JPS59126257A - Signal processing in autoradiography - Google Patents

Abstract

PURPOSE:To easily determine the base arrangement of a DNA specimen with high accuracy, by photoelectrically reading the 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)T and 4)C of a DNA specimen are transferred to a photographic photosensitive material 1. This photosensitive material 1 is mounted to a rotary drum 2 moving 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 with a signal processing circuit 10 through an amplifier 8 and an AD converter 25 and sampling masks around the basic sampling points of the separated and developed lines 1)-4) are set while the numbers of signals each equal to or more than the threshold value among them are detected and an exclusive principle is applied to perform identification. By this method, 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 using a photosensitive material for determining the base sequence of DNA or a partial decomposition product into DNA.

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, after attaching a radioactive label to a macromolecular substance derived from a biological body such as a protein or a nucleic acid, the radioactively labeled macromolecular substance, its derivative, or its decomposition product (hereinafter referred to as radioactive (also called a labeling substance) on a gel-like support medium for electrophoresis, etc. 1 [By performing separation and development through operation, forming a separation and development column (however, invisible) of the radiolabeled substance on the support medium,
The positional information of the radiolabeled substance is obtained from the autoradiograph obtained by transferring and visualizing this separation and development array onto a radiographic film. Furthermore, methods for separating and identifying the polymeric substance, or evaluating the molecular weight and characteristics of the polymeric substance based on the obtained positional information of the radiolabeled substance have already been developed and are currently in use. .

Particularly in recent years, autoradiography has been
It is effectively used to determine the base sequence of jA (or a partial decomposition product of DNA, etc., hereinafter the same).

By using this autoradiography, DN
Methods for determining the base sequence of A include the Maxam-Gilbert method and the Sanger-C'oulson method.
) 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 of GC and AT, to determine its base sequence.

For example, the Maxam-Gilbert method is implemented in the following manner.

First, phosphorus (
By bonding a group containing a radioactive isotope of P), the target substance becomes a radioactive labeling substance, and then the bonds between each constituent unit of the chain molecule are specifically cleaved using chemical means. . Next, the DNA obtained by this operation or a mixture of many base-specific cleavage products of the DNA decomposition product was separated and developed by gel electrophoresis, and the many cleavage products were separated by forming bands. You get a separate expansion column (but you can't see it visually). Then, this separated and developed array was visualized on an X-ray film to obtain an autoradiograph. Based on the autoradiograph and each base-specific cutting means, bases located in a certain positional relationship from the end of the chain molecule to which the radioactive isotope is bound are sequentially determined, and in this way all bases of the target object are The sequence is determined.

By the way, as mentioned above, in autoradiography that uses conventional radiography, in order to obtain positional information of a radiolabeled substance, it is essential to visualize an autoradiograph containing this positional information on a radiographic film. It becomes.

Therefore, researchers determine the distribution of the radiolabeled substance on the support medium by visually observing the visualized autoradiograph. That is, the DNA base sequence is determined by visually determining the separation and development position of each of the base-specific cleavage products or mixtures thereof to which a radioactive label has been added, and then determining the separation and development sequence of these base-specific cleavage products. are determined by comparing them with each other.

However, in conventional autoradiography, the analysis work relies on the human eye as mentioned above, so the position information of the radiolabeled substance obtained by analyzing the autoradiograph, which is a visible image, is used for research. There are problems such as discrepancies between people, and there are limits to the accuracy of the information that can be obtained. In particular, autoradiographs visualized on radiographic films have good image quality (sharpness,
If the contrast) is not satisfied, it is difficult to obtain satisfactory information at T4, and the accuracy tends to decrease. Conventionally, in order to improve the accuracy of the positional information sought, a method has been used in which, for example, the visualized autoradiograph is measured using a measuring instrument such as a scanning densitometer. However, there is a limit to the improvement of accuracy in methods that simply use such measuring instruments. ~ For example, if the sample contains radioactively labeled impurities, if the support medium is radioactively contaminated by natural radioactivity, or if the separation and development procedure is insufficient, Since noise tends to appear on the autoradiograph, it becomes difficult to analyze the positional information of the radiolabeled substance, thus reducing the accuracy of the information obtained.

