JPS60233557A - Signal processing method in autoradiography - Google Patents

Abstract

PURPOSE:To obtain positional information on the base of DNA or DNA section as a visible image by using an accumulative phosphor sheet in place of a radiation film of autoradiography and processing the detection signal of the accelerated luminous light thereof. CONSTITUTION:The accumulative phosphor sheet 1 having the positional information obtd. from the development array of the radioactively labeled DNA or DNA section moves to an advanced read-out part 2 for preliminary reading and is scanned with the laser light 5 from a laser light source 4 via an optical deflector 7 via a filter 6 which cuts the wavelength region of the accelerated luminous light. The accelerated luminous light generated therefrom is made incident via a conductive sheet 10 to a photodetector 11, by which only the accelerated luminous light is detected via a filter. The output signal thereof is inputted to a control circuit 13 via an amplifier 12. Such an amplification factor set value (a), recording scale factor (b) and treating condition set value (c) at which the image having excellent density and contrast is obtd. are respectively fed to an amplifier 24, an analog-to-digital converter 25 and a signal processing circuit 26, by which the detection signals obtd. similarly in a read-out part 3 for normal reading are processed and the positional information on the base is obtd. as the image.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a signal processing method in autoradiography.

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

For example, a radioactive label is added to a biologically derived polymeric substance such as a protein or a nucleic acid, and the radiolabeled polymeric substance, its derivative, or its decomposition product is subjected to a separation operation such as gel electrophoresis to form a gel-like support. The gel-like support medium is separated and developed in a medium, and the gel-like support medium and a high-sensitivity X-ray film are overlaid for a certain period of time to expose the film to light, and the position of the radiolabeled substance on the gel-like support medium obtained from the exposed area is determined. Based on this information, methods for separating and identifying the polymeric substances, or evaluating the molecular weight and characteristics of the polymeric substances have also been developed and are in actual use.

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

One of the representative methods for determining the base sequence of DNA using autoradiography is the Sanger-Gaulson method.
The law is known. 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),
It is composed of structural units containing the bases guanine (G), cytosine (C), and thymine (T), and the two chain molecules are cross-linked by hydrogen bonds between these four types of bases. Focusing on the characteristic structure of DNA, in which hydrogen bonds between each structural unit are realized only in two types of combinations, G-C and A-T, we investigated how DNA fragments are formed by DNA synthase. This is a method for determining the base sequence of DNA by skillfully utilizing synthesis, gel electrophoresis, and autoradiography.

In the Sanger-Coursin method, an NA fragment is synthesized that is complementary to the DNA or DNA fragment whose base sequence is to be determined (hereinafter referred to as sample DNA).
There are several methods for this, but basically, single-stranded sample DNA is used as a template, and DNA synthesis enzyme (DNA polymerase) to create D of various lengths that are complementary to the sample DNA.
Synthesize the NA fragment. At this time, some mononucleoside triphosphates should be radiolabeled and the synthesis conditions should be devised to make them specific for any one of the four types of bases. base-specific synthesized NA with a radioactive label
A fragment (DNA composite) is obtained.

Next, the mixture consisting of a large number of DNA compounds obtained by this operation is separated and developed on a support medium by gel electrophoresis (however, it cannot be seen visually). Conventionally, the 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 chain molecule is determined sequentially from the end. In this way, the sequence of all bases of sample 1) NA was determined.

The characteristics and operation of the Sanger-Luzon method summarized above are described, for example, in the following literature.

“Reading genetic information in its original language: An unexpected DNA sequence analysis method” Genichi Miura, Gendai Kagaku, September 1977 issue 4
Pages 6-54 (■Tokyo Kagaku Doujin Series) Sanger, F.
, N1cklen, S., & Coulson,
A, R-IProc, Natl, Acad, S
ci, USA, 74. pp, '5483-54
87 (11117?) As mentioned above, in autoradiography using conventional radiography, it is possible to visualize an autoradiograph containing positional information on a radiographic film in order to obtain positional information of a radiolabeled substance. It is mandatory.

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 base-specific DNA compound or mixture thereof to which a radioactive label has been added, and by determining the separation and development sequence of these base-specific DNA compounds. It is determined by comparing them with each other. Therefore, analysis of the obtained autoradiograph is usually performed through human vision, which requires a great deal of time and effort.

