JPH0160781B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a signal processing method in autoradiography. More specifically, the present invention provides autoradiography, in which:
The present invention relates to a method for determining sampling points in a digital signal processing method for determining the base sequence of DNA or DNA partial decomposition products.

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. In recent years,
Autoradiography is DNA or DNA
It is effectively used to determine the base sequence of partial decomposition products.

The method for determining the base sequence of DNA using this autoradiography is as follows.
Maxam-Gilbert method,
and Sanger Coulson
Coulson) law is known. These methods are
DNA has a double helical structure consisting of two chain molecules, and each of the two chain molecules contains four types of bases: adenine (A), guanine (G), cytosine (C), It is composed of a structural unit having a base called thymine (T), and the two chain molecules are cross-linked by hydrogen bonds between these four types of bases, and each structural unit The hydrogen bonds between G-C and A-T
This method cleverly utilizes the characteristic structure of DNA, which is realized only in combinations of two types, to determine its base sequence.

For example, the Maxam-Gilbert method is implemented by the method described below.

First, the DNA whose base sequence is to be determined
Alternatively, by bonding a group containing a radioactive isotope of phosphorus (P) to one end of a chain molecule of a DNA decomposition product, the target substance is made into a radioactive labeling substance, and then chemical means are applied. It is used to base-specifically cleave the bonds between each constituent unit of a chain molecule. Next, the DNA or DNA obtained by this operation
A mixture of many base-specific cleavage products is separated and developed by gel electrophoresis, and a large number of base-specific cleavage products are separated and developed to form a band shape (however, it is difficult to see visually). (cannot be seen at the target). Then, the autoradiograph of this separation and development array is visualized on an X-ray film, and from the obtained visible image and each base-specific cutting means, it is possible to detect from the end of the chain molecule to which the radioactive isotope is bound. The bases in a certain positional relationship are sequentially determined, and in this way the sequence of all bases in the target object is determined.

In the above-mentioned autoradiography, instead of using the conventional radiographic method using a radiographic film, Japanese Patent Application No. 193418/1983 by the present applicant discloses a radiographic method using a stimulable phosphor sheet. An autoradiographic measurement method that utilizes a conversion method is described.

In the radiation image conversion method, a sample and a stimulable phosphor sheet are overlapped, the radiation energy emitted from the sample is absorbed by the stimulable phosphor sheet, and then this stimulable phosphor sheet is exposed to visible light and By scanning with electromagnetic waves (excitation light) selected from infrared rays, the radiation energy accumulated in the stimulable phosphor sheet is converted into fluorescence (stimulated luminescence).
This fluorescence is photoelectrically read to obtain an electrical signal, and this electrical signal is A/D converted to obtain a digital signal.

Further, the stimulable phosphor sheet contains, for example, a photostimulable phosphor such as a divalent europium-activated alkaline earth metal fluorohalide phosphor. This photostimulable phosphor can be used for X-rays, α-rays, β-rays,
After being irradiated with radiation such as rays, gamma rays, and ultraviolet rays and accumulating a portion of the radiation energy, electromagnetic waves (excitation light) selected from visible light and infrared rays are generated.
When exposed to irradiation, it exhibits stimulated luminescence depending on the accumulated energy.

Autoradiography using the radiation image conversion method described above is capable of obtaining position information of a radiolabeled substance as a digital signal without converting the autoradiograph having position information into an image.

The applicant has also already filed an invention regarding a digital signal processing method for obtaining one-dimensional positional information of a radiolabeled substance as a signal and/or numerical value in autoradiography using a radiation image conversion method. (Special application 1982-
No. 1326).

According to the above signal processing method, the positional information of a radioactive label, which conventionally relied on the visual judgment of researchers, can be automatically and highly accurately obtained as desired symbols and/or numerical values. It is something.

The present invention is an autoradiography method for determining the base sequence of DNA or partial DNA decomposition products. The object of the present invention is to provide a signal processing method for easily and highly accurately determining the base sequence of a partially degraded DNA product.

The above purpose is, when a sample contains a mixture of two or more types of base-specific cleavage products, to first separate the separation and development column of this mixture and at least one type of base-specific cleavage product in this mixture. One type of base-specific cleavage product or a cleavage product consisting of a combination of different types can be obtained by performing a simple numerical operation (for example, subtraction) between the separation and expansion sequence of the cleavage product or mixture of cleavage products. After obtaining a virtual separation and expansion sequence for the mixture,
This is achieved by determining sampling points for the virtual separation expansion sequence and obtaining positional information of desired bases from these sampling points. In other words, instead of determining sampling points for each separation and expansion sequence obtained by separating and developing a sample and then performing numerical operations between each sampling point to obtain positional information about the desired base, After obtaining a virtual separation and expansion sequence for the desired base-specific cleavage product (or mixture of cleavage and decomposition products), perform arithmetic processing such as threshold processing on the virtual separation and expansion sequence to determine sampling points. By doing so, the above objective can be achieved.

