JPS6118874A - Signal processing for autoradiography - Google Patents

Abstract

PURPOSE:To determine base sequence at a high accuracy, by applying a specified signal processing to a digital signal obtained by optically reading out an autoradiograph to correct distortion and deviation of the autoradiograph. CONSTITUTION:Autoradiographs of separate development trains 1-8 of respective base-specified DNA synthetic matters of guanine, adenine, thymine and cytosine each imparted a radioactive label are recorded on a photosensitive material and then, optically read out to be converted into a digital signal. As for the digital signal, first, sampling points of the trains 1-8 are detected. Then, a reference train 0 containing all the synthetic matters is synthesized from the trains 3-6. Subsequently, the train 2 adjacent to the trains 3-5 is collated with the reference train 0 by comparison to identify the sampling points of the train 2. Based on the sampling points identified, new reference sampling points 7 are determined to identify the train 1. Likewise, the trains 7 and 8 are identified.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to signal processing methods in autoradiography. More specifically, the present invention provides D
The present invention relates to a comparative identification method for separating and developing positions of radiolabeled substances in digital signal processing for determining the base sequence of NA or DNA fragments.

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

For example, after attaching a radioactive label to a macromolecular substance derived from a biological body such as a protein or nucleic acid, the radiolabeled macromolecular substance, its derivative, or its decomposition product (hereinafter also referred to as the radiolabeled substance) is used. ) is subjected to a separation operation such as electrophoresis on a gel-like support medium to perform separation and development, thereby forming a separation and development column (however invisible) of the radiolabeled substance on the support medium, and this separation and development column The autoradiograph is captured as a visible image on radiographic film, and position 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 effectively used to determine the base sequence of A (or DNA fragment, hereinafter the same).

One of the representative methods for determining the base sequence of DNA using autoradiography is Sanger-C.
The law is known. In this method, DNA has a double helical structure consisting of two chain molecules, and each of the two chain molecules contains four types of bases, namely adenine (A),
It is composed of structural units containing the bases guanine (G), cytosine (C), and thymine (T), and the two chain molecules are cross-linked by hydrogen bonds between these four types of bases. Focusing on the characteristic structure of DNA, in which hydrogen bonds between each structural unit are realized only in two types of combinations, GC and AT, This method skillfully utilizes the techniques of synthesis, gel electrophoresis, and autoradiography to determine the base sequence of NA.

In the Sanger Fist-Luzon method, there are several methods for synthesizing an NA fragment that is complementary to the DNA or DNA fragment whose base sequence is to be determined (hereinafter referred to as sample DNA). Specifically, single-stranded sample DNA is used as a template, and DNA synthesis enzyme (DNA polymerase) is activated in the presence of mononucleoside triphosphate containing the four types of bases mentioned above to generate complementary DNA to the sample DNA. DNs of various lengths
Synthesize A fragment. At this time, some mononucleoside triphosphates should be radioactively labeled and the synthesis conditions should be devised to make them specific for any one of the four types of bases. As a result, a base-specific synthetic DNA fragment (DNA composite) to which a radioactive label has been added is obtained.

Next, the mixture consisting of a large number of DNA compounds obtained by this operation is separated and developed on a support medium by gel electrophoresis (however, it cannot be seen visually), and the separation on this support medium is performed. The developed array is visualized on a radiographic film such as an X-ray film to obtain an autoradiograph, and based on the obtained autoradiograph, the base sequence of the chain molecule is sequentially determined from the end, and in this way, the sample DNA is determined. The sequence of all bases has been determined.

Note that the characteristics and operation of the Sanger-Cournon method summarized above are described, for example, in the following literature. , “Reading genetic information in its original language: An unexpected DNA sequence analysis method,” Kinbe Miura, Gendai Kagaku, September 1977 issue 4
pp. 6-54 (■Tokyo Kagaku Doujin Publishing) Sanger, F.
, N1cklen, S., & Coulson, ^
, R0゜Proc, Natl, Acad, Sci
, USA, 74. pp, 5483-" In the first traradiography, which utilizes radiography as in a double series, in order to obtain positional information of a radiolabeled substance, an autoradiograph having this positional information is visualized on a radiographic film. It has become essential.

