JPS59126253A - Signal processing in autoradiography - Google Patents

Abstract

PURPOSE:To easily determine the base arrangement of a DNA specimen with high accuracy, by photoelectrically reading autoradiography due to the photographic photosensitive material of the predetermined separated and developed lines of the DNA specimen while applying predetermined processing to the digital signal thereof. CONSTITUTION:Positional informations due to radioactive labels of specific split decomposed and migrated lines 1)G, 2)A, 3)G+A+T+C, 4)T and 5)C of a DNA specimen is transferred to a photographic photosensitive material. This photosensitive material is mounted to a rotary drum 2 which is rotatable and moves to the axial direction thereof and the pervious light of laser beam 5 is detected through a mirror 3 by a photoelectric multiplier 7. The output thereof is processed in a signal processing circuit 10 through an amplifier 8 and an AD converter 9. The separated and developed lines 1), 2), 4), 5) are subjected to interpolation identification by the reference line 3) and the base arrangement of the DNA specimen is easily determined with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a signal processing method in autoradiography. More specifically, the present invention relates to autoradiography using photographic materials,
The present invention relates to a method for comparatively identifying the separation and development position of a radioactive labeling substance in digital signal processing for determining the base sequence of DNA or DNA partial decomposition products.

A woman's hand extends at least in one dimension on the body! 1
Autoradiography is already known as a method for obtaining positional information of radiolabeled substances forming a distribution array.

For example, a radioactive label is attached to a macromolecular substance derived from an organism such as a protein or a nucleic acid, and then the radiolabel is detected, such as a macromolecular substance, its derivative, or its decomposition product (hereinafter referred to as radiolabeled substance). By performing separation and development using a separation operation such as electrophoresis on a shell-shaped support, a separated and developed column of the radiolabeled substance is formed on the supporting medium, and an autoradiograph of this separated and developed column is formed. After being transferred and recorded on a radiation film, it is visualized and the positional information of the radiolabeled substance is obtained from the visible image. 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 used as a stone to determine the base sequence of A (or partial decomposition products such as DNA, hereinafter the same).

By using this autoradiography, DN
Methods for determining the base sequence of A include the Maxam-Gi 1bert method and the Sanger-Coulson method.
The law is known. In these methods, 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), cytosine (C), thymine (T)
The two chain molecules are bridged by hydrogen bonds between these four types of bases, and the hydrogen bonds between each of the constituent units are This method cleverly utilizes the characteristic structure of DNA, which is realized only in two types of combinations: G"-C and AT, to determine its base sequence.

For example, the Maxim-Killhart method is implemented by the method described below.

First, add phosphorus (P) to one end of the chain molecule of the DNA or DNA decomposition product whose base sequence is to be determined.
By bonding a group containing a radioactive isotope, the object becomes a radiolabeled substance, and then chemical means are used to cleave the bonds between each constituent unit of the chain molecule in a base-specific manner. Next, the DNA obtained by this operation or a mixture of a large number of base-specific cleavage products of the DNA degradation product is separated and developed by gel electrophoresis to form a band shape from each of the many base-specific cleavage products. Separate expanded minute # expanded column (but cannot be seen visually)
get. Then, the autoradiograph of this # development sequence is visualized on an X-ray film, and from the obtained visible image and each base-specific cutting means, the end of the chain molecule to which the radioactive isotope is bound is determined. The bases in a certain positional relationship are determined one after another, and in this way the sequence of all the bases of the target object is determined.

As mentioned above, in autoradiography using conventional radiography, in order to obtain positional information of a radiolabeled substance, it is essential to visualize the autoradiograph containing this positional information on a radiographic film. It becomes.

Therefore, researchers determine the distribution of radiolabeled substances on the support medium by visually observing the visualized autoradiograph.

That is, the DNA base sequence is determined by visually determining the development position of each radioactively labeled base-specific cleavage product or mixture thereof, and
It is determined by mutually comparing the separation and development sequences of these cut and decomposed products.