Furthermore, since it relies on the human eye, there are limits to the accuracy of the information obtained, such as the location information obtained by analyzing the autoradiograph differing depending on the researcher. In particular, autoradiographs visualized on radiographic film have poor image quality (sharpness) due to the small amount of sample, the weak radiation energy emitted from radiolabeled substances, and the inability to obtain suitable exposure conditions. , contrast), it is difficult to obtain satisfactory information, and the accuracy tends to decrease.

Conventionally, in order to improve the accuracy of positional information, for example, the visualized autoradiograph has been measured using a measuring instrument such as a scanning densitometer. However, this means that
This increases the time required to analyze an autoradiograph and complicates its operation.

In addition, in order to visualize an autoradiograph having the above-mentioned positional information on a radiographic film, it is necessary to overlap the sample and the radiographic film for a long time (for example, several days) and expose the sample to light. In addition, in order to reduce chemical fog caused by various substances in the sample on the silver salt, which is the photosensitive component of the radiation film, the exposure operation is carried out at a low temperature (0 to -
9000), and there are various restrictions on the exposure conditions. Furthermore, the photosensitive silver salt of radiographic film is easily affected by physical stimuli, that is, it is prone to physical fog, so its handling requires a high degree of skill and care, and therefore autoradiography operations making it even more complicated.

In addition to the chemical fog and physical fog mentioned above, the image quality obtained in this way is also affected by natural radioactivity contained in the sample other than the radiolabeled substance during long exposure operations, or by environmental radioactivity. tends to decrease.

[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. By obtaining an autoradiograph containing positional information of a substance as a digital signal without converting it into a visible image, and applying suitable signal processing to the obtained digital signal, it is possible to easily determine the base sequence of DNA, or DNA fragments. I realized that I decided to.

Furthermore, the present invention has been achieved by realizing that, if desired, a visible image corresponding to an autoradiograph can also be obtained from an electrical signal or a digital signal obtained by using the above radiation image conversion method.

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. l) a base-specific DNA composition containing at least a guanine-specific DNA composition; 2) a base-specific DNA composition containing at least an adenine-specific DNA composition; and 3) at least a thymine-specific DNA composition. 4) a base-specific DNA compound comprising at least a cytosine-specific DNA compound; each of at least four groups of base-specific DNA compounds are arranged in parallel relationship on a support medium. By allowing the stimulable phosphor sheet to absorb the radiation energy emitted from the group of radioactive labeled substances in the separated and expanded array formed by one-dimensional separation and expansion, the stimulable phosphor sheet After accumulating and recording an autoradiograph having positional information of the group of radiolabeled substances, the stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is For the digital signal corresponding to the autoradiograph of each separation development column obtained by photoelectrically reading out, the step of: i) determining the scanning direction for signal processing for each of the separation development column; ii) the separation development column; a step of detecting sampling points in the scanning direction of the column for each of the separation expansion columns; 1ii) matching sampling points between corresponding positions in the scanning direction for each of the four groups of separation expansion columns; The present invention provides a signal processing method in autoradiography, which includes the step of obtaining positional information of each of guanine, adenine, thymine, and cytosine.

Further, the present invention performs signal processing to determine the base sequence of the DNA or DNA fragment, and at the same time performs electrical processing corresponding to the autoradiograph obtained by photoelectrically reading out the stimulated light. The present invention also provides a signal processing method in autoradiography, which consists of obtaining the autoradiograph as a visible image from a signal or a digital signal. Or various information centered on the position of the aggregate, for example, one or more of the information consisting of the location and shape of the aggregate of radioactive substances present in the support medium, the concentration and distribution of the radioactive substance at that position, etc. Refers to various types of information obtained as arbitrary combinations.

Here, the radiation image conversion method in place of conventional radiography refers to a method of converting a stimulable phosphor using a stimulable phosphor sheet, as disclosed in JP-A-55-12145, for example. The stimulable phosphor of the stimulable phosphor sheet absorbs the radiation energy transmitted through the subject or emitted from the subject, and then the stimulable phosphor is exposed to visible light. By scanning with electromagnetic waves (excitation light) such as infrared rays, the radiation energy accumulated in the stimulable phosphor is released as fluorescence (stimulated luminescence), and this fluorescence is read photoelectrically to generate an electrical signal. This electrical signal is then reproduced as a visible image, or A/D converted to obtain a digital signal.