In the above cases, it becomes particularly difficult to analyze the positional information of the radiolabeled substance, and even if the measuring instruments described above are used, the positional information of the separated and expanded radiolabeled substance, that is, the DNA or DNA partial decomposition, cannot be obtained. It is difficult to obtain the base sequence of a substance with sufficient accuracy.

The present inventor has succeeded in obtaining positional information of radiolabeled substances by using a radiation image conversion method using a stimulable phosphor sheet instead of the radiography method using a radiation film used in conventional autoradiography. By obtaining the positional information of an autoradiograph as a digital signal without specifically imaging it, and then applying suitable signal processing to the obtained digital signal, the base sequence of DNA or a partial DNA decomposition product can be easily and highly determined. The present invention has been achieved by achieving accurate determination.

That is, the present invention is a signal processing method in autoradiography for determining the base sequence of DNA or a partially degraded DNA, which
1) Guacon-specific cleavage product, 2) Adenine-specific cleavage product, 3) Thymine-specific cleavage product, 4) Obtained by base-specific cleavage degradation of NA or DNA partial digest. Cytosine-specific cleavage products, each of which contains at least four base-specific cleavage products, are one-dimensionally separated and developed in parallel on a support medium, and a radioactive label is formed in a separation and development array. After recording an autoradiograph having positional information of a substance group on a photosensitive material, the autoradiograph of each separated development column obtained by photoelectrically reading the autoradiograph visualized on the photosensitive material. For the digital signal corresponding to the graph, i) synthesizing an internal standard sequence from the plurality of separation expansion sequences and determining a basic sampling point for the internal standard sequence, il) setting a basic sampling point based on the basic sampling point. A step of comparing the radioactive material signals of each separation expansion column distributed within the sampling mask with each other and detecting the sampling point in each separation expansion column by using the exclusivity of the assumed sampling points between each separation expansion column. The present invention provides a signal processing method in autoradiography including the following.

The present invention also provides D
1) Guanine-specific cleavage product, 2) Adenine-specific cleavage product, 3) Thymine-specific cleavage product, 4) Cytosine-specific cleavage product obtained by base-specific cleavage degradation of NA or DNA partial decomposition product. and 5) at least five base-specific cleavage products comprising a mixture comprising a guanine-specific cleavage product, an adenine-specific cleavage product, a thymine-specific cleavage product, and a cytosine-specific cleavage product. Using separated development columns formed by one-dimensionally separating and developing each one in a parallel relationship on a support medium, for the digital signal corresponding to the autoradiograph of each separation development column, i) 11) Determining basic sampling points for the internal standard sequence using the separation and development sequence of the mixture as an internal standard sequence, 11) Each minute [[ The signals of the radioactive substances in the deployment column are compared with each other, and each minute 1. Name +? Using the exclusivity of the assumed sampling points between the expansion columns? A U
A method of signal processing in autoradiography is also provided, comprising: detecting sampling points outside the range.

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, by performing signal processing on digital image data for an autoradiograph that includes noise, true position information that does not include noise can be easily obtained. In other words, for example, DNA or DNA partial decomposition products can be detected with high precision without being affected by noise generated by radioactively labeled impurities contained in the sample or noise generated by poor separation development conditions. The base sequence can be obtained.

In particular, radiolabeled DNA or base-specific cleavage products of DNA partial decomposition products cannot be visualized in autoradiography, which uses conventional radiography based on measurement by the human eye. Because the base sequence is determined by visually analyzing the radiograph,
Generally, the following combinations are often used to make it easier to visually check the position.

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 In the present invention, In order to automatically determine the base sequence of DNA by signal processing a digital signal corresponding to an autoradiograph of a sample, the following simplest combination can be used.