However, in conventional autoradiography,
-As mentioned above, the analysis work relies on the human eye, so the positional information of the radiolabeled substance obtained by analyzing the autoradiograph, which is a visible image, may differ depending on the researcher. Due to these problems, there are limits to the accuracy of the information that can be obtained. In particular, autoradiographs visualized on radiographic film have good image quality (sharpness,
contrast), it is difficult to obtain satisfactory information, and its accuracy tends to decrease. Conventionally, in order to improve the accuracy of the desired positional information, 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 problem with accuracy in methods that simply use such measuring instruments.
- There is a limit to

Furthermore, for example, during an exposure operation in which a radiation film is electrodeposited on a support medium on which the separation and development rows described in ['rlI have been formed], a shift may occur in the overlay, and in this case, the radiation film may be misaligned. - The separation and development array (e.g., electrophoresis array) obtained as a visible image is not parallel to the length direction of the film, and as a result, it blurs, so it is difficult to visually judge the position information of the radiolabeled substance. Errors tend to occur, and the accuracy tends to decrease. Also,
Depending on the support medium and separation/deployment conditions, it often happens that the resulting separation/deployment rows are not parallel to the length direction of the support medium or are distorted.

For example, when using a gel as a support medium, the gel is not self-supporting, so it is usually spread out by holding both sides with a crow. There may be unevenness, and the radiolabeled substance is not necessarily separated and developed uniformly on the support medium. Similar non-uniformity in separation and development also occurs when air bubbles are included in the gel 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 separation and development conditions may vary locally on the support medium, so the resulting separation and development column Distortion tends to occur.

There is no suitable method for such distortions in the separation/deployment column other than artificial pressure compensation. Therefore, in the above cases, it becomes particularly difficult to analyze the positional information of the radiolabeled substance, and the above-mentioned 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 base sequences of NA partial decomposition products with sufficient accuracy.

In autoradiography that uses radiography, the undiscovered person photoelectrically reads an autoradiograph having positional information of a radiolabeled substance imaged on a photosensitive material and converts it into a digital signal, Then, by further performing suitable signal processing on the sampling points for detecting the position Z (J position) of the obtained radioactively labeled substance, the base sequence of the DNA or DNA partial decomposition product can be determined simply and with high precision. We have realized this and arrived at the present invention.

That is, the present invention is a signal processing method in autoradiography for determining the base sequence of a partially degraded DNA product.
1) Guanine-specific cleavage products, adenine-specific cleavage products, thymine-specific cleavage products, and cytosine-specific cleavage products obtained by specific cleavage degradation of NA or DNA partial degradation products. 2) a mixture of specific cleavage decomposition products comprising four types of base-specific cleavage decomposition products; and 2) at least two base-specific cleavage decomposition products comprising at least one base-specific cleavage product. Each of the cut and degraded samples of
An autoradiograph containing positional information of a group of radiolabeled substances spread one-dimensionally in parallel on a support medium is recorded on a photographic light-sensitive material, and then visualized on the photographic light-sensitive material. Regarding the digital signal corresponding to the autoradiograph of each separation and expansion column obtained by photoelectrically reading the autoradiograph, i) the fraction #development column of the cleavage and decomposition product containing the four types of base-specific cleavage and decomposition products; is a reference sequence, and a step of detecting a reference sampling point for this reference sequence; The present invention provides a signal processing method in autoradiography that includes a step of identifying sampling points of adjacent minute #F expansion sequences by comparing sampling points of adjacent minute #F expansion sequences.

That is, in the present invention, the radiation energy emitted from the test tube 4 is absorbed by the radiation film by overlapping the sample and a photographic material such as a radiation film, and the radiation film is visualized. The image is read photoelectrically to obtain an electric signal, and this electric signal is sent to the A/
This method utilizes a radiation image conversion method that involves performing D conversion and obtaining a digital signal.

1. In the present invention, "position information" refers to various 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 a combination of information such as the concentration and distribution of radioactive substances at that location.

According to the present invention, the positional distortion during separation and development of the radiolabeled substance on the support medium as described above, or the autoradiograph of the radiolabeled substance distributed in the -dimensional direction to form a distribution row. Due to misalignment during the transfer operation of the photosensitive material to the stimulable phosphor sheet,
Second, even when distortions or scratches occur throughout the transcribed autoradiograph, the base sequence of DNA or DNA partial decomposition products can be determined with high accuracy. In particular, it is possible to identify the direction of separation and expansion of each column without correcting the distortion in the direction of separation and expansion. Therefore, it is possible to identify the base sequence with high precision and rationally. can be determined.