Further, the stimulable phosphor sheet contains, for example, a stimulable phosphor such as a divalent europium activated alkaline earth metal fluorohalide phosphor. Stimulable phosphors are irradiated with radiation such as X-rays, α-rays, β-rays, γ-rays, and ultraviolet rays, accumulate some of the radiation energy, and then emit electromagnetic waves (excitation light) such as visible light and infrared rays. When exposed to irradiation, it exhibits stimulated luminescence depending on the accumulated energy.

That is, the present invention utilizes the radiation image conversion method described above in autoradiography to directly obtain positional information of a radiolabeled substance separated and developed on a support medium as a digital signal. Moreover, if desired, based on the digital signal or A/D
Based on the electrical signal before it is converted into a digital signal, an autoradiograph indicating the position of the radiolabeled substance is also obtained as a visible image.

[Effects of the Invention] The radiographic image conversion method described above has a very practical advantage in that a radiographic image can be recorded over an extremely wide radiation exposure range (latitude) compared to conventional radiographic methods. In other words, it has been recognized that in the stimulable phosphor used in this method, the amount of stimulable light emitted by irradiation with excitation light after accumulating the radiation energy is proportional to the amount of radiation exposure over an extremely wide range. It is being Therefore, according to the above method, a highly accurate digital signal corresponding to the autoradiograph of the subject can be directly obtained.

In addition, by setting the reading gain to an appropriate value at the stage of photoelectrically reading the photostimulated light that contains the autoradiograph of the test object and obtaining it as an electrical signal, it is possible to prevent changes in the exposure amount depending on the conditions of the test subject. Even if the level of radiation energy accumulated in the stimulable phosphor sheet varies due to factors such as variations in the sensitivity of the stimulable phosphor and variations in the sensitivity of the photodetector, it is possible to generate digital signals that are not affected by fluctuations in these factors. Obtainable. Furthermore, it has become possible to reduce the amount of radiation emitted from the subject compared to before.
This makes it possible to reduce the amount of radiolabeled substances in samples that are harmful to the health of researchers.

Then, by passing the digital signal containing the positional information of the radiolabeled substance obtained by the radiation image conversion method using the above-mentioned stimulable phosphor sheet through an appropriate signal processing circuit having a signal processing function, the position of the radiolabeled substance is determined. Information can be automatically displayed as desired symbols and/or numerical values without relying on human vision. Then, in the signal processing circuit, the positional information displayed as symbols and/or numerical values is further subjected to appropriate arithmetic processing and other related information to obtain desired information, that is, DNA information without human intervention. It is possible to determine the base sequence.

Therefore, the present invention automates the analysis of autoradiographs by digital signal processing of digital signals corresponding to autoradiographs having positional information of radiolabeled substances, thereby significantly reducing the time and labor required in conventional methods. This allows for significant shortening. It also makes it possible to obtain highly accurate position information.

In addition to the various advantages mentioned above, the present invention makes it possible to obtain the positional information of a radiolabeled substance as symbols and/or numerical values, and at the same time to visualize the autoradiograph with the positional information as an image. It also makes it possible.

This makes it possible to compare the positional information (DNA base sequence) obtained as symbols and/or numerical values through signal processing with the visible image.

It also allows comparison with other visualized autoradiographs. #f so far, −
Radiography is often used for one-tendiogutephy, allowing comparison with visible images obtained by this conventional method. Furthermore, since it can be stored as a visible image, it becomes possible to store records in other formats other than storing them as symbols and/or numerical values on magnetic tape or the like.

Furthermore, since this imaging is performed after obtaining electrical and/or digital signals corresponding to the autoradiograph of the sample, suitable image processing may be applied to the obtained digital signals as necessary. This makes it possible to obtain visible images with particularly excellent observation and interpretation performance.

Another advantage is that the exposure operation of the sample onto the stimulable phosphor sheet can be performed under significantly relaxed exposure conditions (time, temperature, etc.) compared to the exposure operation for obtaining conventional photographic images. It also has In other words, exposure can be performed without reducing the accuracy of the positional information of the radiolabeled substance even under the temperature conditions of the environmental temperature or a temperature in the vicinity thereof. Furthermore, since the stimulable phosphor sheet has high sensitivity, the exposure time can be significantly shortened. In this respect as well, the accuracy of autoradiography is improved and the operation is simplified.

