A in the x direction and the area of band B of the remaining separation expansion column do not overlap,
Let xa be the x-coordinate of the next tracking start point, and add the difference between ya and the y-coordinate of the tracking start point of the column with band A to the y-coordinate of the tracking start point of the column with band B to the next tracking. A method for determining the base sequence of a nucleic acid, comprising the following steps: determining the y-coordinate of the starting point; and (4) tracking each separation and expansion column by sequentially repeating the first to third steps. signal processing methods for. 2 In the first step, (i) for each separation deployment column, on a straight line along the separation deployment direction x from the column tracking start point, the signal level ls and the signal level threshold ln (however,
(n is 0 or a positive integer), and (ii) after determining the area of the band based on the threshold ln, the size of this area is (a) determining that the band is a real band and detecting the band; or (b) determining that the band is not a real band and starting over from step (i) above. 2. A signal processing method for determining the base sequence of a nucleic acid according to claim 1. 3 In step (i) above, regarding the signal level ls at the tracking start point and the signal level threshold ln, (a) When ls≦ln, the signal level is next set to the threshold ln.
or (b) When ls>ln, the signal level is set to the next threshold ln.
Claim 2, characterized in that the point where the signal level becomes equal to or higher than the threshold value ln after falling below the threshold value ln, or the point where the signal level next becomes equal to or higher than the starting level ls without falling below the threshold value ln, is defined as the band detection point. A signal processing method for base sequencing of the described nucleic acid. 4 In step (ii) above, the signal level reaches the threshold ln
After finding a continuous region 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 region, (a) xmin≦xn≦xmax and ymin≦yn≦
When ymax, the band is detected by determining the area separated by the 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,
restart from step (i) along the direction, or (b ₂ ) when xn<xmax or yn<ymax,
After determining that the area separated by distances xn and yn is not a true band, the threshold is set as ln+Δl=ln ₊₁
(i) along the x direction from the tracking start point.
(However, xmin, xmax, ymin, and ymax are each constant, and x that is recognized as a band is
Δl is a constant increment of the threshold value ln). Signal processing method for base sequencing. 5 In step (ii) (b ₂ ) above, when (b ₂₁ ) ln ₊₁ ≦lmax, set the threshold to ln ₊₁ and start over from step (i) along the x direction from the tracking start point, or (b ₂₂ ) When ln ₊₁ ≦lmax, the distances xn and yn
After determining that the area delimited by
5. The signal processing method for determining the base sequence of a nucleic acid according to claim 4, characterized in that the step is restarted from the beginning (lmax is a constant and is the maximum value that the threshold value ln can take). 6. A one-dimensional waveform consisting of a position in the x direction and a signal level is obtained for each separated development column based on the tracking information of the separation development column obtained in the fourth step. A signal processing method for determining the base sequence of a nucleic acid according to item 1. 7 The mixture of the base-specific DNA fragments is (1) a guanine-specific DNA fragment, (2) an adenine-specific DNA fragment, (3) a thymine-specific DNA fragment, and (4) a cytosine-specific DNA fragment. A patent characterized in that the separation and development array consists of four separation and development arrays formed by separating and developing these four types of base-specific DNA fragments on a support medium, respectively. A signal processing method for determining the base sequence of a nucleic acid according to claim 1. 8 A digital signal corresponding to the autoradiograph is transmitted to the autoradiograph of the radiolabeled substance on the support medium by superimposing the support medium and the stimulable phosphor sheet containing the stimulable phosphor on the phosphor sheet. Claim 1, characterized in that the autoradiograph is obtained by accumulating and recording the phosphor sheet, and then photoelectrically reading out the autoradiograph as photostimulated light by irradiating the phosphor sheet with excitation light. A signal processing method for base sequencing of the described nucleic acid. 9 A digital signal corresponding to the above-mentioned autoradiograph is transferred onto the photosensitive material after superimposing the support medium and the photographic light-sensitive material to photosensitively record the autoradiograph of the radiolabeled substance on the support medium on the photosensitive material. 2. The signal processing method for determining the base sequence of a nucleic acid according to claim 1, wherein the signal processing method is obtained by photoelectrically reading a visualized autoradiograph.