Furthermore, in order to conduct experiments efficiently, it is generally done to provide as many separation and development columns as possible on a support medium. As a result, distortions of the expansion sequence such as the above-mentioned smiling effect may occur, but the present invention uses a reference sequence containing all four types of base-specific cleavage products of DNA as an internal standard. By providing a supporting medium
This makes it possible to correct distortions for all of the two separated expansion columns.

An example of a sample used in the present invention is a support medium in which a radiolabeled substance is separated and developed in one dimension. Examples of radioactively labeled substances include biopolymer substances to which radioactive labels have been added, derivatives thereof, and decomposition products thereof.

For example, in the present invention, a biopolymer substance to which a radioactive label is attached is 1. In the case of polymeric substances such as proteins, nucleic acids, their derivatives, and their decomposition products, it is useful for the separation and identification of these biological polymeric substances.

Furthermore, the present invention can be effectively used to analyze the entire or partial molecular weight, molecular structure, or basic unit configuration of these biopolymer substances.

However, the sample that can be used in the present invention is not limited to the above-mentioned sample, but is a support medium containing a radiolabeled substance distributed in at least one dimension, and the radioactive substance is placed on a photosensitive material. Any device may be used as long as it can image an autoradiograph having positional information of a labeling substance.

In addition, methods for separating and developing radiolabeled substances using support media include, for example, gel-like support media (any shape such as layered or columnar), polymer moldings such as acetate, or various types of supports such as filter paper. Representative methods include electrophoresis using a medium and thin layer chromatography using a support medium such as silica gel, but the separation and development method is not limited to these methods.

The photographic material used in the present invention has the following basic structure:
It consists of a support and a photographic emulsion layer. The photographic emulsion layer consists of a binder, such as gelatin, containing and supporting silver halide in a dispersed state. A photosensitive material is one in which a transparent sheet such as polyethylene terephthalate is used as a support, and the above-mentioned photographic emulsion layer is provided on this sheet, and an example thereof is a radiation film such as a high-sensitivity X-ray film. be able to.

In the present invention, the photosensitive operation (exposure operation) of a photographic light-sensitive material with radiation emitted from a support medium containing a radiolabeled substance is carried out by overlapping the support medium and the photographic light-sensitive material for a period of time. - This is carried out by absorbing at least a portion of the radiation emitted from the radiolabeled substance into the photosensitive substance in the photographic light-sensitive material. This exposure operation can be carried out by placing the medium and the photographic material in an electrodeposited state and leaving them in this state for several days at a low temperature, for example, below freezing. In the exposure operation, sensitization may be performed by using an intensifying screen or by applying pre-exposure such as Franche exposure. .

Incidentally, the exposure operation of a sample to a photographic light-sensitive material in autoradiography and the development treatment of the light-sensitive material are already well known, and these operations and treatments are described, for example, in the following documents. There is.

Biochemistry Experiment Course 6 Tracer Experiment Method (Part 1) 271-2
89 pages, li'8. Autoradiography J Toru Sueyoshi, Akiyo Shigesue (1977, published by Tokyo Kagaku Dojin) Next, in the present invention, one-dimensional positional information of a radiolabeled substance on a support medium recorded on a photographic light-sensitive material is provided. A method for reading an autoradiograph and converting it into a digital signal will be briefly described with reference to an example of a reading device shown in FIG. 1 of Appendix A.1.

FIG. 1 shows a schematic diagram of an example of an image reading device for reading an autoradiograph having one-dimensional positional information of a radiolabeled substance recorded as a visible image on a photographic light-sensitive material 1.

A hollow rotating drum 2 is equipped with a photosensitive material l having a visible image on its outside. This rotating tram 2 is -
・It rotates at a constant speed and at the same time moves at a partial pitch in the axial direction. 1. A mirror 3 is inserted into the rotating drum 2, and a light beam 5 from a light source 4 is emitted from the rotating drum 2.
enters through the lens 6. A light beam 5 from this light source 4 is reflected upward by a mirror 3, passes through a photosensitive material 1 mounted on a transparent drum 2, and enters a photomultiplier tube 7. In this way, the screen of the photosensitive material 1 is scanned in the X and Y directions by the light spot of the light beam 5.

The photomultiplier tube 7 converts the transmitted light at each point of the photosensitive material 1 into an electrical signal. This electrical signal is amplified by an amplifier 8 and then input to an A/D converter 9. An A/D converter 9 converts the electrical signal into a digital signal. In addition,
Details of the image reading operation are described in, for example, Japanese Patent Laid-Open No. 121043/1983.