[Structure of the Invention] An example of a sample used in the present invention is a sample synthesized using DNA or a DNA fragment as a template and using a radioactively labeled deoxynucleoside triphosphate (dNTP) and a DNA synthesizing enzyme. Examples include a support medium in which each base-specific DNA compound and/or a mixture thereof is separated and developed in a -dimensional manner to form a separation and development array.

In particular, when this base-specific DNA compound is synthesized by the dideoxy method (chain termination method) commonly used in the Sanger-Coursin method, dideoxynucleoside triphuosine, which has the function of stopping the DNA' synthesis reaction, is used. By using phate (ddNTP), an exclusive combination of the following four types can generally be obtained.

1) Guanine-specific DNA composite 2) Adenine-specific DNA composite 3) Thymine-specific DNA composite 4) Cytosine-specific DNA composite In addition, for separating and developing the above radiolabeled substance using a support medium. Methods include, for example, gel-like support media (
Examples include electrophoresis using various support media such as a polymer molded body such as acetate or filter paper, and thin layer chromatography using a support medium such as silica gel. Of these,
Electrophoresis using a gel-like support medium is a typical separation development method and is preferred for practicing the present invention.

The basic structure of the stimulable phosphor sheet used in the present invention is a support, a phosphor layer, and a transparent protective film. The phosphor layer is made of a binder containing and supporting a stimulable phosphor in a dispersed state. For example, divalent europium-activated barium fluoride bromide (BaFBr:Eu2+) phosphor particles are combined with nitrocellulose and linear polyester. It is obtained by dispersing it in a mixture. 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. .

In the present invention, the transfer and accumulation operation (exposure operation) of the radiation energy emitted from the support medium containing the radiolabeled substance onto the stimulable phosphor sheet is performed by overlapping the support medium and the stimulable phosphor sheet for a certain period of time. Accordingly, at least a portion of the radiation emitted from the radiolabeled substance on the support medium is absorbed by the stimulable I-photon 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 of the sample preparation method, stimulable phosphor sheet, and exposure operation in autoradiography using the Sanger-Luzon method, please refer to the patent application filed by the applicant in 1973.
8-201231 in detail.

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 is shown 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). The apparatus is comprised of a main reading output section 3 having a function of reading out the autoradiograph stored on the phosphor sheet 1 in order to output the positional information of the radiolabeled substance.

In the prefetch successive section 2, the following prefetch operation is performed.

The laser light 5 generated by the laser light source 4 or 4 passes through the filter 6, whereby a portion of the wavelength range corresponding to the wavelength range of stimulated luminescence generated from the stimulable phosphor sheet 1 in response to excitation by the laser light 5 is detected. is cut. Next, the laser beam is deflected by a light deflector 7 such as a galvanometer 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 stimulated luminescence emitted from the phosphor q-t 1.

The phosphor sheet 1 is transported in the direction of arrow 9 under irradiation with the above-mentioned polarized laser light. Therefore, the entire surface of the phosphor sheet l is irradiated with the polarized laser light. Note that 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 phosphor sheet l
The transfer speed is adjusted so that the energy of the laser beam 5 for the pre-reading operation is smaller than the energy used for the main reading operation.

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 light guide sheet 10 for pre-reading. do. The light guide sheet IO has a linear incident surface and is arranged close to the scanning line on the phosphor sheet 1 so as to face it, and its exit surface forms a ring and is a photomultiplier or the like. It is connected to the light receiving surface of the photodetector 11. This light-guiding sheet 1O 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. It is configured to The stimulated luminescence from the phosphor sheet 1 is guided through the light guide sheet 10 and reaches the exit surface, is emitted from the exit surface, and is 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. The stimulated luminescence detected by the photodetector 11 is converted into an electrical signal, and further transmitted to the amplifier 12.
is amplified 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, so that a signal of an appropriate level can be obtained according to the obtained accumulated recording information, and an image with excellent density and contrast can be obtained.
The recording scale factor b and the reproduction image processing condition setting value C are output.