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

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

Traditionally, in order to improve the accuracy of the desired location information,
For example, a method is also used in which the visualized autoradiograph is measured using a measuring instrument such as a scanning densitometer. However, there is a limit to the improvement of accuracy in methods that simply use such measuring instruments.

For example, during an exposure operation in which the radiation film is brought into close contact with the support medium in which the separation/development rows have been formed, a misalignment may occur in the overlay, and in this case, a visible image may be obtained on the radiation film. The separation and development array (e.g., electrophoresis array) that is used is not parallel to the length of the film, but rather deviates from it, making it easy for errors to occur when visually determining the positional information of the radiolabeled substance. Its accuracy tends to decrease. Further, depending on the support medium and the separation and development conditions, the obtained separation and development rows often are not parallel to the length direction of the support medium or are distorted.

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

In the above cases, it becomes particularly difficult to analyze the positional information of the radiolabeled substance, and even if the above-mentioned measuring instruments are used, the positional information of the separated and expanded radiolabeled substance, that is, DNA or DNA fragments, cannot be analyzed. It is difficult to obtain the base sequence of nucleotides with sufficient accuracy.

[Summary of the Invention] In autoradiography using radiography, the present inventor photoelectrically reads an autoradiograph having positional information of a radiolabeled substance imaged on a photographic light-sensitive material and generates a digital signal. By converting the digital signal into a digital signal and subjecting this digital signal to specific signal processing, the present invention has been achieved by realizing the simple and highly accurate determination of the base sequence of DNA or DNA fragments.

That is, the present invention is a signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, which has a complementary base sequence to the DNA or DNA fragment, and has a radioactive signature. 1) a base-specific DNA composition comprising at least a guanine-specific DNA composition; 2) a base-specific DNA composition comprising at least an adenine-specific DNA composition; 3) at least a thymine-specific DNA composition. A base-specific DNA compound containing a substance.

4) a base-specific DNA compound comprising at least a cytosine-specific DNA compound; Each separation obtained by recording an autoradiograph having positional information of a group of separated and developed radiolabeled substances on a photographic light-sensitive material and then photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material. Regarding the digital signal corresponding to the autoradiograph of the expanded column,
1) Detecting sampling points for each of the separation and expansion columns; ii) composing a reference column from a plurality of separation and expansion columns and using the sampling points of this reference column as reference sampling points; iii) Detecting sampling points for each of the separation and expansion columns; By comparing and matching the sampling points of the separation and expansion column adjacent to the separation and expansion column used for synthesis with the reference sampling points of the reference column, the sampling points of the adjacent separation and expansion column are identified, and the identified points are identified here. ii) the step of newly determining a reference sampling point based on the sampled point determined in step iii) above, and the separation expansion column adjacent to the separation expansion column in which the reference sampling point was determined; The present invention provides a signal processing method in autoradiography, which includes the step of identifying a sampling point of an adjacent separation expansion sequence by comparing and collating the sampling point with the sampling point of the separation expansion sequence.

That is, the present invention records the radiation energy emitted from the sample as an autoradiograph on the photographic light-sensitive material by overlapping the sample and the photographic light-sensitive material,
It is based on the fact that this autoradiograph is read photoelectrically to obtain an electrical signal, and this electrical signal is A/D converted to obtain a digital signal.

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, for example, the position 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 shape, concentration of radioactive substances at that location, distribution, etc.

Furthermore, in the present invention, the reference column refers to a column ejected to the separation and development column containing all of the base-specific DNA compounds of four types of DNA bases, namely guanine, adenine, thymine, and cytosine; In determining the base sequence of DNA, base-specific DNA that has been given a radioactive label
It serves as an internal standard for a separation and development column in which a compound is separated and developed on a support medium.

The radiolabeled DNA used to obtain the reference string may be made using sample DNA, or alternatively, may be made using NA. The former is more preferable.