That is, the present invention is a signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, which method involves base-specific cleavage and decomposition of radioactively labeled DNA or DNA partial decomposition products. 1) A base-specific cleavage product consisting of one to three types of guanine-specific cleavage product, adenine-specific cleavage product, thymine-specific cleavage product, and cytosine-specific cleavage product, or 2) At least two groups of base-specific cleavage products containing a mixture of two to four types of base-specific cleavage products, including at least one type of base-specific cleavage product included in 1) above. Alternatively, each of the cut and decomposed product mixtures is separated and developed in a one-dimensional manner on a support medium in a parallel relationship, and the radiation energy emitted from the radiolabeled substance group of the separated and developed column is applied to the stimulable phosphor sheet. By absorbing the stimulable phosphor sheet, an autoradiograph containing positional information of the radiolabeled substance group is stored and recorded, and then the stimulable phosphor sheet is scanned with electromagnetic waves to record the autoradiograph. Regarding the digital signal corresponding to the autoradiograph of each separated and expanded array obtained by emitting photostimulated light and reading out this photostimulated light photoelectrically, (i) the base-specific cleavage product of 1) above; Alternatively, a virtual The present invention provides a signal processing method in autoradiography, which includes the steps of: obtaining a virtual separation expansion sequence; (ii) determining sampling points for the virtual separation expansion sequence;

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 combination of information such as the concentration and distribution of radioactive substances at that location.

According to the present invention, when a sample contains a mixture of two or more types of base-specific cleavage products, simple numerical calculations are first performed between the separation and development arrays of the base-specific cleavage products and the mixture of cleavage products. After obtaining a virtual separation and expansion sequence for one type of base-specific cleavage product or a mixture of different combinations of cleavage and decomposition products, arithmetic processing is performed on the virtual separation and expansion sequence to determine the sampling points. By determining , it is possible to directly obtain positional information of a desired base from this sampling point. Since the arithmetic processing is performed on the virtual separation and expansion sequence of the desired base-specific cleavage and decomposition products (or the mixture of cleavage and decomposition products), it becomes possible to suitably perform the arithmetic processing and determine the sampling points. This allows the base sequence of DNA or DNA partial decomposition products to be determined easily and with high accuracy.

In addition, even if the area of each distribution site of a radiolabeled substance is small, it is possible to detect the distribution position with high accuracy, so the absolute amount of radiolabeled substance used in one autoradiography can be can be reduced. or,
The number of separation and development columns can be increased without increasing the width of the support medium, and more information can be obtained with a single autoradiography operation than before.

Examples of samples used in the present invention include:
Name a support medium in which each base-specific cleavage product or a mixture thereof obtained by base-specific cleavage and decomposition of radioactively labeled DNA or a DNA partial decomposition product is separated and developed in a one-dimensional direction. Can be done.

In addition, methods for separating and developing the above-mentioned radioactively labeled substance using a support medium include, for example, a gel-like support medium (the shape may be arbitrary, such as layered or columnar), a polymer molded body such as acetate, or various types such as filter paper. Representative methods include electrophoresis using a support medium and thin layer chromatography using a support medium 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 protective film. The phosphor layer is made of a binder containing and supporting the stimulable phosphor in a dispersed state,
For example, it can be obtained by dispersing divalent europium-activated barium fluoride bromide (BaFBr: Eu ²⁺ ) phosphor particles 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. .

In the present invention, the operation (exposure operation) of transferring and accumulating radiation energy emitted from a support medium containing a radiolabeled substance onto a stimulable phosphor sheet is performed as follows:
By overlapping the support medium and the stimulable phosphor sheet for a certain period of time, 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 of the stimulable phosphor sheet and the exposure operation, please refer to the patent application No. 57-193418 filed by the present applicant.
It is stated in the specification of the No.

Next, in the present invention, a method for reading out one-dimensional positional information of a radiolabeled substance on a support medium transferred and accumulated 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. Part 2
1 shows a schematic diagram of an example of a reading device comprising a main reading reading section 3 having a function of reading out an autoradiograph stored and recorded on a phosphor sheet 1 in order to output positional information of a radiolabeled substance. There is.