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

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

Laser light 1 emitted from the main reading laser light source 14
5, the beam diameter is strictly adjusted by a beam expander 17 after passing through a filter 16 having the same function as the filter 6 described above.Next, the laser beam is passed through an optical deflector such as a galvanometer mirror. 18 and reflected by a plane reflecting mirror 19, the light is one-dimensionally deflected and incident on the phosphor sheet l. 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 transported in the direction of the 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 l 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 light guiding sheet 22 for book reading is a light guiding sheet 22 for pre-reading)
It has the same material and structure as 10, and the stimulated luminescence that is guided through the interior of the reading light guiding sheet 22 through repeated total reflection is emitted from the exit surface and is received by the photodetector 23. Ru. Note that a filter that selectively transmits only the wavelength range of stimulated luminescence is attached to the light receiving surface of the photodetector 23, so that the photodetector 23 detects 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 that has been transferred and accumulated on the stimulable phosphor sheet in the present invention, the readout operation consisting of the pre-reading operation and the main reading operation has been described above. Readout operations that can be utilized in the present invention are not limited to the above examples, for example, the amount of radiolabeled material on the support medium and the exposure time of the stimulable phosphor sheet for that support medium. If known in advance, it is also possible to omit the look-ahead operation in the above example.

Further, in the present invention, the method for reading out the positional information of the radiolabeled substance on the support medium that has been transferred and accumulated on the stimulable phosphor sheet is not limited to those exemplified above.

The digital signal corresponding to the autoradiograph of the radiolabeled substance thus obtained is then input to the signal processing circuit 26. The signal processing circuit 26 performs signal processing to convert the positional information of the radiolabeled substance into symbols and/or numerical values, and to determine the base sequence of the target DNA. That is, after determining the scanning direction for signal processing, the sampling points are detected, and then the sampling points are compared and identified. Furthermore, signal processing for imaging based on the reproduced image processing condition setting value C is performed as necessary.

Hereinafter, embodiments of signal processing in autoradiography for DNA base sequencing using the signal processing method of the present invention will be described below, taking as an example the case where the dideoxy method of the Sanger-Coursin method is used. A migration column formed by a combination of different types of base-specific DNA compounds (
A case using a separate expansion column) will be explained.

1) Guanine (G) specific DNA compound 2) Adenine (
A) Specific DNA compound 3) Thymine (T) specific DNA
Synthesis product 4) Cytosine (C)-specific DNA composition First, according to a conventional method, a -terminal strand of the sample DNA (Tembray DNA) and a short DNA fragment complementary to a part of this DNA (
After preparing primers and hybridizing the primers to DNA, DNA polymerase is activated to convert the template using four types of deoxynucleoside triphosphates (dNTPs) and one type of dideoxynucleoside triphosphates (ddNTPs). D
Complementary to NA, NA is synthesized. Four types of dNTPs
By using at least one of them labeled with a radioactive element such as 32p, base-specific DNA compounds of the above four groups to which a radioactive label has been added are prepared. For example, guanine-specific DNA compounds are obtained by using ddGTP as the ddNTP. Specific DNA compounds for other bases are obtained in a similar manner by using the corresponding ddNTPs (ie ddATP, ddTTP, or ddcTP).

This is separated and developed by electrophoresis on a gel-like support medium to obtain a sample. Next, this sample (supporting medium) and a stimulable phosphor sheet are overlapped for several minutes at room temperature to carry out an exposure operation, and the DNA separation and development arrays formed on the sample are transferred to the stimulable phosphor sheet using autoradiation. Accumulate and record as a graph.

FIG. 2 shows an autoradiograph of the above-mentioned four types of electrophoresis column in which a ring group-specific DNA compound to which a radioactive label has been added is separated and developed. That is, the 2nd v! The first to fourth columns of J are (1)-” (G) Specific DN
Each column (electrophoresis column) of A compound (2) - (A) specific DNA compound (3) - (T) specific DNA compound (4) - (C) specific DNA compound is shown.

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 digital signal input to the signal processing circuit 26 is
The address (x, y) expressed in the coordinate system fixed on the stimulable phosphor sheet and the signal level (2
), and the level of the signal corresponds to the amount of photostimulated light. That is, the digital signal corresponds to the autoradiograph of FIG. Therefore, digital image data having positional information of the radiolabeled substance is input to the signal processing circuit 26. As used herein, digital image data refers to a collection of digital signals corresponding to an autoradiograph of a radiolabeled substance.

First, the scanning direction for digital signal processing is determined for the digital signal.Here, if the vertical direction (separation development direction) is the y-axis direction and the horizontal direction is the x-axis direction in FIG. The direction can be determined, for example, as follows.