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. Even if the entire autoradiograph transferred and recorded on the photosensitive material is distorted or misaligned due to positional misalignment during the transfer operation of the photosensitive material onto the photosensitive material, high accuracy < DNA or DNA
The base sequence of the A fragment can be determined.

In particular, with respect to distortion in the direction of separation and expansion, it is possible to identify the separation and expansion operation of each column without correcting the distortion between separation and expansion columns, and therefore it is possible to identify the base with high precision and rationally. The sequence can be determined.

In addition, as mentioned above, it is possible to automatically compare sampling points between separation and development rows on digital image data, so even if each separation and development site of a radiolabeled substance is reduced, the accuracy is high. It becomes possible to identify the separation and development site (sampling point) of the radiolabeled substance.

In other words, the absolute amount of radiolabeled substance used in -times of autoradiography can be reduced. Alternatively, it becomes possible to increase the number of separation and development rows in the separation and development operation without expanding the width of the support medium, and it becomes possible to obtain more information than before with - times of autoradiographic measurement operations.

[Structure of the Invention] As an example of a sample used in the present invention, DNA or a DNA fragment is used as a template, and a radioactively labeled deoxynucleoside triphosphate (dNTP) and a DNA synthase are used. Examples include a support medium in which each base-specific DNA compound to be synthesized and a mixture thereof is spread in the -dimensional direction to form a separated spread column.Used for radioactive labeling in the present invention. Radioactive elements are
Any nuclide may be used as long as it emits radiation (α rays, β rays, γ rays, neutron rays, X rays, etc.), and a specific example is "P.

Examples include 1'4C, 35S, 3H1t2sI, and the like.

In addition, the above radiolabeled substance was added for 111 minutes using a support medium.
1! Methods for opening include, for example, electrophoresis using various support media such as gel support media (any shape such as layered or columnar), polymer moldings such as acetate, or cancer paper, and supports such as silica gel. A typical method is thin layer chromatography using a medium.

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

The basic structure of the photographic material 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 exposure 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 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 support medium and the photographic light-sensitive material in close contact with each other and allowing them to remain 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
89 pages, r8. Autoradiography” Toru Sueyoshi,
Akiyo Shigesue (1977, published by Tokyo Kagaku Dojin) Next, in the present invention, an autoradiograph containing one-dimensional positional information of a radiolabeled substance on a support medium recorded on a true photosensitive material is read. The method for converting into a digital signal will be briefly described with reference to the example of a reading device shown in FIG. 1 of the accompanying drawings.

FIG. 11 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 the 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 the signal processing circuit 10 shown in FIG. Signal processing circuit 10
Then, the base sequence of the target DNA is determined by converting the one-dimensional positional information of the radiolabeled substance into symbols and/or numerical values.

Hereinafter, embodiments of signal processing in autoradiography for DNA base sequencing using the signal processing method of the present invention will be explained using the above-mentioned Sanger-Cournon method as an example, and two different types of DNA will be described. , cases using the following four groups of base-specific DNA compounds will be explained.

l) Guanine-specific DNA compound 2) Adenine-specific DNA compound 3) Thymine-specific DNA compound 4) Cytosine-specific DNA compound First, a radioactive label (32F) is added using two types of sample DNA as templates. By base-specific synthesis using a mononucleoside triphosphate and a DNA polymerase by a conventional method, two sets of the above l) to 4 are synthesized.
) to obtain four groups of base-specific DNA compounds.

Next, two sets of base-specific DNA compounds of the four groups described above are separated and developed by electrophoresis on a gel support medium to obtain respective separation and development columns (electrophoresis columns). However, the separation and development rows of four groups of base-specific DNA compounds for one type of DNA are divided into sections, placed adjacent to each other at two locations on the support medium, and the sets of adjacent separation and development rows are The separation and development operations are performed by arranging the separation and development columns of four groups of base-specific DNA compounds for one other type of DNA so as to sandwich them therebetween.

Next, an exposure operation was carried out by superimposing 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 exposed to radiation. Transfer and record on film.