In addition, regarding the method of reading an autoradiograph having positional information of a radiolabeled substance recorded on a photographic light-sensitive material in the present invention, the reading operation by a light transmission method using a light beam has been explained above. Reading operations using reflection methods can also be applied. The reading operation that can be used in the present invention is not limited to the above example, and various methods such as reading operation using a television camera are possible.

The digital signal corresponding to the radiolabeled substance thus obtained is then input to the signal processing circuit 10 shown in FIG. 1. (In the No. a processing circuit 10, 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, the implementation of the signal processing in autoradiography using the signal processing power method of undeveloped IJJ-1 will be explained by taking as an example the DNA base sequencing method using the above-mentioned Tumaxam Kill method. The case where the following five types of cleavage and decomposition product samples are used as a typical combination of base-specific cleavage and decomposition products for base sequencing will be explained.

■) Guanine-specific cleavage product + adenine-specific cleavage product + thymine-specific cleavage product + cytosine-specific cleavage product, 2) Guacon-specific cleavage product, 3) Adenine-specific cleavage product, 4) Thymine-specific cleavage product, 5) Cytosine-specific cleavage product. First, the sample was prepared using a conventional method to prepare the above-mentioned Itsuki cleavage product and cleavage product mixture that had been given a radioactive label with 3′2P. It can be obtained by electrophoretic development in a gel support medium for several minutes. Next, this sample (support medium) and a radiation film are superimposed on each other at a low temperature of -70 to -90°C for several days to carry out an exposure operation, and the autoradiograph of the sample is transferred and recorded on the radiation film.

FIG. 2 shows an autoradiograph of the above-mentioned five types of electrophoresis arrays in which radiolabeled DNA is spread in minutes. That is, the first to fifth columns in FIG. Cleavage product + (A) specific cleavage product + (T) specific cleavage product + (C) specific cleavage product (4) - (T) specific cleavage product (5) - (C) specific The electrophoresis rows of each target cleavage product are shown. The third column contains all base-specific cleavage products of (G, A, T, C) and is an internal standard column (reference column) for base sequencing.

By loading the autoradiograph transferred and recorded on the radiographic film into the reading device shown in FIG.
The address expressed in the coordinate system fixed to the radiographic film (
x, y) and a signal level (Z) at that address, and the signal level corresponds to the amount of stimulated light. That is, the digital signal is present in the autoradiograph of FIG. Therefore, digital image data having positional information of the radiolabeled substance is manually input to the signal processing circuit 10. In the present invention, digital image data and l± refer to a collection of Tental No. 118 corresponding to an autoradiograph of a radioactive label iCj.

First, on the digital image data, detect the position of separation and development of the radiolabeled substance for each of the 11 records,
Let these be the sampling points. For example, sampling points can be detected as follows.

By scanning different positions on the digital image data once across the one-dimensional 4i direction of the radiolabeled substance with respect to the digital signal described in , the radiolabeled substance in each row is detected on each scanning area. (The scan to detect this distribution point is called a preliminary scan), connect the two distribution points for each column to obtain five straight lines, and use the obtained straight lines to detect the sampling points in each column. l of
This is the scanning direction of the grain.

In the signal processing method of the present invention, the digital signal obtained by reading the radiation film is processed by the signal processing circuit 10.
(i.e., stored in a non-volatile memory such as /Henfer memory or a magnetic disk).

In signal processing, scanning digital image data means that only the digital signal at this scanned point is transferred from memory.
It means to take it out selectively.

Next, by scanning the digital image data "2" 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, for example by convolution using an appropriate filter function, and the function g (
w) is obtained. Next, threshold processing is performed on this function g (w). That is, for the threshold value (α0), when g(w)≧&0, g(w)=1g (w)
When <α0, the function g(w) is converted into a continuous function of 1 or 0 by processing g(w)−0. The sampling points are detected by setting each midpoint of the area of g(w)=1. Note that the value (irj (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 in the scanning area.

In this way, sampling points Skn can be detected for each column. Here, k is a fixed integer and represents the number of each column, and n is a positive integer and represents the number of sampling points. Note that the method for detecting sampling points is not limited to the above method.