1) Guanine-specific cleavage product, 2) Adenine-specific cleavage product, 3) Thymine-specific cleavage product, 4) Sil-'yn-specific cleavage product, that is, the above four are mutually exclusive. Therefore, the obtained digital signal can be rationalized by taking advantage of the exclusivity that only one group of base-specific cleavage products always exists in the direction perpendicular to the direction of separation and development of a group of radiolabeled substances. The position of each base can be determined by a majority vote. Therefore, it is possible to obtain the base sequence more accurately than human visual judgment.

In addition, since it is possible to reasonably detect the separation and development site (sampling point) of the radiolabeled substance on digital image data as described above, it is possible to detect the separation and development site of the radiolabeled substance one by one even if the separation and development site of the radiolabel substance is reduced. It becomes possible to accurately determine the separation development site (sampling point). That is, it is possible to reduce the absolute amount of radiolabeled substance used in -times of OI radiography. Alternatively, it is possible to increase the number of separation development rows at the separation development site without expanding the width of the support medium, and it is possible to obtain more information than before with -1 autoradiography operations. Become.

Examples of samples used in the present invention include radioactive
Each base-specific cleavage product and/or a mixture thereof obtained by specifically cleaving and decomposing a labeled DNA or a DNA partial decomposition product between bases that are its constituent units is Mention may be made of support media that are separated and spread out to form separate spread rows.

Methods for separating and developing the above-mentioned radioactively labeled substances using support media include, for example, gel support media (any shape such as layered or columnar), polymer moldings such as acetate, or various support media such as filter paper. Typical methods include electrophoresis using silica gel, 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 for carrying out the present invention.

The basic structure of the photographic light-sensitive material 4 used in the present invention is 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. I can do it.

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 certain period of time. This is carried out by absorbing the radiation emitted from the above radioactive label into the photosensitive substance in the photographic light-sensitive material. This exposure operation can be carried out by arranging the support 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 flash exposure.

In addition, 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 processing are described, for example, in the following documents. .

Biochemistry Experiment Course 6 Tracer Experiment Method (Part 1) 271-2
p. 89, "8. Autoradiography j Toru Sueyoshi, Akiyo Shigesue (1977, published by Tokyo Kagaku Dojin) Next, in the present invention, one-dimensional analysis of the radiolabeled substance on the support medium recorded on the photographic light-sensitive material. A method for reading an autoradiograph having specific positional information 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 the accompanying drawings.

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 1 having a visible image on its outside. The rotating drum 2 is configured to rotate at a constant speed and at the same time move in the axial direction at a partial pitch. Further, a mirror 3 is inserted into the rotating drum 2, and a light beam 5 from a light source 4 enters through a lens 6. A light beam 5 from this light source 4
is reflected upward by the mirror 3, passes through the photosensitive material 1 mounted on the transparent drum 2, and enters the photomultiplier tube 7.

In this way, the screen of the photosensitive material 1 is scanned in the X and Y directions by the light spots 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. The 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 autoradiograph of the radiolabeled substance thus obtained is then input to a signal processing circuit 10 shown in FIG. Signal processing circuit lO
Then, the base sequence of the target DNA is determined by converting the one-dimensional positional information of the radiolabeled substance into symbols and/or one-dimensional numbers.

Hereinafter, embodiments of signal processing in autoradiography for DNA base sequencing using the signal processing method of the present invention will be described, taking as an example the case where the above-mentioned Maxam-Gilbert method is used. The case where a specific cleavage product is used will be explained.

1) Guanine-specific cleavage product 2) Adenine-specific cleavage product 3) Thymine-specific cleavage product 4) Cytosine-specific cleavage product First, radioactively labeled (32p) DNA was cleaved to each base using a conventional method. By cutting in units, the above l) to 4
) to obtain four groups of base-specific cleavage products.

Next, the above-mentioned four groups of base-specific cleavage products are separated and developed by electrophoresis on a gel support medium to obtain respective separation and development arrays.

Next, an exposure operation was performed by overlapping this sample (gel-like support medium on which separation and development rows were formed) and a radiation film at a low temperature of -70 to -90°C for several days, and the autoradiograph of the sample was transferred to the radiation film. transcribed and recorded.