That is, obtained. Regarding digital image data, y
An arbitrary point in the axial direction (located at y=ya) is numerically scanned in the X-axis direction to find the X coordinate (X&) at which the signal level has a maximum value. However, a is a positive integer and represents the number of each column.

At this time, the scanning width in the y-axis direction needs to be large enough to include at least one separation and development site of the radiolabeled substance (usually referred to as a "migration zone"). When this scanning width is made large enough to include only one migration band, one maximum value is usually found,
This corresponds to the X coordinate of any one of the four migration columns described above. Next, by changing the value of Va and repeating the above scanning and finding the maximum value at least four times, it is possible to determine the X coordinates corresponding to the four different electrophoresis rows.

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

Therefore, the X coordinate (X &) is, for example, when the digital signal within the above scanning width is repeatedly extracted in the y-axis direction for each X coordinate and the signal levels are added, and the level of the obtained signal is the maximum. The X coordinate (Xa) can be set as follows. Alternatively, after extracting the digital signal within the above scanning width in the X-axis direction and finding the X-coordinate where the signal level is maximum for each X-coordinate, calculate the average coordinate of these X-coordinates, This can be set as the X coordinate (Xa). In order to avoid peaks of noise mixed in during such scanning, the signal level is binarized using a preset threshold. You can leave it there.

In this way, the scanning direction of each column can be determined as a straight line passing through the detected X coordinate (Xa) and parallel to the y-axis direction as the scanning direction for the following signal 0 signal processing.

Next, the sampling point for detecting the separation and development site of the radiolabeled substance is detected at the sampling point in the scanning direction, and when only the digital signal in the scanning direction is extracted, the level of the signal is at its maximum. It can be all points where .

In the above scanning, it is desirable to scan with a constant width. Therefore, the maximum point of the signal level is, for example, the position (y) in the scanning direction on the horizontal axis, and the position (y) within the scanning width on the vertical axis. When expressed in a graph of the average value (z) of the signal level, it means all the peak points that appear on this graph.

Hereinafter, the average value of the signal level at each position in the scanning direction will be simply referred to as the signal level at that position.

Figure 3 shows a graph in which the horizontal axis represents the position (y) in the scanning direction and the vertical axis represents the signal level (=) for one electrophoresis column shown in Figure 2, as described above. ing.

In this way, the coordinate point and the level of the signal at this coordinate point CX & + '11L n r Z & n
) is determined. Here, n is a positive integer and represents the number of sampling points.

By subjecting the digital signals to signal processing as described above, it is possible to express the one-dimensional positional information of the radiolabeled substance for each electrophoresis column by the one-dimensional position and the signal level at that position (Y & n + Zan). can. Here, the signal level (Z an) at each position can be considered to represent the relative amount (concentration) of the radiolabeled substance.

Alternatively, for example, by recording the starting position for separating and developing a radiolabeled substance on a stimulable phosphor sheet with a marker containing a radiolabeled substance, this starting position (yo) can be recorded on the digital image data. can be detected in the same manner as above. Also, start position (
y o) can also be detected by, for example, processing the stimulable phosphor sheet itself using physical means such as punching holes, and then setting the starting position during the exposure operation. be. Then, by subtracting (Van Vo=Van') using this yo, the above position information can be calculated as the moving distance from the separation deployment start position and the signal level at that position (Y an' +
Z an).

Furthermore, regarding the evaluation of the relative amount of the radiolabeled substance, in addition to the signal level at the above-mentioned sampling points, various calculations such as the integral value near each maximum point are possible.

Next, the positions (Van) of the above-mentioned convex markings are compared with each other and arranged in descending order of Van. For example, we can obtain the diagram shown below.

S 11 * S 4 * * S 3 Z + 32
1 + 342 +” 12 + S32 + S22
r S23 +...In the above diagram, 5
By replacing sn=G, 52n=A, San=T, and S4H=C, the following diagram is obtained.

G, -C-T-A-C-G-T-A-A-...
In this way, the base sequence of the NA chain molecule complementary to the sample DNA can be determined. Note that the information about the obtained DNA base sequence is not limited to the above display format, and any display format is possible. For example, if desired, the signal level (Z an) at the sampling point in the above-mentioned signal processing can be set at the same time as
It is also possible to display the relative amounts of each separated and developed compound. Alternatively, a diagram as shown in FIG. 4 is also possible.

