JPS6118868A - Signal processing for autoradiography - Google Patents

Abstract

PURPOSE:To determine base sequence, by making a storage phosphor sheet absorb radiation energies from five separate development trains made up of two sets of samples for forming a reference train respectively comprising two types of base-specified DNA synthetic matters and a DNA synthetic matter to process. CONSTITUTION:Radioactive energies from five separate development trains made up of two sets of samples for forming a reference train respectively comprising two types of base-specified DNA synthetic matters or synthetic matter mixtures combined as desired to include all of guanine-specified DNA synthetic matter, adenine-specified DNA synthetic matter, thymine-specified DNA synthetic matter and cytosine-specified DNA synthetic matter and a base-specified DNA synthetic matter or synthetic matter mixture are absorbed by a storage phosphor sheet 1, scanned in an electromagnetic wave 15 with a light deflector 18 to be read out photoelectrically with a photo detector 23 whereby sampling points of the respective separate development trains are detected. Individual reference sampling points of the samples for forming a reference train are detected with a control circuit 13, an A/D converter 25, a signal processing circuit 26 and the like and the sampling points of the respective separate development trains are identified by comparison depending on a plurality of continuous lines connecting these points.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a signal processing method in autoradiography. More specifically, the present invention relates to a method for comparative identification of the separation and deployment position of a radiolabeled substance in digital signal processing for base sequencing of DNA or DNA fragments in autoradiography.

[Background of the invention] Autoradiography is already known as a method for obtaining positional information of a radiolabeled substance that is distributed in at least one dimension on a support medium to form a distribution array.

For example, after attaching a radioactive label to a macromolecular substance derived from a biological body such as a protein or nucleic acid, the radiolabeled macromolecular substance, its derivative, or its decomposition product (hereinafter also referred to as the radiolabeled substance) is used. ) by subjecting it to separation operations such as electrophoresis on a gel-like support medium to develop molecules 4.

1 forms a separated and developed column of the radiolabeled substance (however, it is not visible in 11) on the surface supporting body, an autoradiograph of this separated and developed column is acquired as a visible image on a radiographic film, and from that visible image. Information on the location of radioactively labeled substances has been obtained. Furthermore, methods for separating and identifying the polymeric substance, or evaluating the molecular weight and characteristics of the polymeric substance based on the obtained positional information of the radiolabeled substance have already been developed and are currently in use. .

Particularly in recent years, autoradiography has been
It is effectively used to determine the base sequence of NA (or DNA fragments, hereinafter the same).

The Sanger-Coulson method is known as one of the representative methods for determining the base sequence of DNA using autoradiography. In this method, DNA has a double helical structure consisting of two chain molecules, and each of the two chain molecules contains four types of bases, namely adenine (A), guanine (G), and cytosine. (C) and thymine (T), and the two chain molecules are cross-linked by hydrogen bonds between these four types of bases, and each Focusing on the characteristic structure of DNA, in which hydrogen bonds between structural units are realized only in two types of combinations: G-C and A,-T, we developed a method for synthesizing DNA fragments using DNA synthase and using gel electrolysis. This is a method of determining the base sequence of DNA by skillfully utilizing the means of electrophoresis and autoradiography.

In the Sanker-Rielson method, there are several methods for synthesizing an NA fragment that is complementary to the DNA or DNA fragment whose base sequence is to be determined (hereinafter referred to as sample DNA). Specifically, single-stranded sample DNA is used as a template, and a DNA polymerase (DNA polymerase) is activated in the presence of mononucleoside triphosate containing the four types of bases mentioned above to generate complementary DNA to the sample DNA. D' of various lengths
Synthesize the NA fragment. At this time, some mononucleoside triphosphates should be given no radioactive label, and the synthesis conditions should be modified to make them specific for any of the four types of bases. By doing so, base-specific synthesis N
A fragment (DNA synthesis product) is obtained.

Next, the mixture consisting of a large number of DNA compounds obtained by this operation is divided into supporting media by gel electrophoresis.
I expand (but cannot be seen visually)
. Conventionally, this separation and development array on the support medium is visualized on an X-ray film to obtain an autoradiograph, and based on the obtained autoradiograph, the base sequence of the five chain chains is sequentially determined from the end. In this way, sample D
The sequence of all bases of NA has been determined.

Note that the characteristics and operation of the Sanger Φ Coulson method summarized above are described, for example, in the following document.

rReading genetic information in its original language: Surprising DNA base sequence analysis method j Kenro Miura, Gendai Kagaku, September 1977 issue 4
pp. 6-54 (■Tokyo Kagaku Doujin Publishing) Sanger, F.
, N1cklen, S., & Coulson,
A.R.

Proc, Natl, Acad, Sci, US
A. 74. pp, 5483-'' In autoradiography, which utilizes conventional radiography methods such as duplication, an autoradiograph with positional information is visualized on a radiographic film in order to obtain positional information of a radiolabeled substance. is required.

Therefore, by judging the visualized autoradiograph with their own eyes, researchers can measure the distribution of radiolabeled substances in the sample and obtain knowledge of the location of specific radiolabeled substances. I am getting . That is, the DNA base sequence is determined by visually determining the separation and development position for each radioactively labeled base-specific DNA compound or mixture thereof, and by mutually aligning the separation and development columns of these base-specific DNA compounds. It is determined by comparing the Therefore, analysis of the obtained autoradiograph is usually done through human vision.
A great deal of time and effort is spent on this.

Furthermore, since it relies on the human eye, there are limits to the accuracy of the information obtained, such as the positional information obtained by analyzing the autoradiograph differing depending on the researcher. Particularly when the autoradiograph visualized on radiographic film does not have good image quality (sharpness, contrast), it is difficult to obtain satisfactory information and its accuracy tends to decrease. There is a problem.

Conventionally, in order to improve the accuracy of the positional information to be obtained, a method has been used in which, for example, the visualized autoradiograph is measured using a measuring instrument such as a scanning densitometer. However, there is a limit to the improvement of accuracy in methods that simply use such measuring instruments.

For example, during an exposure operation in which a radiation film is brought into close contact with a support medium on which the separation and development rows are formed, a shift may occur in the overlapping of the two, and in this case, a visible image may appear on the radiation film. The resulting separation and development array (e.g., migration array) is W in the longitudinal direction of the film.
This results in shifts rather than lines, making it easy for errors to occur when visually determining the position information of radiolabeled substances.
Its accuracy tends to decrease. Further, depending on the support medium and the shunt development conditions, the resulting separated and developed rows often are not parallel to the length direction of the support medium or are distorted.

Furthermore, when using gel as a support medium, this gel does not have self-supporting properties, so it is usually separated and developed with both sides sandwiched between glass or the like, but the thickness of the gel may be uneven due to deformation of the coating. The radiolabeled substance may not necessarily be separated and developed uniformly on the support medium. Similar non-uniform separation and development also occurs when the gel contains air bubbles or when the composition of the gel is non-uniform. For this reason, a so-called smiling effect often appears, in which, for example, the moving distance of the separation and deployment rows at both ends is relatively short compared to the movement distance of the separation and deployment rows near the center of the support medium. Alternatively, when performing separation and development by electrophoresis, the voltage may not be applied uniformly to the support medium, and even in such cases, the voltage may not be uniformly applied to the support medium.
Since the I development conditions differ locally on the supporting medium,
Distortion tends to occur in the resulting separated and expanded array.

There is no suitable method for such distortions in the separation and expansion array other than artificial correction, and therefore, in such cases, it becomes particularly difficult to analyze the position information of the radiolabeled substance, and it is difficult to analyze the position information of the radiolabeled substance. Even if a standard measuring instrument is used, the location information of the separated and developed radioactive label substance, i.e., DNA or D
It is difficult to obtain the base sequence of an NA fragment with sufficient accuracy.

[Summary of the Invention] The present inventor has developed a method for converting radioactive labels by using a radiographic image conversion method using a stimulable phosphor sheet instead of a radiographic method using a radiographic film used in conventional autoradiography. Data indicating the distribution position of a radiolabeled substance obtained by obtaining the positional information as a digital signal from an autoradiograph containing the positional information of the substance without specifically converting it into an image, and then applying signal processing to this digital signal. By further performing appropriate signal processing, it was realized that the base sequence of DNA or DNA fragments could be determined simply and with high accuracy, and the present invention was achieved.

That is, the present invention is a signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, which has a base sequence complementary to the DNA or DNA fragment, and has a radioactive label. 1) at least two guanine-specific DNA compounds, an adenine-specific DNA compound, a thymine-specific DNA compound, and a cytosine-specific DNA compound, arbitrarily combined to include all of them as a whole; at least two sets of reference train formation samples each consisting of a base-specific DNA compound or a mixture of base-specific DNA compounds of the species; and 2) a base-specific DNA compound or a base-specific DNA compound mixture.
Each of the at least one composite mixture is placed on a support medium, and each of the two sets of reference row forming samples of item 1) is placed on the supporting medium.
DN of the above 2) in which at least one separation expansion column of 1- is provided.
A radiolabeled substance constituting at least five separated and developed columns formed by one-dimensionally separating and developing the molecules of the A compound or base-specific DNA compound mixture in parallel relation to each other so as to sandwich the aK open column. By causing the stimulable phosphor sheet to absorb radiation energy emitted from the group, 2) After recording an autoradiograph having positional information of the radiolabeled substance group on the stimulable phosphor sheet, the accumulation The photosensitive phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is read out photoelectrically to obtain a digital image corresponding to the autoradiograph of each separated and expanded array. Regarding the signal, i) detecting a sampling point for each of the separation and development rows; ii) synthesizing each reference row from the separation and development rows on each tool of each reference row forming sample; Step of detecting sampling points: 1) setting a plurality of continuous lines consisting of straight lines, polygonal lines, or curves connecting corresponding reference sampling points between the plurality of reference columns, and i) using the continuous lines, The present invention provides a signal processing method in autoradiography, which includes the step of comparing and identifying sampling points of each separation expansion sequence.

That is, in the present invention, the radiation energy emitted from the sample is absorbed by the stimulable phosphor sheet by overlapping the sample and the stimulable phosphor sheet, and then the stimulable phosphor sheet is exposed to visible light, infrared rays, etc. By scanning with electromagnetic waves (excitation light), the radiation energy stored in the stimulable phosphor sheet is emitted as fluorescence (fR total emission), and this fluorescence is read photoelectrically to obtain an electrical signal. This method utilizes a radiation image conversion method that involves A/D converting a signal to obtain a digital signal.

The radiation image conversion method is described in, for example, Japanese Unexamined Patent Publication No. 12445/1983.

The stimulable phosphor sheet used in the present invention is, for example,
It contains a stimulable phosphor such as a divalent europium-activated alkaline earth metal fluorohalide phosphor. This stimulable phosphor is irradiated with radiation such as X-rays, alpha-rays, beta-rays, gamma-rays, and ultraviolet rays and accumulates a portion of the radiation energy. ) has the property of exhibiting stimulated luminescence depending on the accumulated energy.

According to the present invention, the positional information of a radiolabeled substance is directly obtained as a digital signal without going through any imaging process, using the radiation image conversion method using the above-mentioned stimulable phosphor sheet.

In the present invention, "position information" refers to various types of information centered on the position of a radiolabeled substance or an aggregate thereof in a sample, such as the location and shape of an aggregate of radioactive substances present in a support medium. , refers to various types of information obtained as one or any auxiliary combination of information such as the concentration and distribution of radioactive substances at that location.

In addition, in the present invention, the reference column refers to a column corresponding to a separation and development column containing all of the base-specific DNA compounds of four types of DNA bases, guanine, adenine, thymine, and cytosine. DNA or D
In determining the base sequence of an NA fragment, a radioactively labeled base-specific DNA compound is separated and developed on a support medium and serves as an internal standard for MU opening.

The labeled DNA used to obtain the reference column was sample D.
It may be made using NA or another DNA, but the former is more preferable.

According to the undiscovered invention, positional distortion during separation and development of a radiolabeled substance on a support medium as described above, or -
Due to misalignment during the operation of forming an autoradiograph of a radiolabeled substance that has been separated and developed in the dimensional direction to form a separated and developed column on the stimulable phosphor sheet, the autoradiograph is accumulated and recorded on the stimulable phosphor sheet. Even when distortion or deviation occurs throughout the autoradiograph, the base sequence of DNA or DNA fragments can be determined with high accuracy.

In particular, with respect to the distortion of the separated development site, by synthesizing multiple reference columns that serve as internal standards from multiple separation development columns, it is possible to eliminate the distortion in the digital image data based on this reference column. After measuring and correcting the obtained distortion for each column, it becomes possible to identify the separation development site of each column. Therefore, the target base sequence can be determined with high precision and rationality.

[Structure of the Invention] As an example of a sample used in the present invention, DNA or a DNA fragment is assembled into a template, and a deoxynucleoside triphosphate (dNTP) to which 0 radioactive labels have been given and a DNA synthase are combined. Each base-specific DNA compound synthesized using -
Minutes in the dimensional direction! li[support media which are expanded to form separate expanded columns.

In the present invention, the radioactive element used in the radioactive label may be any nuclide as long as it emits radiation (α rays, β rays, γ rays, neutron rays, X rays, etc.), and its specific As an example, 32p, 14C, 35S, 3)(,
125I and the like.

In addition, methods for separating and developing the -F radiolabeled substance using a support medium include, for example, a gel-like support medium (the shape can be arbitrary, such as layered or columnar), a polymer molded body such as acetate, or a filter paper. Representative methods include electrophoresis using various support media and thin layer chromatography using support media such as silica gel.

Among these, electrophoresis using a gel-like support medium is a typical separation and development method and is preferred.

The basic structure of the stimulable phosphor sheet used in the present invention is a support, a phosphor layer, and a transparent protection layer. The phosphor layer is made of a binder containing and supporting the stimulable phosphor in a dispersed state, for example, divalent europium activated helium fluoride bromide 1, (B aFB r : E
u2'+) phosphor particles are dispersed in a mixture of nitrocellulose and linear polyester. A stimulable phosphor sheet is, for example, one in which a sheet of polyethylene terephthalate or the like is used as a support, the above-mentioned phosphor layer is provided on this sheet, and a polyethylene terephthalate sheet or the like is further provided as a protective film on the phosphor layer. .