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. 2 are (1) -
(G) Specific cleavage and decomposition product (2) - (A) Specific cleavage and decomposition product (3) - (T) Specific cleavage and decomposition product (4) - (C) Each minute # development column of specific cleavage and decomposition product shows.

By loading the autoradiograph transferred and recorded on the radiographic film into the reading device shown in FIG. The given address (x
, , y) and the signal level (Z) at that address, and the signal level corresponds to the amount of stimulated 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 10. Herein is meant a collection of digital signals corresponding to an autoradiograph of a substance.

First, an internal standard (reference) sequence can be obtained, for example, by the following signal processing. That is, on the digital image data, the scanning direction of the signal-processing stage is determined for each of the separated and expanded rows of the intaglios. That is, by scanning the digital signal twice at different positions on the digital image data across the one-dimensional distribution direction (separation development direction) of the radiolabeled substance, each column on each scanning area is The scan used to detect the distribution points of the radiolabeled substance is called the preliminary scan).
, for each separation expansion column, two distribution points are connected to obtain four straight lines, and the obtained straight lines are respectively taken as the scanning direction for the sampling inspection-mountain in each column.

In addition, in the signal processing method of the present invention, the digital signal obtained by reading out the radiation film is transmitted to the signal processing circuit 1.
0, it is stored in the -H memory (that is, it is stored in a non-volatile memory such as a buffer memory or 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 each scanning direction, the position (W) in the scanning direction is virtually plotted on the horizontal axis for each column, and the signal level (z') is plotted on the vertical axis. Obtain the graph taken. Here, the position in the scanning direction is preferably expressed by the migration distance from the migration start position, for example, by detecting the migration start position (Wko) of each column using a marker. However, k is a positive integer and represents the number of each column.

For the obtained graphs in the first to fourth columns, by extracting and synthesizing the level values of the signals in the columns where the signal level (z) shows the maximum value at the same position (W) in each scanning direction. , (G) specific cleavage degradation product, (A, ) specific cleavage degradation product, (T) specific cleavage degradation product, and (C)
A graph containing all four types of base-specific cleavage decomposition products is obtained. The obtained graph is a graph regarding a column that should also be called an internal standard (reference) column.

In addition, instead of synthesizing the internal standard column from the separation and expansion column of each base-specific cleavage product as described above, we actually prepared an electrophoresis column containing all of the above four types of base-specific cleavage products in advance, This may be used as an internal standard column.

Next, the basic sampling points for the internal standard sequence can be determined, for example, by the following signal processing. In other words, if the graph corresponding to the above internal standard column is expressed as a function f ('w) [w represents the position in the scanning direction], then this function f (w) can be controlled by using, for example, an appropriate filter function. Smoothing processing is performed by performing volution to obtain a function g(w). Next, threshold processing is performed on this function g (w). That is, for the threshold value (α0), when g(w)≧α0, g (w) = 1g (w)
When <α0, the function g(w) is converted into a continuous function of 1 or O by performing processing to set g(w)=0. Sampling candidate points Son are detected by setting each midpoint of the region g (w)=,1. However, 0
represents an internal standard sequence, n is a positive integer, and represents the number of the sampling point corresponding to the candidate point. Note that the threshold value (α0) in the above threshold processing is, for example,
For digital signals on the scanning area, the signal level and
It can be determined from the relationship with the frequency, that is, from the histogram.

Next, the obtained sampling candidate points S. For n,
Perform statistical processing. In other words, the radiolabeled substances present at sampling candidate points on the internal standard column are arranged in order of decreasing migration distance, that is, in order of increasing sampling point number, the number of constituent units containing one of the four types of bases is 1. It has been experimentally determined that there is a linear relationship between the migration distance of these radiolabeled substances and the logarithm of the molecular weight of the radiolabeled substance. Therefore, statistical processing can be performed by approximating the sampling candidate points using the following function.