Alternatively, it is also possible to display the base sequences of both two stranded molecules of DNA. That is, by providing the information A-T, G-C1C→G, and T4A as combinations corresponding to each base in the diagram represented by the above symbols, DNA represented by the following diagram can be obtained.
Obtain the base sequence of

G-C-T-A-C-G-T-A-A-...C
-G-A-T-G-C-A-T-T-... Information about the DNA base sequence determined by the above signal processing is output from the signal processing circuit 26. Thereafter, the data is transmitted to a recording device (not shown) either directly or, if necessary, via a storage means such as a magnetic tape.

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

Furthermore, in the present invention, an autoradiograph of the radiolabeled substance on the support medium can be imaged from the obtained digital signal or the electric signal before A/D conversion, if desired.

Note that the signal processing circuit 26 performs signal processing (image processing) on the digital signal based on the reproduction image processing condition setting value C so that a visible image with appropriate density and contrast and excellent observation and interpretation performance is obtained. Examples of this image processing include spatial frequency processing, gradation processing, averaging processing, reduction processing, and enlargement processing.

The digital signal subjected to image processing is transmitted to the reproducing/recording apparatus shown in FIG. 5 via a storage means such as a magnetic tape, if necessary.

Next, Section 5 describes a method for imaging a digital signal having image-processed position information of a radiolabeled substance.
A brief description will be given with reference to the example of the reproducing/recording device shown in the figure.

FIG. 5 shows a schematic diagram of an example of a reproducing/recording device for converting the digital signal outputted from the reading device of FIG. 1 into a (visible) image.

In order to image the positional information of the radiolabeled substance, the following reproduction/recording operation is performed.

The digital signal input to the D/A converter 27 is converted into an analog signal representing the concentration, and is input to the optical modulator 28. The optical modulator 28 is modulated by this analog signal. A laser beam 30 from a recording laser light source 29 is modulated by the optical modulator 28, and then scanned by a scanning mirror 31 onto a photosensitive material 32 such as photographic film, and an image is reproduced on the photosensitive material 32. .

Note that the method for reproducing and recording the digital signal is not limited to the above method, and for example, a method using the above-mentioned recording device may be used.

Furthermore, the visible image of the autoradiograph can also be obtained by reproducing and recording the electric signal before the digital signal is A/D converted. That is, it can also be obtained by directly transmitting the electrical signal obtained by the amplifier 24 in the reproduction apparatus shown in FIG. 1 to the optical modulator 28 of the reproducing/recording apparatus shown in FIG.

The DNA base sequencing method using the above combination of (G, A, T, C) is an example of the DNA base sequencing method, and the signal processing method of the present invention uses the above combination. The combinations are not limited to the above, and various combinations are possible, and the base sequence can be determined in a similar manner using the combinations described above.

For example, even if you use the combination obtained by the plus-minus method, which is an alternative method of the Sanger-Cournon method, D
The base sequence of NA can be determined. or,
at least one group of base-specific DNA compounds and a suitable reference material (e.g., a mixture of each base-specific DNA compound)
It is also possible to determine the sequence of a specific base from the combination.

Furthermore, in the above example, the explanation was given using four rows of radiolabeled substance groups that are separated and developed in a one-dimensional direction on the support medium, but the separation and development rows are not limited to four rows. It may be more than a convex engraving, or it may be less than that. Alternatively, it is also possible to simultaneously determine the base sequences of two or more types of DNA using one support medium.

In addition to the information obtained above,
For example, it is also possible to perform genetic linguistic information processing such as comparing with other DNA base sequences that have already been recorded and stored.

[Brief explanation of the drawing]

FIG. 1 shows an example of a follow-up device (or reading device) for reading out positional information of a radiolabeled substance in a sample stored and recorded on a stimulable phosphor sheet in the present invention. l: stimulable phosphor sheet, 22 reading part for pre-reading, 3: continuous reading part for main reading, 4: laser light source, 5: laser light, 6
: filter, 7: optical deflector, 8: plane reflecting mirror, 9: transport direction, lO: light guiding sheet for pre-reading, 11: photodetector, 12: amplifier, 13: control circuit, 14: laser light source, 15 Elm f-light, 16 Nifilter, 17: Beam expander, 18: Optical deflector, 19: Plane reflecting mirror, 20: fθ lens, 21: Transfer direction, 22 Light guiding sheet for dual reading, 23: Light Detector, 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 DNA is separated and developed on a gel support medium. FIG. 3 is a graph showing an example of the relationship between the position in the scanning direction and the level of the digital signal for signal processing. FIG. 4 shows the DN determined by the signal processing method of the present invention.
1 is a diagram schematically showing an example of the base sequence of A. FIG. 5 shows an example of a reproducing/recording apparatus for converting into an image a digital signal having positional information of a radiolabeled substance in a sample stored and recorded on a stimulable phosphor sheet in the present invention. 27: D/A converter, 28: Optical modulator, 29: Recording laser light source, 30: Laser light, 3: Scanning mirror,
32: Photosensitive material patent applicant Fuji Photo Film Co., Ltd. Agent Patent attorney Yasuo Yanagawa