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

The laser light 5 generated from the laser light source 4 passes through the filter 6, whereby a portion of the wavelength range corresponding to the wavelength range of stimulated luminescence generated from the phosphor sheet 1 in response to excitation by the laser light 5 is cut out. Ru. Next, the laser beam is deflected by an optical deflector 7 such as a galvano mirror, reflected by a flat reflector 8, and then reflected by a phosphor sheet 1.
It is deflected one-dimensionally and incident on the top. 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 sheet 1.

The phosphor sheet 1 is transported in the direction of the arrow 9 under irradiation with the above-mentioned polarized 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 light guide sheet 10 for prereading. 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, allowing light such as photomal It communicates with the light receiving surface of the detector 11. The light guide sheet 10 is made by processing a transparent thermoplastic resin sheet such as acrylic synthetic resin, and allows light incident from the incident surface to be transmitted to the exit surface while being totally reflected inside. 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 stimulated emission wavelength range and cuts out light in the excitation light (laser light) wavelength range.
It is designed to detect only stimulated luminescence. 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 outputs an amplification factor setting value a and a recording scale factor b so that a signal at an appropriate level can be obtained according to the obtained accumulated recording information.

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

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 14 passes through a filter 16 having the same function as the filter 6 described above, and then the beam
The beam diameter is precisely adjusted by the expander 17. Next, the laser beam is deflected by a light deflector 18 such as a galvano mirror, reflected by a plane reflecting mirror 19, and then one-dimensionally deflected and incident on the phosphor sheet 1. Note that an fθ lens 2 is provided between the optical deflector 18 and the plane reflecting mirror 19.
0, etc., so that when the polarized laser beam scans the phosphor sheet 1, a uniform beam speed is always maintained.

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 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 light guide sheet 22 for book reading has the same material and structure as the light guide sheet 10 for book reading, and the stimulated luminescence guided through the interior of the light guide sheet 22 for book reading while repeating total reflection. The light is emitted from the exit surface and is received by the photodetector 23. Note that a filter that selectively transmits only the wavelength region 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 electrical signal at an appropriate level in the amplifier 24 whose sensitivity is set according to the amplification factor setting value a, and then A/D converted. The signal is input to the device 25. The A/D converter 25 converts the signal into a digital signal using a scale factor suitable for the signal fluctuation width according to the recorded scale factor setting value b.

Regarding 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 in the present invention, the readout 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, the look-ahead operation can be omitted in the above example.

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

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. In the signal processing circuit 26, 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.

Hereinafter, embodiments of signal processing in autoradiography for DNA base sequencing using the signal processing method of the present invention will be described.
Using the Gilbert method as an example, we will explain the case where the following four groups of base-specific cleavage products are used as typical combinations of base-specific cleavage products for base sequencing. do.

1) Guanine-specific cleavage product 2) Guanine-specific cleavage product + adenine-specific cleavage product 3) Thymine-specific cleavage product + cytosine-specific cleavage product 4) Cytosine-specific cleavage product First, radioactive label DNA with ( ³² P) added
By cutting each base unit by a conventional method,
The base-specific cleavage products of the four groups 1) to 4) above are obtained. Next, each of the above four groups of base-specific cleavage products is separated and developed by electrophoresis on a gel support medium to obtain respective separation and development arrays.

Next, an exposure operation is performed by overlapping this sample (gel-like support medium with separation and development columns 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. Transcription and accumulation. Details of the above exposure operation are described in the above-mentioned Japanese Patent Application No. 193418/1983.

FIG. 2 shows an example of an autoradiograph of a separation and development column (electrophoresis column) in which each base-specific cleavage product of radiolabeled DNA is separated and developed.

That is, the first to fourth columns in FIG. (1)-(G) Specific cleavage product (2)-(G) Specific cleavage product +(A) Specific cleavage product (3)-(T) Specific cleavage product +(C) Specific cleavage product (4)-(C) Specific cleavage product shows each separation expansion column.

By loading the autoradiograph transferred and accumulated 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 fixed on the stimulable phosphor sheet. It has an address (x, y) expressed in a coordinate system and a signal level (z) at that address, and the signal level corresponds to the amount of photostimulated light. That is, the digital signal obtained 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.

Detection of sampling points, which is a characteristic requirement of the present invention, is performed, for example, as follows.