Figure 2 shows an autoradiograph of a separation and development column (electrophoresis column) consisting of the above four groups for two different types of DNA, in which a radioactively labeled base-specific DNA compound of sample DNA is separated and developed. An example of a graph is shown.

That is, the first column to the eighth column in FIG. 2 are (1) -
(G) Specific DNA compound (2)-(A) Specific DNA compound (3)-CG) Specific DNA compound (4)-'(A) Specific DNA compound (5)-(T ) Specific DNA compound (6) - (C) @ Different DNA compound (7) - (T) Specific DNA compound (s: + - (C) Each separation and expansion column of specific DNA compound The 1.2nd column, the 7.8th column, and the 3rd to 6th columns are a group of separation and development columns for mutually different DNAs.

By loading the autoradiograph transferred and recorded on the radiation film into the reading device shown in Figure 1 and reading it, the digital signal input to the signal processing circuit IO is expressed in the coordinate system fixed on the radiation film. The given address (
x, 'y) and a signal level (2) at that address, and the signal level corresponds to the amount of transmitted light. That is, the digital signal corresponds to the autoradiograph of FIG. Therefore, the signal processing circuit 10
Digital image data having the positional information of the radiolabeled substance is inputted to the . As used herein, digital image data refers to a collection of digital signals corresponding to an autoradiograph of a radiolabeled substance.

First, on the digital image data, the separation and development positions of the radiolabeled substance are detected for each of the separation and development rows in the above rows, and these are used as sampling points. The sampling point can be detected, for example, as follows.

'By scanning different positions on the digital image data twice across the one-dimensional distribution direction (separation development direction) of the radiolabeled substance for the bipedal digital signal, each column on each scanning area is The scan to detect the distribution point of the radioactively labeled substance is called a preliminary scan).The two distribution points of each separation development column are connected to obtain a Yagi straight line, and the obtained straight line is This is the scanning direction for sampling point detection in a column.

In addition, in the signal processing method of the present invention, the digital signal obtained by reading out the radiation film is processed by the signal processing circuit 10.
-H memory (that is, stored in a buffer memory or non-volatile 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, 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. Then, this function f(w) is subjected to smoothing processing by, for example, convolution using an appropriate filter function, and the function g(w) is
get. Next, threshold processing is performed on this function g (w). That is, by processing the threshold value (α0) such that when g(w)≧α0, g(w)=1, and when g(w) <αO, g(w) is 20, the function g( w) into a continuous function of 1 or O. The sampling points are detected by setting each midpoint of the area of g(w)=1. Note that the threshold value (α0) in the above threshold processing can be determined, for example, from the relationship between the signal level and its frequency, that is, the histogram, for a digital signal on the scanning area.

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

Next, the comparative identification between each column specifically includes the task of searching for the same separation expansion between the reference column and the remaining separation expansion columns [for example, (G)+: (A)+ ( T) + (
When comparing column C) and column (G), use (G)+
Find the separated expansion that is an element of (G,) from the column (A) + (T) + (C)]. However, when this comparative identification is distorted, the positions of the equivalent separation and 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, distortion can be automatically corrected without relying on humans, and accurate comparison and identification between columns can be performed. This will be explained based on FIGS. 2 and 3. In FIG. 2, the distortion exists in the first to eighth columns, but if attention is paid to the narrow area of the third and sixth columns, the effect of this distortion is small.

Therefore, by logically adding the sampling points from the third column to the sixth column, we newly obtain (G) specific DNA compound, (A) specific DNA compound, (T) specific DNA compound, and (C) a sequence of sampling points including all four types of base-specific DNA compounds,
That is, a reference (internal standard) sequence is synthesized. Assuming that the sampling point Son of this reference sequence is the reference sampling point,
The reference sampling point 5Ofi is the sampling point S3n
, 34111. It is composed of S5n and Sen.

However, 0 represents 1 for the reference column.

For example, this composition can be expressed as an operation as follows. That is, (So'n)= (S3'n)U (San)U (S
sn) Ll (Ssn) Here, () represents a set of sampling points, and U represents a logical sum operator.