Claims (1)

[Scope of Claims] 1. A signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, comprising a base sequence complementary to the DNA or DNA fragment, and a radioactive 1) A base-specific DNA compound containing at least a guanine-specific DNA compound, to which a label is attached. ) a base-specific DNA compound comprising at least an adenine-specific DNA compound; 3) a base-specific DNA compound comprising at least a thymine-specific DNA compound; 4) a base-specific DNA compound comprising at least a cytosine-specific DNA compound. Each of at least four groups of base-specific DNA compounds, including a compound, is released from a group of radiolabeled substances in a separation and development array formed by one-dimensional separation and development in a parallel relationship on a support medium. By absorbing 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 exposed to electromagnetic waves. With regard to the digital signals corresponding to the autoradiographs of each separated development column obtained by scanning the autoradiograph with a light source to emit photostimulated light and reading out this photostimulated light photoelectrically, i) the separated determining a scanning direction for signal processing for each of the expanded columns; ii) detecting sampling points in the scanning direction of the separated expanded columns for each of the separated expanded columns; 1ii) separation of at least the four groups. A signal processing method in autoradiography, comprising the step of obtaining positional information for each of guanine, adenine, thymine, and cytosine by matching sampling points between corresponding positions in the scanning direction for each of the development rows. 2゜Base-specific DNA of the above DNA or DNA fragment
A compound is a base-specific compound of at least four groups consisting of: 1) a guanine-specific DNA compound, 2) an adenine-specific DNA compound, 3) a thymine-specific DNA compound, and 4) a cytosine-specific DNA compound. The signal processing method in autoradiography according to claim 1, characterized in that the method comprises a DNA composite. 3. Signal processing in autoradiography according to claim 1, characterized in that in step 11), all points where the signal level is maximum in the scanning direction are taken as sampling points in the scanning direction. Method. 4. 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 provided with a radioactive label. 1) a base-specific DNA compound comprising at least a guanine-specific DNA compound; 2) a base-specific DNA compound comprising at least an adenine-specific DNA compound; 3) a base-specific DNA compound comprising at least a thymine-specific DNA compound. 4) a base-specific DNA compound comprising at least a cytosine-specific DNA compound; By causing the stimulable phosphor sheet to absorb the radiation energy emitted from the radiolabeled substance group in the separated and developed column formed by one-dimensional separation and development, the stimulable phosphor sheet After accumulating and recording an autoradiograph having positional information, the stimulable phosphor sheet is scanned with electromagnetic waves to emit the autoradiograph as photostimulated light, and this photostimulated light is read out photoelectrically. For the obtained digital signal corresponding to the autoradiograph of each separated development column, i) determining a scanning direction for signal processing for each of said separation development column, li) said separation development column 1ii) Detecting sampling points in the scanning direction for each of the separation expansion columns; 1ii) By comparing sampling points between corresponding positions in the scanning direction for each of the four groups of separation expansion columns; , obtaining position information of each of guanine, adenine, thymine, and cytosine. 1. A signal processing method in autoradiography comprising obtaining the autoradiograph as a visible image from an electric signal or digital signal corresponding to the autoradiograph obtained by photoelectrically reading out light. 5゜Base-specific DNA of the above DNA or DNA fragment
A compound is a base-specific compound of at least four groups consisting of: 1) a guanine-specific DNA compound, 2) an adenine-specific DNA compound, 3) a thymine-specific DNA compound, and 4) a cytosine-specific DNA compound. 5. The signal processing method in autoradiography according to claim 4, which comprises a DNA composite. 6. Signal processing in autoradiography according to claim 4, characterized in that in step ii), all points where the signal level is maximum in the scanning direction are taken as sampling points in the scanning direction. Method.