By scanning different positions on the digital image data twice across the one-dimensional distribution direction (separation and development direction) of the radiolabeled substance, each column on each scanning area is Detect the distribution points of the radiolabeled substance (the scan to detect these distribution points is called a preliminary scan), connect the two distribution points for each separation development column to obtain four straight lines, and use the obtained straight lines as This is the 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 temporarily stored in a memory in the signal processing circuit 26 (that is, in a buffer memory or a nonvolatile memory such as a magnetic disk). ). 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 of each of the four separation and development columns, a function f _k (w) representing the level of the signal on the scanning area [w is the position in the scanning direction] is obtained. where k represents the number of the separation expansion column] can be obtained.

The following numerical calculations are performed on the separation and expansion columns of base-specific cleavage and decomposition product groups in the first to fourth columns. Using the function f ₁ (w) representing the first column and the function f _{2 (w) representing the second column, perform the subtraction as follows: f 2 (} w) - _{f 1} (w) = f ₅ (w) As a result, the fifth column is virtually obtained. This fifth column is a virtual separation and expansion column corresponding only to adenine (A)-specific cleavage products.

If the above relationship f _k (w) is expressed in the form of a graph, it can be shown as shown in Fig. 3 A to C. 3A and 3B show the relationship between the position in the scanning direction and the signal level for the second column and the first column, respectively, and C shows the relationship between the virtual fifth column obtained by the subtraction process. It shows the relationship between the position of a column in the scanning direction and the signal level.

In addition, in the above-mentioned numerical calculation, if the signal levels in each separation expansion column are considerably different from each other, for example, by setting the amplification factor for each separation expansion column according to the accumulated record information obtained in the pre-read operation, It is preferable that the signal levels of each separation and expansion column be equal.

By performing similar subtraction processing using the third and fourth columns, a virtual sixth column corresponding only to the thymine (T)-specific cleavage product expressed by the function f ₆ (w) can be obtained. obtain.

In this way, a separation and expansion column (including a virtual separation and expansion column) consisting of four columns that does not contain a mixture of base-specific cleavage products as shown below is obtained.

(1)-(G) Specific cleavage and decomposition product (4)-(C) Specific cleavage and decomposition product (5)-(A) Specific cleavage and decomposition product (6)-(T) Specific cleavage and decomposition product (4) The function f _k (w) obtained for the column is subjected to smoothing processing, for example, by convolution using an appropriate filter function, to obtain the function g _k (w). Then this function g _k
(w) is subjected to threshold processing. That is, the threshold (α ₀ )
, when g _k (w)≧α ₀ , g _k (w)=1 when g _k (w) < α ₀ , g _k (w)=0, so that the function g _k (w) to 1
Or convert it to a continuous function of 0. The sampling points are detected by setting each midpoint of the region g _k (w)=1. Note that the threshold value (α ₀ ) in the above threshold processing is determined based on, for example, the relationship between the signal level and its frequency for digital signals on the scanning area,
That is, it can be determined from the histogram.

In this way for each column the sampling point
S _ko 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 of each column. That is, S _ko means the n-th sampling point of the k-th column.

Note that the method for detecting the sampling point after obtaining four separation and expansion columns consisting of specific cleavage and decomposition products of one type of different base through the above subtraction process is not limited to the above method. .

From the detected sampling points of each column, the target DNA base sequence can be determined, for example, as follows.

For each sampling point S _ko in the first column, fourth column, fifth column, and sixth column, if the sampling points S _ko are arranged in a line in descending order of the position (W _ko ) in the scanning direction, for example, We can obtain the following diagram.

S ₃₁ , S ₄₁ , S ₆₁ , S ₃₂ , S ₅₁ , S ₅₂ , S ₆₂ , S ₃₃ , S ₄₂ ,
... In the above diagram, S _3o = G, S _4o = C, S _5o =
By replacing A, S _6o =T, we obtain the following diagram.

G-C-T-G-A-A-T-G-C-... In this way, the base sequence of one chain molecule of DNA can be determined.

Note that the determination of the DNA base sequence after detecting the sampling points of each separation and expansion column is not limited to the above method; for example, if the separation and expansion column is distorted, one set of Standard (internal standard) from the separation and development column of base-specific cleavage products
It is also possible to use a method of synthesizing sequences and correcting the distortion based on the reference sequence to determine the base sequence. Considering the distortion of such a separation expansion sequence,
The signal processing method for DNA base sequencing is
For example, it is described in Japanese Patent Application No. 58-1331 and Japanese Patent Application No. 58-1333 by the present applicant.