W□n=a-blog(A+Mn) (1) (However, a and b are numerical values determined experimentally according to the electrophoresis conditions, and A and M are related to the molecular weight of the base-specific cleavage product of DNA. ) We decided to perform statistical processing by substituting the migration distance Won of each sampling candidate point Son and the number n of the sampling point corresponding to each into equation (1), and the most probable values a□ and b
By calculating o and substituting a□ and bo into equation (1), the most probable migration distance (W o, n'
) is determined by determining the basic sampling point son'''.

Based on this basic sampling point Son'', in each scan of each of the four columns, among the digital signals existing within a certain width centered on the basic sampling point, for each basic sampling point, Calculate the number of digital signals that exhibit a signal level equal to or higher than the threshold determined by the threshold processing.In other words, for each basic sampling point, set a constant width (sampling mask) centered at that position (Won'). , calculate the number of digital signals equal to or greater than the threshold value for each sampling mask on each of the four columns in the scanning direction.

FIG. 3 shows the DN detected by the signal processing method of the present invention.
FIG. 3 is a diagram schematically showing a sampling mask and a portion of sampling points on the separation and expansion column of the base-specific cleavage product of A. The area surrounded by a square in FIG. 3 is a sampling mask.

In the present invention, since the different salt cleavage and decomposition products contained in each of the above four columns are mutually exclusive, the basic number of sampling points is the sum of the sampling points in each of the above four columns. Match. This means that one sampling point corresponding to one basic sampling point is detected in any one of the four columns. Therefore, in the same sampling mask, the sampling point to be detected must be detected in only one of the four columns.

For each of the sampling masks by exploiting the exclusivity between each column above.

A column with the highest evaluation value of the signal level within the same sampling mask is selected, and it is determined that there is a sampling point in this column, and it is determined that there is no sampling point in the other samples. Here, the evaluation value of the signal level refers to the integral value of the signal level included in the mask, or the number of signals whose level exceeds the threshold value when threshold processing is performed.

In this way, sampling points are detected in any one of the four water motion columns for all sampling masks. Therefore, a sampling point is detected in any of the above four columns corresponding to the basic sampling point of the internal standard column.

Therefore, by the above process, each column has the most probable migration distance '
The basic sampling point So with cW on ')
n'' set (Son''2), where k represents the column number.

Regarding the basic sampling point SOn', 1) (Son'
) r is the sampling point belonging to G11) (Son'')
Sampling point belonging to 4) (S on
")"The sampling points belonging to 5 are AiV)(Son'
) 6 is replaced with T and then arranged in order of sampling number, the following diagram is obtained.

a -c -c -c -p, -A, -T,, -
c, −c −−・・'−i・In this way, D'N
The base sequence of one chain molecule of A (or its partial decomposition product) can be determined. Note that the display of information regarding the obtained DNA base sequence is not limited to the above display format, and various arbitrary display formats are possible. For example, if desired, it is possible to display the relative amount of each separated and developed base-specific cleavage product by arbitrarily processing the signal levels in the scanning direction of each column.

Alternatively, it is also possible to display the base sequences of both DN'A chain molecules. -i.e.
By providing information such as A7T, G+C, C+, G, and T+A as combinations corresponding to each base in the diagram represented by the above symbols, a DNA base sequence represented by the following diagram is obtained.

G'-C-G-C-A-A-T-G-C-...
C-, G-C-G 7 T-T-A-C-G-
...Also, in the above example, the explanation was made using a group of rotating radiolabeled substances that are separated and deployed in a one-dimensional direction on the support medium. There is no limitation, and it may be more than the number of turns, or may be less than the number of turns. Or maybe one. Wow, that's a tough job. . 143 Yu. Wow. . ,stomach.

It is also possible to determine the base sequence.

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 in Section 2 may be subjected to genetic linguistic information processing, such as comparing it with other DNA base sequences that have already been recorded. It is also possible.

[Brief explanation of the drawing]

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 tube,
8: Amplifier, 9: A-/D converter, 10: Signal processing circuit Figure 2 shows an example of an autoradiograph of a sample in which a base-specific cleavage product of DNA was separated and developed on a gel support medium. It is a diagram. FIG. 3 shows the DN detected by the signal processing method of the present invention.
FIG. 3 is a diagram stratigraphically showing a portion of the sampling mask, samples, and ring points on the separation and development column of the base-specific cleavage product of A. Patent Applicant Fuji Photo Film Co., Ltd. Agent Patent Attorney Yasuo Yanagawa 1 2 3 4 Teito Sale 1 Seisho January 25, 1980 Commissioner of the Patent Office Kazuo Wakasugi Tono t Jr. Patent application filed on January 8, 1982 (23) 2゜Name of the invention: Signal processing method in autoradiography 3゜Relationship with the case of persons making amendments Address of the person who issued the patent (520) Fuji Photo Film Co., Ltd. Name Representative: Minoru Ohnishi 4゜Agent procedure Director-General of the Office of Amendments Kazuo Wakasugi Indication of the case 1978 Patent essay No. 1343 2, Title of the invention Signal processing method in autoradiography 3 Relationship with the case of the person making the amendment Patent applicant 6 Increase due to the amendment Number of inventions: None 7,
1 Detailed explanation of the invention in the specification to be amended Conventional → Delete (3) io Page line 5 to page 10 - confining pressure By using a radiographic image conversion method using a fluorescent phosphor sheet, an autoradiograph containing the positional information of a radiolabeled substance can be converted into an image without specifically converting the positional information into an image.
(4) Page 23, line 9 Reading device → Reading device (5) Page 23, line 1o3'7 By outputting,
- Take ≦ -5 2 J - (6) Page 23, line 14 Stimulated light → From transmitted light, page 5, line 5 Part or more - -393~

Claims (1)

[Scope of Claims] 1. A signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, the method comprising DNA or DNA to which a radioactive label has been added.
Obtained by base-specific cleavage decomposition of A partial decomposition product, l) guanine-specific cleavage decomposition product, 2) adenine-specific cleavage decomposition product, l--■--□-drops-seki--- 3) thymine 4) a cytosine-specific cleavage product; each of at least four base-specific cleavage products including: 4) a cytosine-specific cleavage product; After recording an autoradiograph having the positional information of the radiolabeled substance group in the separated and developed column on a photographic light-sensitive material, it is obtained by photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material. For the digital signal corresponding to the autoradiograph of each separated expansion column, i) composing an internal standard column from the plurality of separation expansion columns and determining basic sampling points for the internal standard column; 11) the basic sampling The radioactive material signals of each separation expansion column distributed within the sampling mask set based on the points are compared with each other, and sampling in each separation expansion column is performed using the exclusivity of the assumed sampling points between each separation expansion column. A signal processing method in autoradiography that includes the step of detecting points. 2-0 basic sampling point is determined by performing smoothing, thresholding and statistical processing on digital image data. 3. A signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, which uses radioactively labeled DNA or DNA.
Obtained by base-specific cleavage of A partial decomposition product: 1) guanine-specific cleavage product, 2) adenine-specific cleavage product, 3) thymine-specific cleavage product, 4) cytosine-specific cleavage product. , and 5) at least five base-specific cleavage products comprising a mixture comprising a guanine-specific cleavage product, an adenine-specific cleavage product, a thymine-specific cleavage product, and a cytosine-specific cleavage product. Each of these is one-dimensionally separated and expanded in parallel on a support medium, and an autoradiograph is recorded on a photographic light-sensitive material, which has positional information of a group of radiolabeled substances in a sub-array. Thereafter, with regard to the digital signals corresponding to the autoradiographs of each separated development column obtained by photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material, i) the separation development column of the mixture; as an internal standard column, determining basic sampling points for this internal standard column, lI) determining the signals of radioactive substances of each separation development column distributed within the sampling mask set based on the basic sampling points; A signal processing method in autoradiography, comprising the steps of: detecting sampling points in each separation sequence by comparing them with each other and utilizing the exclusivity of assumed sampling points between each separation expansion sequence. 4. The signal processing method in autoradiography according to claim 3, wherein the 4° basic sampling point is determined by performing smoothing, threshold processing, and statistical processing on digital image data.