Next, comparative identification between each column specifically involves searching for the same separation expansion between each column (for example, (
When comparing the column of 'G)+ (A) and the column of (G), find the separated expansion that is an element of (G) from the column of (G)+ (A)). However, in this comparative identification, when there is the above-mentioned distortion, the positions of the equivalent component # expansion columns of each column are not necessarily equal on the X coordinate as shown in FIG. Conventionally, correction of such distortions has been left to human visual judgment. However, according to the method of the present invention, by using a reference column and a reference sampling point, it is possible to automatically correct distortion and perform accurate comparative identification between columns without relying on humans.

This will be explained based on FIG.

FIG. 3 is a diagram showing a portion of the reference column (third column) and the fourth column. Here, the black angle of view represents the sampling and ring points in each row corresponding to the separation and deployment site of the radiolabeled substance, and the hollow square represents the interpolated reference sampling point.

The sampling point 33n of the third column, which is the reference column, is used as a quasi-sampling point and is compared with the sampling point s4n of the adjacent fourth column to interpolate the reference sampling point for the fourth column. .

For example, for the sampling point S41 in the fourth column,
The position K (X 41 ) of the sampling point 341 is compared with the position of the reference sampling point S32 (×3□) and the position of the reference sampling point S32 (X33) in the third column. For example, if lX32 In this way, all the sampling points in the fourth column are assigned to one of the reference sampling points in order. Then, based on the assigned sampling point s4n of the fourth column, by interpolating those of the remaining reference sampling points of the third column into the fourth column, tentative reference sampling is performed in the fourth column. A set of points (Sa.

). However, m is a positive integer and matches the number n of the sampling point in the third column. In this way, a virtual reference column can be obtained by moving the reference column obtained in the third column to the position of the fourth column.

Next, based on the created standard sampling point s4m in the fourth column, the substrate sampling point S
3n and further interpolates the reference sampling point s4m in the fourth column to create a virtual set of reference sampling points (35m) in the fifth column.

In this way, based on the reference sampling points S3n of the reference column, a set of virtual reference sampling points (Sklfl) is created in each column in turn, and all sampling points Skn are set to any of the reference sampling points S3n. Attribute to crab.

That is, the above method includes: 1) a mixture of four types of cleavage products: a guanine-specific cleavage product, an adenine-specific cleavage product, a thymine-specific cleavage product, and a cytosine-specific cleavage product; Each of at least two groups of cleavage decomposition product samples comprising a base-specific cleavage decomposition product,
An autoradiograph having positional information of a group of radiolabeled substances separated and developed one-dimensionally in a parallel relationship on a "supporting medium" is recorded on a photographic light-sensitive material, and then visualized on the photographic light-sensitive material. Regarding the digital signals corresponding to the autoradiographs of the respective separation and development columns obtained by photoelectrically reading out the autoradiographs, i) the separation and development column of the cleavage products containing the four types of base-specific cleavage products; 11) detecting sampling points for each separation expansion column other than the reference column; 111) adjacent to the reference column with reference to the reference sampling point of the reference column; The process of identifying the sampling points of the separation expansion sequence adjacent to the sample of the separation expansion sequence, and determining a new reference sampling point based on the identified sampling points, 1v) - 111) identifying the sampling point of the adjacent separation expansion sequence by comparing and matching the reference sampling point determined by the standard sampling point with the sampling point of the separation expansion sequence adjacent to the separation expansion sequence in which the reference sampling point is determined; It can be summarized as a signal processing method that includes.

Next, regarding the first column, second column, fourth column, and fifth column,
The virtual reference sampling point (skm) in each column is compared with the actual sampling point Skn in that column in descending order of m, and when they match, it corresponds to m in the reference column (third column). The sampling point 53m is replaced with the matching sampling point Skn. and,
If we follow the reference row in order of the smallest mouth, we can obtain, for example, the following diagram.

S++・5511S411S12. S21゜S2
2, S 4 2 + Sl 3 +
S5 □ ,...In the above diagram, S, n
=G, 52n=A.

By replacing 54n=T and 55n-C, the following diagram is obtained.

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 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, it is also possible to display the relative amount of each separated and developed cut and decomposed product by arbitrarily processing the level of the signal in the scanning direction of each column.

Alternatively, it is also possible to display the base sequences of both two stranded molecules of DNA. In other words, by providing information such as AT, G+C1C-G, and T+A as targets 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-G-A-A-T-(,-C-...
C-G-A-C-T-T-A-C-G-...It should be noted that by the signal processing method of the present invention, (G+A+
DNA using combinations of T+C, G, A, T, C)
The base sequencing method is an example of a DNA base sequencing method, and the signal processing method of the present invention is not limited to the combinations described above, but various combinations are possible. Furthermore, by using the combination, the base sequence can be similarly determined by a method similar to the above method.