In addition, information about the obtained DNA base sequence is not limited to the above display format,
Any display format is possible. For example, if desired, the relative amount of each separated and developed base-specific cleavage product can also be displayed by arbitrarily processing the signal level in the scanning direction of each column.

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

G-C-T-G-A-A-T-G-C-... C-G-A-C-T-T-A-C-G-... The signal processing method of the present invention includes: Above (G,
There is no limitation to the combinations of G+A, T+C, and C), and various combinations are possible.
In the above example, explanation was given using a group of radiolabeled substances constituting four separation and development columns consisting of one type of DNA, but the separation and development column is not limited to four columns, and multiple types of DNA Alternatively, the base sequence of a DNA partial decomposition product can also be determined at the same time.

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

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

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

[Brief explanation of drawings]

FIG. 1 shows an example of a reading device (or reading device) for reading positional information of a radiolabeled substance in a sample that has been transferred and accumulated on a stimulable phosphor sheet in the present invention. 1: stimulable phosphor sheet, 2: readout section for pre-reading, 3: readout section for main reading, 4: laser light source,
5: Laser light, 6: Filter, 7: Optical deflector, 8: Planar reflector, 9: Transfer direction, 10: Light guide sheet for prereading, 11: Photodetector, 12: Amplifier, 13: Control circuit, 14: Laser light source, 1
5: laser beam, 16: filter, 17: beam expander, 18: optical deflector, 19: plane reflecting mirror, 20: fθ lens, 21: transport direction, 2
2: Light guide sheet for main reading, 23: Photodetector, 2
4: Amplifier, 25: A/D converter, 26: Signal processing circuit Figure 2 is a diagram showing an example of an autoradiograph of a sample in which base-specific cleavage products of DNA are separated and developed on a gel support medium. It is. Figure 3 A is the second
It is a graph showing the relationship between the position in the scanning direction and the signal level for the second column of the figure, and b is the graph for the second column.
This is a graph showing the relationship between the position in the scanning direction and the signal level for the first column in the figure, and C is a graph for the fifth column in the scanning direction obtained by subtracting the graph in B from the graph in A. It is a graph showing the relationship between the upper position and the signal level.

Claims (1)

[Scope of Claims] 1. A signal processing method in autoradiography for determining the base sequence of DNA or a partially degraded DNA product, which is provided with a radioactive label.
1) One of a guanine-specific cleavage product, an adenine-specific cleavage product, a thymine-specific cleavage product, and a cytosine-specific cleavage product obtained by base-specific cleavage degradation of DNA or DNA partial decomposition products. 2) A mixture of two to four base-specific cleavage products containing at least one type of base-specific cleavage product included in 1) above. At least two groups of base-specific cleavage decomposition products or cleavage decomposition product mixtures containing at least two groups are one-dimensionally separated and developed on a support medium in a parallel relationship. By absorbing the emitted radiation energy in the stimulable phosphor sheet, an autoradiograph having positional information of the group of radiolabeled substances is stored and recorded on the stimulable phosphor sheet, and then the stimulable phosphor sheet is For the digital signal corresponding to the autoradiograph of each separated development column obtained by scanning the sheet with electromagnetic waves to cause the autoradiograph to emit photostimulated light and photoelectrically reading out this photostimulated light, ( i) Correspondence in the scanning direction between the separation and development column for base-specific cleavage products or mixtures of cleavage products in 1) above and the separation and development column for base-specific cleavage and degradation product mixtures in 2) above. 1. A signal processing method in autoradiography, comprising: obtaining a virtual separation expansion sequence by performing numerical calculations between positions; (ii) determining sampling points for the virtual separation expansion sequence. 2. The autoradiograph according to claim 1, wherein the sampling points are determined by performing smoothing and/or threshold processing on digital image data regarding a virtual separation expansion sequence. Signal processing method in E. 3 The mixture of base-specific cleavage products in 2) above consists of the base-specific cleavage product or the mixture of cleavage products in 1) above and one type of base-specific cleavage product other than the base-specific cleavage product in 1) above, and 3. The signal processing method for autoradiography according to claim 1 or 2, wherein the numerical calculation in step i) is subtraction. 4 The combination of the base-specific cleavage product of 1) and the base-specific cleavage product mixture of 2) is 1) guanine-specific cleavage product and 2) guanine-specific cleavage product + adenine-specific cleavage product. and/or a combination of: 1) a cytosine-specific cleavage product; 2) a combination of a thymine-specific cleavage product + a cytosine-specific cleavage product; A signal processing method in autoradiography according to item 3.