For details of the support medium and stimulable phosphor sheet used in the present invention, please refer to the Japanese Patent Application No. 1983-1980 filed by Mizu Applicant.
It is described in the specification of No. 193419.

In the present invention, the operation of accumulating and recording radiation energy emitted from a support medium containing a radiolabeled substance onto a stimulable phosphor sheet ('R light operation) is performed by overlapping the support medium and the stimulable phosphor sheet for a certain period of time. By combining, at least a portion of the radiation emitted from the radiolabeled substance on the support medium is absorbed by the stimulable phosphor sheet. This exposure operation can be carried out by placing the support medium and the stimulable phosphor sheet in close proximity to each other, for example, by keeping them in this state for at least several seconds at room temperature or low temperature.

For details on the sample preparation method, stimulable phosphor sheet, and exposure operation in autoradiography using the Sanger e-Coulson method, please refer to the patent application filed in 1973 by the applicant.
8-201231.

Next, in the present invention, a method for reading out one-dimensional positional information of a radiolabeled substance on a support medium stored and recorded on a stimulable phosphor sheet and converting it into a digital signal will be described in FIG. 1 of the attached drawings. This will be briefly described with reference to an example of the reading device (or reading device) shown in FIG. .

Figure 1 shows a pre-reading system for temporarily reading out one-dimensional positional information of a radiolabeled substance accumulated and recorded on a stimulable phosphor sheet (hereinafter sometimes abbreviated as phosphor sheet) 1. The apparatus is comprised of a main reading section 2 and a main reading output section 3 having a function of reading out the autoradiograph stored on the photoreceptor sheet 1 in order to output positional information of the radiolabeled substance.

In the prefetch reading unit 2, the following prefetch operation is performed.

By passing the laser light 5 generated from the laser light source 4 through the filter 6, a portion of the wavelength range corresponding to the wavelength range of the photostimulation destination generated from the phosphor sheet 1 in response to excitation by the laser light 5 is canted. Ru. Next, the laser beam is deflected by an optical deflector 7 such as a carpano mirror, reflected by a plane reflecting mirror 8, and then one-dimensionally deflected and incident on the phosphor sheet 1. The laser light source 4 used here is selected so that the wavelength range of its laser light 5 does not overlap with the main wavelength range of the stimulable destination emitted from the phosphor sheet 1.

The phosphor sheet 1 is transported in the direction of arrow 9 under irradiation with the polarized Ω laser light. Therefore, the entire surface of the phosphor sheet 1 is irradiated with the polarized laser light. Note that regarding the output of the laser light source 4, the beam diameter of the laser light 5, the scanning speed of the laser light 5, and the transport speed of the phosphor sheet 1, the energy of the laser light 5 for the pre-reading operation is smaller than the energy used for the main reading operation. It will be adjusted so that

When the phosphor sheet 1 is irradiated with the laser light as described above, it exhibits stimulated luminescence with an amount of light proportional to the accumulated and recorded radiation energy, and this light enters the pre-reading light guiding sheet IO. The light guide sheet 10 has a linear incident surface and is placed close to the scanning line on the phosphor sheet 1 so as to face it, and its exit surface forms a ring.・Connect to the light-receiving surface of the photodetector 11, such as a circle. This light guiding sheet 10 is made by processing a transparent thermoplastic resin sheet such as acrylic synthetic resin, and the light incident from the incident surface is totally reflected inside and transmitted to the exit surface. is configured to be Stimulated luminescence from the phosphor sheet 1 is guided through the light-guiding sheet 10 and reaches the exit surface, where it is emitted and received by the photodetector 11.

A filter is attached to the light-receiving surface of the photodetector 11, which transmits only light in the wavelength region of stimulated luminescence and cuts light in the wavelength region of excitation light (laser light), and detects only stimulated luminescence. It is made to be wet. Stimulated luminescence detected by the photodetector 11 is converted into an electrical signal, which is further amplified by the amplifier 12 and output. The accumulated recording information output from the amplifier 12 is input to the control circuit 13 of the main reading reading section 3. The control circuit 13 sets the amplification factor setting value a according to the obtained accumulated recording information so that a signal of an appropriate level is obtained.
and output the recording scale factor b.

Phosphor sheet 1 whose pre-reading operation has been completed as described above
is transferred to the reading section 3 for main reading.

In the main reading reading section 3, the following main reading operation is performed.

The laser beam 15 emitted from the main reading laser light source 214 passes through a filter 16 having the same function as the filter 6 described above, and then a beam expander 17.
The laser beam is then deflected by an optical deflector 18 such as a galvano mirror, reflected by a plane reflector 19, and then one-dimensionally applied to the phosphor sheet 1-A. It is deflected and incident on the target. Note that an fθ lens 20 or the like is arranged between the optical deflector 18 and the plane reflecting mirror 19, so that when the deflected laser beam scans the phosphor sheet 1, a uniform beam speed is always maintained. has been done.

The phosphor sheet 1 is exposed in the direction of arrow 21 under irradiation with the above-mentioned polarized laser light. Therefore, as in the pre-reading operation, the entire surface of the phosphor sheet 1 is irradiated with the polarized laser light.

When the phosphor sheet 1 is irradiated with laser light as described above, it emits stimulated luminescence with an amount of light proportional to the accumulated and recorded radiation energy, as in the pre-reading operation, and this light is used as a guide for main reading. The light enters the optical sheet 22.

This main reading light guide sheet (22) has the same material and structure as the preread light guide sheet 10. The emitted light is emitted from the emission surface and is received by the photodetector 23.A filter that selectively transmits only the wavelength range of stimulated luminescence is attached to the light receiving surface of the photodetector 23.
The photodetector 23 is designed to detect only stimulated luminescence.

The stimulated luminescence detected by the photodetector 23 is converted into an electrical signal, which is amplified to an appropriate level electrical signal in the amplifier 24 whose sensitivity is set according to the amplification factor setting value a, and then sent to the A/D converter 25. is input. A/D converter 2
5 is converted into a digital signal with a scale factor suitable for the signal fluctuation range according to the recording scale factor setting value.

Regarding the method for reading the positional information of the radiolabeled substance on the support medium stored and recorded on the stimulable phosphor sheet in the present invention, the reading operation consisting of the pre-reading operation and the main reading operation has been described above. , the read operations that can be utilized in the present invention are not limited to the above examples. For example, if the amount of radiolabeled substance on the support medium and the exposure time of the stimulable phosphor sheet for the support medium are known in advance, it is possible to omit the pre-reading operation in the example described in -1-. .

The stimulable phosphor sheet used in the present invention can be reused by irradiating the sheet with appropriate light or heating it to erase residual radiation energy after the above readout operation is completed.

Furthermore, as a method for reading the positional information of the radiolabeled substance on the support medium stored and recorded on the stimulable phosphor sheet in the present invention, it is of course possible to use any method other than those exemplified above. be.

The digital signal corresponding to the autoradiograph of the radiolabeled substance thus obtained is then input to the signal processing circuit 26 shown in FIG. Signal processing circuit 26
Then, the base sequence of the target DNA is determined by converting the one-dimensional positional information of the radiolabeled substance into symbols and/or numerical values.

Below, 1! of DNA using the signal processing method of the present invention! ! Taking as an example the embodiment of signal processing in autoradiography for base sequence determination using the above-mentioned Sanger-Cournon method, the following four groups of base-specific DNA compounds and combinations of DNA compound mixtures are used. A case will be described in which three different types of sample DNA are electrophoresed on the same support medium using the following method.

1) Guanine-specific DNA composition + Abii7-specific DNA composition, 2) Thymine-specific DNA composition + Cytosine-specific DNA composition, 3) Guanine-specific DNA composition, 4) Cytosine-specific DNA synthesis First, using the sample DNA as a template, a radioactive label (3
By base-specific synthesis using a mononucleoside triphosphate to which 2F) has been added and a DNA polymerase, the base-specific DNA compounds and DNA of the four groups of steps) to 4) above are obtained, respectively. Triplicates of composite mixtures are obtained.

Next, base-specific DNA compounds and DNA of 2.4 groups.
Each of the three sets of composite mixtures is electrophoretically separated on a gel support medium to obtain a respective separation column. However, at least the separation and development column of the base-specific DNA compound mixtures of the three groups 1) and 2) above can be used as a support medium.
(2) arranged adjacent to each other in at least two places;
Separation and development operations are performed by arranging adjacent sets of separation and development columns so as to sandwich the separation and development columns of the three groups of base-specific DNA compounds in 3) and 4) above.

Next, an exposure operation is performed by overlapping this sample (gel-like support medium with separation and development rows formed) and a stimulable phosphor sheet at room temperature for several minutes, and the autoradiograph of the sample is transferred to the stimulable phosphor sheet. Accumulate and record. For details of the exposure operation mentioned above, please refer to the above-mentioned patent application No. 58-20123.
It is stated in the specification of No. 1.

FIG. 2 shows an example of an autoradiograph of the above-mentioned four groups of electrophoretic arrays in which each DNA base-specific DNA compound and a DNA compound mixture are separated and developed using radioactive labels. That is, the 1st to 12th columns in FIG. Composite + (C) Specific DNA compound (3) - CG) Specific DNA compound (4) - (C) Specific DNA compound (5) - (G) Specific DNA compound (6) - (G) Specific DNA compound + (A) Specific DNA compound (2) - (r: + Specific DNA compound + (C) Specific DNA compound (8) - (C) Specific DNA synthesis Product (9) - (G) Specific DNA compound (10) - (C) Specific product (11) - (
G) Specific DNA compound + (A) Specific DNA compound (12)' - (T) Specific DNA compound + (C) Specific DNA compound each column (electrophoresis column) is shown. The 1st to 4th columns, the 5th to 8th columns, and the 9th to 12th columns are sample DNs that are different from each other.
This is the separated deployment position of A. Also, the first and second rows, and the sixth row
The column and the 7th column, and the 11th column and the 12th column are sets of samples for forming a reference column, respectively.

By loading the autoradiograph stored and recorded on the stimulable phosphor sheet into the reading device shown in FIG. 1 and reading it out, the signal processing circuit 26. The input digital signal is the address (x, y) expressed in the coordinate system fixed to the stimulable phosphor sheet and the signal level (Z) at that address.
The signal level corresponds to the amount of photostimulated light. That is, the digital signal corresponds to the autoradiograph of FIG. Therefore, the signal processing circuit 2
6, digital image data having position information of the radiolabeled substance is input. As used herein, digital image data refers to a collection of digital signals corresponding to an autoradiograph of a radiolabeled substance.

First, the separation and development positions of the radiolabeled substances are detected for each of the above 12 separation and development columns using the digital image data, and these are used as sampling points. The sampling points can be obtained, for example, as follows.

The digital image data is scanned twice in parallel at two different positions across the one-dimensional distribution direction (separation and development row direction) of the radiolabeled substance on each scanning area. The scanning to detect the distribution points of the radiolabeled substance in each row is called a preliminary scan), and the two distribution points for each row are connected to form a
Two straight lines are obtained, and each of the obtained straight lines is used as a scanning direction for detecting sampling points in each column.

In the signal processing method of the present invention, the digital signal obtained by reading out the stimulable phosphor sheet is stored in the signal processing circuit 26 in a base memory (i.e., in a buffer memory or a non-volatile memory such as a magnetic disk). stored in sexual memory). In signal processing, scanning digital image data means selectively retrieving only the digital signal at this scanning location from memory.

Next, by scanning the digital image data along the scanning direction, a function f (w) representing the level of the signal on the scanning area [w represents the position in the scanning direction] can be obtained. This function f(w) is then subjected to smoothing processing by, for example, performing convolution using an appropriate filter function to obtain a function g(w). Next, threshold processing is performed on this function g (w).

That is, for the threshold value (α0), when g(w)≧α0, g (w) = 1g (w)
<00, by processing g(w)−〇, the function g(w) is set to iI! of 1 or 0! Convert to continuation function. The sampling points are detected by setting each midpoint of the area of g(w)=1. Note that the threshold value (α0) in the above threshold processing can be determined, for example, from the relationship between the signal level and its frequency, that is, the histogram, for the digital signal on the scanning area.

In this way, for each column, the set of sampling points jS
Kn (Xkn + y)Cn + Zkn)) can be detected. Here, k is a positive integer and represents the number of each column, and n is a positive integer and represents the number of the sampling point. Note that the method for detecting sampling points is not limited to the method described in "2."

Next, by logically adding the sampling points in the first column and the sampling points in the second column, a new (G) singular D
NA Compounds, (A) % Heterogeneous DNA Compounds, (T) Specific DNA Compounds, and (C) Four Types of Base-Specific DNA Compounds I) Sample Points Containing All of the NA Compounds The reference (internal standard) column (S+z.) is obtained. For example, this composition can be expressed as an operation as follows.

(S I3 nl = (S t n)
U (Sznl, where () represents a set of sampling points, and U represents a logical sum operator. This reference column is set so that it exists virtually between the first column and the second column. , the coordinates of the reference sampling point SL3° are newly set.

By performing similar processing on the sampling points of the 6th column and the sampling points of the 7th column, a reference column having the reference sampling points (S14n) is synthesized, and the 1st, 11th
By performing similar processing on the sampling points of the column and the sampling points of the 12th column, a reference column having the & quasi sampling point (St5n) is synthesized and obtained. In this way, it is possible to virtually provide reference rows of beads on the digital image data at both ends and in the center of the eleven migration rows obtained by separation JJ.

A plurality of straight lines (or polygonal lines) are obtained by connecting corresponding reference sampling points in each reference column, that is, reference sampling points having the same sampling point number n. For example, the farthest (n=1) reference sampling points 3131, S14+, and S15. By connecting them to obtain a polygonal line, and then sequentially obtaining straight lines (or polygonal lines) for n=2, 3, etc., as many straight lines (or polygonal lines) as there are reference sampling points in each reference column. (line). Furthermore, the obtained polygonal line can also be approximated by an appropriate curve. A continuous line group consisting of these straight lines, polygonal lines, or curved lines can be regarded as a contour line group (Ln) regarding the separation development position. However, n matches the reference sampling point number n.

FIG. 3 is a diagram showing a portion of the contour line approximated by the curve obtained as described above.

Based on this contour line, comparative identification is performed for the sampling points in the third column. For example, the third column sampling point S
31, the position of sampling point S31 (X3
+ , y3 t) is closest to which contour line in the contour group (Ln) is determined. Now, if it is true, the sampling point S31 can belong to the reference sampling point S day 1. In this way, all the sampling points in the third column are assigned to one of the reference sampling points in order. That is, the sampling points in the third column are a subset (SL3n) of the reference sampling points.
It is represented by 3.

By the operation described in -, the sampling point Skn expressed by the y coordinate (ykn) can be represented by a contour line Ln, and since this contour line can also be called the electrophoresis coordinate, this operation can be performed from the xy coordinate to the electrophoresis coordinate. It can be regarded as a coordinate transformation to .

Furthermore, the set of sampling points in the third column is the set of sampling points in the first column (S+nl
(S+3nl 3n (S 1 n) = (S+sn
), a virtual 16th column with a new set of sampling points ('S + e n ) expressed by the logical AND operation is obtained, and the obtained 16th column is adenine in the set of columns 1 to 4. It corresponds to a separation and development column for specific DNA compounds.

Perform the same operation between the 4th column and the 2nd column,
The first column corresponds to the separation and development column for thymine-specific DNA compounds.
Get 7 columns. Next, for the 3rd column, 4th column, 16th column, and 17th column, the reference sampling points 5 are set in descending order of n.
13n and the sampling point 5scn are compared, and when they match, the reference sampling point 313n is replaced with the matching sampling point Skn. Then, by tracing the reference columns in ascending order of n, it is possible to obtain, for example, a linear diagram.

S3 sad+S4t+5I7t+S32. S+s kan.

In the above diagram, 53n=G, 54n=C1S +e
By replacing n = A and S 17 n = T, we obtain the following diagram.

G-C-T-G-A-・Old... In this way, in the set of columns 1 to 4, the sample D
The base sequence of a chain molecule of a base-specific DNA compound having a base sequence complementary to NA can be determined.

Furthermore, by performing the same process on the set of columns 5 to 8 and the set of columns 9 to 12, the sample DN
The base sequence of each base-specific DNA composite chain molecule having a base sequence complementary to A can be determined.

Information regarding the base sequence of the obtained DNA is 1.
- The display format is not limited to the above two, and any display format is possible. For example, if desired, it is also possible to display the relative amount of each separated and expanded DNA compound by arbitrarily performing subarithmetic processing on the signal levels in the scanning direction -H of each column.

Alternatively, it is also possible to display the base sequences of both Miki DNA chain molecules. In other words, by providing the information A-T, a-c, C+G, and T+A as possible combinations for each base in the diagram represented by the above symbols, the DNA base sequence represented by the following diagram can be obtained. get.

G-C-T -G -A-...C-G-A-C
-T-... It should be noted that the signal processing method of the present invention allows the above (G+A,
The DNA base sequencing method using the T+C, G, C) (2) combination is one example of the DNA base sequencing method, and the signal processing method of the present invention uses the above combination. Various combinations are possible without being limited to these, and the base sequence can be similarly determined by a method similar to the method described in -h using such combinations.

However, in any combination, G, A, 'T,
It is necessary to have a combination that can be obtained by synthesizing a reference (internal standard) train consisting of all base-specific DNA compounds of C.

Furthermore, the separated 1111 expansion columns of the sample for forming the reference array must be arranged closely, and the set of separation expansion columns for obtaining the reference array must contain the base-specific DNA to be sequenced. It is necessary to perform the separation and development by being arranged so as to sandwich the separation and development column of the compound.

In order to determine the DNA base sequence with high precision, it is preferable that the separation and development positions of the separation and development arrays of the sample for forming the reference array be as far apart as possible. That is, when this set consists of two sets, it is preferable to arrange it at both ends of a plurality of separation deployment rows, and when it consists of three sets, it is preferable to arrange it at both ends and the center of a plurality of separation deployment rows. is preferred. When there are more than three sets, it is preferable that they be arranged at appropriate intervals between the separation and deployment rows of detection targets. The installation of three or more sets is particularly effective when the above-mentioned smiling effect occurs in the support medium in which the radiolabeled substance is separated and developed, and the DNA base sequence can be determined with high precision. The more the number of sets of separation and development arrays of the sample for forming reference arrays to be installed increases, the more accurate the contour lines obtained become, and therefore it becomes possible to determine the base sequence of DNA with high precision.

This sample for forming a reference array not only forms a reference array, but also participates in determining the base sequence of the DNA as a separate expanded array. Therefore, the greater the number of separation and expansion columns, the more reference columns can be synthesized, thereby making it possible to obtain accurate contour lines. In other words, the base sequence can be determined with even higher accuracy.

] - In the above example, the explanation was given using 12 rows of radiolabeled substance groups separated and developed in a one-dimensional direction on the support medium-L, but the number of separated and developed rows is not limited to 12 rows. . Alternatively, it is also possible to determine the base sequences of several types of DNA or more at the same time.

Information about the DNA base sequence determined by the signal processing method described above is output from the signal processing circuit 26 and then stored directly or, if necessary, via a storage means such as a magnetic tape. recording device (not shown)
transmitted to.

Recording devices include, for example, those that optically record by scanning a photosensitive material with a laser beam, those that electronically display on a CRT, etc., and those that record symbols and numbers displayed on a CRT, etc. with a video printer, etc. Recording devices based on various principles can be used, such as those that record on a heat-sensitive recording material using heat rays, and those that record on a heat-sensitive recording material using heat rays.

In addition, the information obtained as described in Section 2 can also be subjected to genetic linguistic information processing, such as comparing it with other DNA base sequences that have already been recorded. It is.

[Brief explanation of the drawing]

FIG. 1 shows an example of a reading device (or reading device) for reading out positional information of a radioactive substance in a sample stored and recorded on a stimulable phosphor sheet in the present invention. 1: stimulable phosphor sheet, 2: readout section for pre-reading, readout section for 30-line reading, 4: laser light source, °5: laser light,
6: Filter, 7: Optical deflector, 8: Plane reflector, 9:
Transfer direction, 10: Pre-reading light guide sheet, 11: Photodetector, 12: Amplifier, 13: Control circuit, 14: Laser light source, 15: Laser light, 16: Filter, 17: Beam expander, 18: Optical deflector, 19: Planar reflecting mirror, 20: fθ lens, 21: Transfer direction, 22 Light guide sheet for dual reading, 23: Photodetector, 24: Amplifier, 25
: A/D converter, 26: Signal processing circuit FIG. 2 is a diagram showing an example of an autoradiograph of a sample in which a base-specific DNA compound of sample DNA is separated and developed on a gel support medium. FIG. 3 is a diagram showing a portion of a contour line connecting reference sampling points approximated by one curve.

Claims (1)

[Scope of Claims] 1. A signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, which has a base sequence complementary to the DNA or DNA fragment and is radioactive. Labeled: 1) Guanine-specific DNA compound, adenine-specific DNA
A compound, a thymine-specific DNA compound, and a cytosine-specific DNA compound. at least two sets of samples for forming a reference array, and 2) a base-specific DNA compound or a base-specific DNA.
At least one type of synthetic compound mixture is used for the base-specific DNA synthesis of 2) above, in which at least one row of separation and development rows for each of the two sets of reference row forming samples of 1) above is formed on a support medium. From a group of radiolabeled substances constituting at least five separation and development rows formed by one-dimensional separation and development in parallel with each other so as to sandwich the separation and development rows of compound- or base-specific DNA compound mixtures. By absorbing the emitted radiation energy into the stimulable phosphor sheet, an autoradiograph having positional information of the radiolabeled substance group is stored and recorded on the stimulable phosphor sheet, and then the stimulable phosphor sheet is Regarding the digital signal corresponding to the autoradiograph of each separated development column obtained by scanning the autoradiograph with electromagnetic waves to emit photostimulated light and reading out this photostimulated light photoelectrically, i) Separation. a step of detecting a sampling point for each of the developed columns; ii) a step of synthesizing each reference column from two or more separated developed columns of each reference column forming sample and detecting a reference sampling point for each of the reference columns; iii) setting a plurality of continuous lines consisting of straight lines, polygonal lines, or curved lines connecting corresponding reference sampling points between the plurality of reference columns, and iv) connecting the sampling points of each separation expansion column with the continuous lines. A signal processing method in autoradiography, comprising the step of comparing and identifying. 2. At least three sets of two or more separation and development rows of each of the reference row forming samples of 1) above are arranged, and at least one separation and development row of the two adjacent sets of reference row formation samples is arranged. 2. The signal processing method in autoradiography according to claim 1, wherein the signal processing method is arranged so as to sandwich a separation and development column of a DNA compound or a mixture of DNA compounds. 3. Claim 1, characterized in that the sampling points are detected by performing smoothing and/or threshold processing on digital image data in the scanning direction of each of the plurality of separation and development columns. Or the second
Signal processing method in autoradiography as described in section. 4. A set of samples for forming reference rows consists of two types: 1) guanine-specific DNA compound + adenine-specific DNA compound, and 2) thymine-specific DNA compound + cytosine-specific DNA compound. 4. A signal processing method in autoradiography according to any one of claims 1 to 3, characterized in that the signal processing method is comprised of a mixture of base-specific DNA compounds.