However, in any combination, as an internal standard,
It is necessary to provide a column (reference column) of a mixture of all salt-specific cleavage products of G, A, T, and C. The separation and development position of this reference column does not necessarily need to be placed in the center of the support medium as described above, but in order to determine the DNA base sequence with higher accuracy, it is necessary to place it in the center of the support medium. is preferable. In addition, the reference string is not applied only to the combination of specific cleavage products of the - set of DNA,
If the same type of DNA is used, it can be applied to each of the groups of radiolabeled substances separated and developed on the support medium.

In the uninvention, for example, the base sequence of DNA can also be determined using a combination of (G, A, T, C). Alternatively, it is also possible to determine the sequence for a particular base from a combination of at least one salt-specific cleavage product and a suitable reference material (e.g., a mixture of each salt-specific cleavage product). be.

Furthermore, in the above example, the explanation was given using five rows of radiolabeled substance groups that are separated and developed in a one-dimensional direction on the support medium, but the number of separated and developed rows is not limited to five rows. The signal processing method of the present invention is particularly effective when there are more than five columns. That is, since the present invention is a method of performing identification while sequentially correcting deviations in the direction of minute 1 III expansion with the reference column as the center, the present invention can be effectively utilized as the number of separated expansion columns increases. It can be done. Alternatively, there may be two or fewer simultaneous numbers. Alternatively, it is also possible to simultaneously determine the base sequences of two or more types of DNA using one support medium.

Information about the DNA base sequence determined by the signal processing method described above is output from the signal processing circuit 10 and then stored in a recording device directly or, if necessary, via a storage means such as a magnetic tape. (not shown).

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 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.

In addition, the information obtained as described above may also be used to collect other DNA information that has already been recorded.
It is also possible to perform genetic linguistic information processing such as matching with base sequences.

[Brief explanation of drawings]

FIG. 1 shows an example of a reading device for reading an autoradiograph having positional information of a radiolabeled substance in a sample recorded on a photosensitive material in the present invention. 1. Photographic material, 2: Transparent drum, 3: Mirror, 4:
Light source, 5: Light beam, 6: Lens, 7: Photomultiplier,
8: Amplifier, 9: A/D converter, 10: Signal processing circuit Figure 2 shows an example of an autoradiograph of a DNA base-specific cleavage product or a sample separated and developed in a gel support medium. It is a diagram. FIG. 3 is a partially enlarged view of the autoradiograph of FIG. 2. Patent applicant: Fuji Photo Film Co., Ltd. Agent Patent attorney: Engraving of the drawing by Yasuo Yanagawa (no changes in content) Figure 1 Figure 2 Figure 3 Figure 1, - 4 Teitouri Ne Ichishosho January 25, 1982 Kazuo Wakasugi, Commissioner of the Japan Patent Office 2゜Name of the invention Signal processing method in autoradiography 3゜Relationship with the case of persons making amendments Patent applicant address (520) Fuji Photo Film Co., Ltd. Name Representative Minoru Ohnishi 4゜Representative Written Amendment to Personal Procedures dated October 1st, 1948, Director-General of the Permanent Office, Mr. Kazuo Wakasugi, 1981, Patent Essay No. 1339, No. 2, Title of Invention: Signal Processing Method in Autoradiography, 3. Relationship with the Amendment Person Case Patent Applicant (J- Name (Name) (520) Nshi Photo Film Co., Ltd. We will amend the "Detailed Description of the Invention" column of the specification as follows. Kiichi Yuzuki (1) page 9, line 19 Previous → deleted (
2) +5 page 15th line Radiation image conversion → Delete (
3) Page 18, line 8, stimulable phosphor sea → 纙凰感X
Material 1・ (4) Page 25, line 1 Reading device → Reading device (5) Page 25, line 2 By outputting, -take) 6
Inferior to Shihi” (6) page 25 line 6 Keijinkou
- Transmitted light (7) Page 30, line 9, interpolate 34m
-Kotoa (No.3m interpolation 3m (9) page 32, line 19 m -”n
(10) Page 34, line 14 Salt asymmetric cleavage and decomposition → base-specific decomposition product (11) page 35, line 14 Salt anomalous cleavage and decomposition → base-specific decomposition product (+2) Page 35, line 8, each salt-specific cleavage component → Decomposition products from Akutabase Kasaguro, etc. (+3) 3f, page 3, line 1, two types at the same time, → Tamayo Iko and below (14) Drawing No. Attached are the corrected drawings in Figure 3. Third issue above (--