By comparing and identifying the reference sampling point Son of the obtained reference column with the sampling point 92n of the second column adjacent to the third column, which is a component of the reference column, one of the reference sampling points of the second column is identified. Do the insertion.

For example, for the second column sampling point 522,
The position of sampling point S22 (X22) and the position of reference sampling point SO3 of the reference column (Xo3) and Se4
(x o 4). For example, if lXO3Xzzl=a IXoa

In this way, all the sampling points in the second column are assigned to one of the reference sampling points in order. Then, by interpolating each of the reference sampling points of the remaining reference columns into the second column based on the assigned sampling point S2n of the second column, a set of virtual reference sampling points is created in the second column. (S 2 m) is created. However, m is a positive integer and matches the number n of the reference sampling point in the reference column. In this way, a virtual reference column can be obtained by moving the reference column (column 0) obtained by combining the third to sixth columns to the position of the second column.

FIG. 3 is a diagram showing a portion of the reference column and the second column synthesized with digital image data-L. Here, the black angle of view represents the sampling point in each column corresponding to the separation and deployment site of the radiolabeled substance, and the hollow square represents the interpolated reference sampling point.

Based on the created reference sampling point 52m in the second column, the adjacent sampling point SlH in the first column is assigned to the reference sampling point Son via the reference sampling point 32m.

In this way, based on the reference sampling point Son of the reference column, while creating a virtual set of reference sampling points (Sym) in each column in turn, all the sampling points Skn are assigned to any of the reference sampling points Son. Attribute to crab.

Next, regarding the first, second, seventh and eighth columns,
The virtual reference sampling point (m in S) in each column is compared with the actual sampling point Skn in that column, and when they match, the reference column (0th column)
The sampling point Son corresponding to n is replaced with the matching sampling point Skn. Then, by tracing the reference columns in ascending order of n, the following diagram can be obtained, for example.

5lIIS211S8 Seal *”j-2+571+S 72
+ S El 2 r S* 3 r 522 +
``''''・In the above diagram, S,, =G, 5zn=
By replacing A, 57n=T, 56n=C,
We get a diagram like this:

G-A-C-G-T-T-C-G-A-...base sequence of a DNA chain molecule that has a base sequence complementary to the sample DNA in a cloudy manner 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 side-to-side amount of each separated and developed DNA compound 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, by providing information such as A, T, G+C1C+G, and T+A as combinations corresponding to the customer bases in the diagram represented by the above symbols, a DNA base sequence represented by the following diagram is obtained.

G-A-C-G-T-T-C-G-A-...C
-T-G-C-A-A-G-C-T-...The signal processing method of the present invention allows the above (G, A, T, C)
A DNA base sequencing method using a combination of
The signal processing method of the present invention, which is an example of the base sequencing method of A, is not limited to the above combinations, and various combinations are possible, and the signal processing method of the present invention is not limited to the above combinations. Then, the base sequence can be determined in the same manner by a method similar to the above method. However, in any combination, G
, A, T, and C. The combination must be such that a standard (internal standard) train consisting of all base-specific DNA compounds of A, T, and C can be obtained. Here, the reference array does not necessarily have to be synthesized from a separation and expansion array of four types of base-specific DNA compounds, but can also be synthesized from a separation and expansion array of a mixture of two or three types of DNA compounds.

In addition, in the example in section 3, the explanation was given using a group of radiolabeled substances constituting eight separated and developed rows that are separated and developed in one-dimensional direction on the support medium. It is not limited. As long as a reference column can be synthesized from a plurality of separated and expanded columns, the signal processing method of the present invention can be applied even if the number of separated and expanded columns is eight or less or more. Furthermore, since the present invention is a method of performing identification while sequentially correcting deviations in the direction of separation development, the more the number of separation development columns is, the more effectively the present invention can be utilized. be.

Alternatively, it is also possible to simultaneously determine the base sequences of two or more types of DNA using one support medium.