Claims (1)

[Claims] ■. A signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, the method comprising: radioactively labeled DNA or D
■) Four types of guanine-specific cleavage products, atenine-specific 9-cleavage products, thymine-specific cleavage products, and cytosine-specific cleavage products obtained by specific cleavage degradation of NA partial decomposition products. 2) a base-specific cleavage product mixture containing a base-specific cleavage product; and 2) each of at least two cleavage product samples comprising:
An autoradiograph having positional information of a group of radiolabeled substances spread one-dimensionally in parallel on a support medium is recorded on a photographic light-sensitive material, and then visualized on the photographic light-sensitive material. i) for the digital signal corresponding to the autoradiograph of each minute # expansion column obtained by photoelectrically reading the autoradiograph
11) Detecting sampling points for separation and expansion columns other than the reference column, with the separation and expansion column of the cleavage products containing the four types of base-specific cleavage and degradation products as a reference column, and detecting reference sampling points for this reference column; and 111) identifying the sampling points of the adjacent separation expansion sequence by comparing and collating the sampling points of the adjacent separation expansion sequence using the reference sampling point of the reference sequence as a reference. Signal processing methods in graphics. Claim 1, characterized in that the 2° sampling point is detected by performing smoothing and/or threshold processing on digital image data in the scanning direction of each of the plurality of separation expansion columns. Signal processing methods in autoradiography described. 3. A signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, which involves specific cleavage and decomposition of radioactively labeled DNA or I) NA partial decomposition products. 1) A mixture of four types of cleavage products: 1) a guanine-specific cleavage product, an adenine-specific dino-cleavage product, a thymine-specific cleavage product, and a cytosine-specific cleavage product, and 2) each of the following: at least two base-specific cleavage products containing at least one type of base-specific cleavage product that are different from each other;
An autoradiograph showing the positional information of a group of radiolabeled substances separated and developed one-dimensionally in parallel on a support medium is recorded on a photographic light-sensitive material, and then visualized on the photographic light-sensitive material. Regarding the digital signals corresponding to the autoradiographs of each separated development column obtained by photoelectrically reading the autoradiographs. 1) A step of detecting a reference sampling point for each separation and development column other than the reference column by using the separation and development column of the four types of cut and decomposition product mixtures as a reference column; 2) A step of detecting sampling points for each separation and development column other than the reference column; 111 ) By comparing and collating the sampling points of the separation deployment position adjacent to the reference column 11 using the reference sampling point of the reference column as a reference, the sampling point of the adjacent separation deployment column is identified, and the sampling point identified here is [Determine a new virtual reference sampling point based on the reference sampling point] Step 1, 1v) - The reference sampling point newly determined in the step 111) and the reference sampling point in the determined separation expansion sequence A signal processing method in autoradiography, comprising the step of identifying a sampling point of an adjacent separation expansion sequence by comparing the sampling points with the sampling points of the adjacent separation expansion sequence. 4゜, (arranging # development rows other than the reference row on both sides of the outer mold, and carrying out the process of 1.1ii) for both the separated development rows on both sides. A signal processing method in autoradiography according to scope 3. Claim 3, characterized in that the 5° sampling point is detected by performing smoothing and/or threshold processing on digital image data in the scanning direction of each of a plurality of separation expansion columns. Or the signal processing method in autoradiography according to item 4. 6. A base-specific cleavage product of DNA or a DNA partial decomposition product; 2) Guanine-specific cleavage product + adenine-specific cleavage product + thymine-specific cleavage product + cytosine-specific cleavage product; 2) Guanine-specific cleavage product + cytosine-specific cleavage product; Base-specific cleavage products including specific cleavage products 3) Base-specific cleavage products including adenine-specific cleavage products 4) Base-specific cleavage products including thymine-specific cleavage products 5) Cytosine-specific cleavage products The method according to any one of claims 3 to 5, comprising at least three groups of nine cleavage product samples consisting of base-specific cleavage products including cleavage products. Signal processing method in radiography.