Therefore, the obtained standard sequence is not only applied to the combination of base-specific DNA compounds of the DNA set, but also applies to the combination of two or more types of DNA as described above, which can be separated on a support medium. It can be applied to each of the developed groups of radiolabeled substances.

In this case, base half moons can be synthesized using any of the adjacent rows on the support medium, but in order to determine the base sequence of DNA with higher accuracy, Preferably, the reference column is obtained using a plurality of separated expanded columns of the parts, and the separated expanded columns for synthesizing the reference columns are preferably adjacent to each other. Then, centering on the separated expanded column constituting the reference column, the separated expanded columns adjacent to the separated expanded column can be sequentially identified.

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 devices based on various principles can be used, such as those that record on heat-sensitive recording materials (using heat rays), and those that record on heat-sensitive recording materials.

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 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: Amplification group, 9: A/D converter, 10: Signal processing circuit Figure 2 shows an example of an autoradiograph of a sample in which base-specific DNA compounds of sample DNA are separated and developed on a gel support medium. FIG. FIG. 3 is a diagram showing a portion of the reference row and the second separation development row. Patent Applicant Fuji Photo Film Co., Ltd. Agent Patent Attorney Yasuo Yanagawa Figure 1 Issun Figure 2 Figure 3 Figure 2. Procedural Amendment September 10, 1980 Patent Application No. 140911 2, Name of the Invention Signal Processing Method in Autoradiography 3 Person making the amendment) Part 1''A with 5 cases Patent Applicant Name Name
, (520) Fuji Photo Film Co., Ltd. 4, Agent

Claims (1)

[Claims] 1. A signal processing method in autoradiography for determining the base sequence of DNA or a DNA fragment, which has a base sequence complementary to the DNA or DNA fragment and is radioactive. A label is attached to: 1) a base-specific DNA composition comprising at least a guanine-specific DNA composition; 2) a base-specific DNA composition comprising at least an adenine-specific DNA composition; 3) at least a thymine-specific DNA composition. 4) base-specific DNA compounds comprising at least a cytosine-specific DNA compound, each of at least four groups of base-specific DNA compounds comprising: After recording an autoradiograph containing positional information of a group of radiolabeled substances separated and developed one-dimensionally in relation to each other on a photographic light-sensitive material, the autoradiograph visualized on the photographic light-sensitive material is photoelectrically recorded. Regarding the digital signal corresponding to the autoradiograph of each separated expansion column obtained by reading the
i) detecting sampling points for each of the separation and expansion columns; ii) composing a reference column from a plurality of separation and expansion columns and using the sampling points of this reference column as reference sampling points; iii) detecting the sampling points of the reference column. By comparing and matching the sampling points of the separation and expansion column adjacent to the separation and expansion column used for synthesis with the reference sampling points of the reference column, the sampling points of the adjacent separation and expansion column are identified, and the identified points are identified here. iv) a step of newly determining a reference sampling point based on the sampled point, and iv) a reference sampling point newly determined in step iii) above and a separation expansion column adjacent to the separation expansion column in which the reference sampling point was determined; A signal processing method in autoradiography, comprising the step of identifying a sampling point of an adjacent separation expansion sequence by comparing and collating the sampling point with the sampling point of . 2. Separated expansion columns are arranged on both sides of a separation expansion column used for synthesizing a reference column, and the step iii) is performed for both of the adjacent separation expansion columns. The signal processing method in autoradiography according to item 1. 3. Claim 1, characterized in that the sampling points are detected by performing smoothing and/or threshold processing on the digital image data in the scanning direction of each of the plurality of separation expansion columns. Or the second
Signal processing methods in autoradiography as described in section. 4. Base-specific DNA synthesis of DNA or DNA fragments includes: 1) guanine-specific DNA synthesis, 2) adenine-specific DNA synthesis, 3) thymine-specific DNA synthesis, 4) cytosine-specific DNA synthesis. The signal processing method in autoradiography according to any one of claims 1 to 3, characterized in that the signal processing method is a base-specific DNA compound of at least four groups comprising: