JPS6118873A - 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 a separate development train 3 of respective base-specified DNA synthetic matters of guanine, adenine, thymine and cytosine each imparted a radioactive label and separate development trains 1, 2, 4 and 5 of synthetic matter mixtures containing all of these synthetic matters are recorded on a photosensitive material and then, read out optically to be converted into a digital signal. As for the digital signal, first, sampling points of the trains 1-5 are detected. Then, with the train 3 as reference, the trains 2 and 5 adjacent to the train 3 are collated by comparison to identify the sampling points of the trains 2 and 5. Moreover, assumed reference sampling points are determined based on the sampling points identified to identify the sampling points of the trains 1 and 5.

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 relates to autoradiography using photographic materials,
The present invention relates to a comparative identification method for separating and developing positions of radiolabeled substances in digital signal processing for base sequencing of DNA 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 (1. invisible to the naked eye) of the radiolabeled substance on the support medium, and this separation The autoradiograph of the developed column 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 not being used in practice. ing.

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

The Sanger-Coulson method is known as one of the representative methods for determining the base sequence of DNA using autoradiography. In this method, DNA has a double helical structure consisting of two stranded molecules, and each of the two stranded molecules contains four types of bases, namely adenine (, A),
It is composed of structural units having the bases guanine (G), cytosine (C), and thymine (T), and
These two chain molecules are cross-linked by hydrogen bonds between these four types of bases, and the hydrogen and hydrogen bonds between each constituent unit are formed by two types of groups: G-C and A-T. A method of determining the base sequence of DNA by focusing on the characteristic structure of DNA, which is realized only by combining, and skillfully utilizing the synthesis of DNA fragments using DNA synthase, gel electrophoresis, and autoradiography. It is.

In the Sanger-Cournon 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), but basically By using single-stranded sample DNA as a template and allowing DNA polymerase to act in the presence of mononucleoside triphosphate containing the above four types of bases, various types complementary to the sample DNA are synthesized. DN of length
Synthesize A fragment. At this time, some of the mononucleoside triphosphates are radioactively labeled, and the synthesis conditions are refined to make them specific for any one of the four types of bases. base-specific synthetic NA with a radioactive label
A fragment (DNA composite) is obtained.

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

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

“A DNA sequence analysis method that uses a unique table to read genetic information in its original language” by Yoshifumi Miura, Gendai Kagaku, September 1977 issue 4
pp. 6-54 (-published by Tokyo Kagaku Dojin) Sanger, F.
, N1cklen, S., & Coulson,
A.R.

Proc, Natl, Acad, Sci, US
A. 74. pp, 54H-5487 (187?) As mentioned above, in autoradiography using radiography, in order to obtain positional information of a radiolabeled substance, an autoradiograph containing 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 positional information about specific radiolabeled substances. We have obtained the following knowledge. That is, the base sequence of DNi is for each radioactively labeled base-specific DNA compound or a mixture thereof.

Visually judge the separation development position and identify those base-specific DNs.
It is determined by comparing the separation and development arrays of A compounds with each other. Therefore, analysis of the obtained autoradiograph is usually performed through human vision, which requires a great deal of time and effort.

Furthermore, since it relies on the human eye, there are limits to the accuracy of the information obtained, such as the positional information obtained by analyzing the autoradiograph differing depending on the researcher. In particular, if the autoradiograph rendered on radiographic film does not have good image quality (sharpness, contrast), it is difficult to obtain satisfactory information and its accuracy tends to decrease. There is a problem.

Conventionally, in order to improve the accuracy of the positional information to be obtained, 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, during an exposure operation in which the support medium on which the separation development rows are formed and the radiation film are brought into close contact with each other, a gap may occur in the superposition, and in this case, a visible image may be obtained on the radiation film. The separation and development column (e.g., electrophoresis column) that is used is not parallel to the length of the film, but is shifted, making it easy for errors to occur when visually determining the position information of the radiolabeled substance. 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 gel is used as a support medium, separation and development is usually carried out with both sides sandwiched between glass or the like because gel T4 does not have self-supporting properties, but the thickness of the gel may be uneven due to deformation of the covering. may occur, and the radiolabeled substance is not necessarily separated and developed uniformly 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, for example, the movement distance of the separation and development rows at both ends is relatively short compared to the movement distance of the separation and development rows near the center of the support medium. A so-called smiling effect often appears. 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 that can be obtained may vary locally on the support medium, resulting in Distortion tends to occur in q.・There is no suitable method other than artificially correcting such distortion of the separation and expansion sequence, so in the above case,
It has become particularly difficult to analyze the positional information of radiolabeled substances, and even with the use of measuring instruments such as those mentioned above, it is difficult to obtain the positional information of separated and expanded radiolabeled substances, that is, the base sequence of DNA or DNA fragments, with sufficient accuracy. It is difficult to obtain.

[Summary of the Invention] In autoradiography using radiography, the present inventor photoelectrically reads an autoradiograph containing positional information of a radioactive label imaged on a photosensitive material and converts it into a digital image. The base sequence of DNA or DNA fragments can be determined simply and with high precision by converting into a signal and further performing suitable signal processing on the sampling points for detecting the distribution position of the obtained radiolabeled substance. We have achieved this and arrived at the present invention.

Specifically, the present invention is a signal processing method in autoradiography for determining the base sequence of DNA or DNA fragments, which comprises a method for determining the base sequence of DNA or DNA fragments, which has a base sequence complementary to the DNA or DNA fragment, and has a radioactive label. granted 2
．． 1) at least one type of base-specific DNA
2) a DNA compound mixture consisting of a guanine-specific DNA compound, an adenine-specific DNA compound, a thymine-specific DNA compound, and a cytosine-specific DNA compound; Each of at least two groups of base-specific DNA compounds and a DNA compound mixture containing at least two groups of base-specific DNA compounds and a DNA compound mixture containing an automate having positional information of a group of radiolabeled substances separated and developed one-dimensionally in a parallel relationship on a support medium. After recording a radiograph on a photographic light-sensitive material, the digital signal corresponding to the autoradiograph of each separated development column obtained by photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material: i ) detecting a reference sampling point for the separation and development column of the DNA compound mixture as a reference column; 100) detecting a sampling point for a separation and development column other than the reference column; A signal in autoradiography, comprising the step of comparing and collating the sampling points of the separation expansion column adjacent to the reference column using the reference sampling point of the column as a reference, thereby identifying the sampling point of the separation expansion column adjacent to the reference column. It provides a processing method.

That is, the present invention allows the radiation energy emitted from the sample to be absorbed by the radiation film by overlapping the sample and a photographic light-sensitive material such as a radiation film.
This method utilizes a method consisting of visualizing this radiation film, photoelectrically reading the visible image to obtain an electrical signal, and A/D converting this electrical signal to obtain a digital signal.

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.

In the present invention, the reference sequence refers to a separation and development sequence that includes all of the base-specific DNA compounds of four types of DNA bases, namely guanine, adenine, thymine, and cytosine, and is a base sequence of the sample DNA. In the determination of , the base-specific DNA compound to which the radioactive label has been added serves as an internal standard for a separation and development column in which the radioactively labeled base-specific DNA compound is developed on a support medium.

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

According to the present invention, the positional distortion during the spread of the radiolabeled substance on the support medium as described above, or -
The autoradiograph of the radiolabeled substance, which is distributed in a dimensional direction to form a distribution row, may be transferred to the photographic material due to positional deviations during the transfer operation to the photographic material. Even when distortions and deviations occur throughout the graph, the base sequence of DNA or DNA fragments can be determined with high accuracy.

In particular, for distortions in the direction of separation and development, it is possible to identify the direction of separation and development in each row without correcting the distortion between the separation and development rows, thus achieving high accuracy.

The base sequence can be determined simultaneously and rationally.

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 in the separation development array, such as the above-mentioned smiling effect, are likely to occur. However, the present invention provides a standard array containing all four types of base-specific DNA compounds of DNA as an internal standard. , it is possible to correct the distortion of all of the separation development arrays on the support medium.

[Structure of the Invention] Examples of samples used in the present invention include DNA or DNA fragments as templates, and deoxynucleotide cytophosphates (dNTPs) to which a radioactive label has been added and a DNA synthesis enzyme. Each base-specific DNA compound and mixture thereof to be synthesized is 2. −
Examples include support media that are separated and expanded in a dimensional direction to form separated and expanded rows.

Furthermore, in the present invention, the radioactive element used for the radioactive label may be any nuclide as long as it emits radiation (α rays, β rays, γ rays, neutron rays, X rays, etc.). , specific examples are 32F, 14C, 35S,
3H, 125, etc. were punched.

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. 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 is performed by arranging the support medium and the photographic material in close contact with each other.6. For example, this can be done by leaving it in this state for several days at a low temperature, such as 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" 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 in the L direction 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.

Furthermore, regarding the method of reading an autoradiograph having positional information of a radiolabeled substance recorded on a photographic light-sensitive material according to the present invention, the reading operation using a light transmission method using a partial beam has been described above. , reading operation by light reflection method 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, the embodiment of the signal processing in autoradiography for determining the base sequence of bNA using the signal processing method of the present invention will be described, taking as an example the case where the above-mentioned Sanger Φ Kuhn method is used. A case will be described using an electrophoresis example (separation development example) formed by a combination of a specific DNA compound and a DNA-A compound mixture.

1) Guanine-specific DNA compound, 2) Adenine-specific DNA compound, 3) Thymine-specific DNA compound, 4) Cytosine-specific DNA compound, 5) Guanine-specific DNA compound + adenine-specific DNA compound , composite + thymine-specific DNA composite + cytosine-specific DNA composite, First, radioactive labeling (3
By a conventional method using a mononucleoside triphosphate to which 2'P, ) has been added and D'NA polymerase,
By base-specific synthesis, base-specific DNA compounds and DNA compound mixtures of the five groups 1) to 5) above are obtained.

Next, the five groups of base-specific DNA compounds and the DNA compound mixtures are separated and developed by electrophoresis on a gel support medium to obtain respective separation and development columns.

Next, this sample (supporting medium) and the radiation film were heated at -7
An exposure operation is carried out by overlapping the samples at a low temperature of 0 to -90°C for several days, and an autoradiograph of the sample is transferred and recorded on a radiation film.

Figure 2 shows an autoradiograph of a separation and development column (electrophoresis column) consisting of the five types mentioned above, in which each base-specific DNA compound and DNA compound mixture of sample DNA, which has been given a radioactive label, is separated and developed. show. That is, the first to fifth columns of FIG. substance + (A) specific DNA compound + (T) specific DNA compound +, (C) specific DNA compound (4) -, (T) specific DNA compound (5) - (C) specific Each separation and expansion column of the target DNA* product is shown. The third column is (G, A, T, C)
It contains all base-specific DNA compounds and is an internal standard column (reference column) for base sequencing.

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

First, the separation and development positions of the radiolabeled substance are detected for each of the above-mentioned one separation and development row on the digital image data, 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 and development direction) of the radiolabeled substance with respect to the digital signal,
The distribution point of the radiolabeled substance in each row is detected on each scan area. The scan to detect this distribution point is called a preliminary scan)
, ``Five straight lines are obtained by connecting the two distribution points for each separation expansion column, and each of the obtained straight lines is used as the scanning direction for sampling point detection in each column.

In the signal processing method of the present invention, the digital signal obtained by reading out the radiation film is once stored in the memory in the signal processing circuit 10 (i.e.), in the non-volatile memory such as a Zuffer memory or a magnetic disk. be remembered). 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 interval number f (w) is subjected to smoothing processing by convolution using an appropriate filter function, for example, and the interval number 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 0 by performing processing to set g(w)=0. The sampling points are detected by taking each midpoint of the region g(w)=1. Note that the threshold value (α0) in the above R value processing can be determined, for example, from the relationship between the signal level and its frequency, that is, the histogram, for the 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 )+((
:) column and (G) column, if (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 FIG.

FIG. 3 is a diagram showing a portion of the reference column (third column) and the fourth column. Here, the black squares represent sampling points in each column corresponding to the separation and development site of the radiolabeled substance, and the hollow squares represent interpolated reference samples and ring points.

Using the sampling point S3n of the third column, which is the reference column, as the reference sampling point, the sampling point S3n of the adjacent fourth column
By performing comparison identification with 4n, reference sampling points are interpolated for the fourth column.

For example, regarding the sampling point S41 in the fourth column,
The position of the sampling point Sat (Xai) and the position of the reference sampling point 332 in the third column (X32) and 533 (
7) Compare with position (X33). For example, if lX32 Xa11=a lX33 Xatl=b, in this case a>b, so the sampling point S41 is assigned to have the same X coordinate as the reference sampling point 333. 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 each of the remaining reference sampling points of the third column into the fourth column, a set of virtual reference sampling points is created in the fourth column. Create (Sam). 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 sam in the fourth column, the adjacent sampling point S5n in the fifth column is assigned to the standard sampling point 33n via the standard sampling point sam, and then the fourth column By interpolating the reference sampling point sam into the fifth column, a virtual set of reference sampling points (35m) is created in the fifth column.

In this way, based on the reference sampling point s3n of the reference column, while creating a virtual reference sampling point set (Skm) in each column in turn, all sampling points 5icn are selected from one of the reference sampling points S3n. Attribute to.

That is, the above method comprises: 1) at least three groups of base-specific DNA compounds each containing at least one type of base-specific DNA compound that is different from each other;
and 2) DNA consisting of a guanine-specific DNA compound, an adenine-specific DNA compound, a thymine-specific DNA compound, and a cytosine-specific DNA compound.
A position of a radiolabeled substance group in which each of at least three groups of base-specific DNA compounds and a DNA compound mixture including a compound mixture is one-dimensionally separated and developed in a parallel relationship on a support medium; After recording an autoradiograph containing information on a photographic light-sensitive material, a digital image corresponding to the autoradiograph of each separated development column obtained by photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material. Regarding the title, i) a step of detecting a reference sampling point for the separation and development column of the DNA compound mixture as a reference column; ii) a step of detecting a sampling point for each separation and development column other than the base column. , iii) By comparing and collating the sampling points of the separation expansion column adjacent to the reference column using the reference sampling point of the reference column as a reference, the sampling points of the adjacent separation expansion column are identified, and the identified here Sambling,
A step of determining a new virtual reference sampling point based on the point, 1) The reference sampling point newly determined in the step iii) above and the separation adjacent to the separation expansion column in which the reference sampling point was determined. This can be summarized as a signal processing method including the step of identifying the sampling point of the adjacent separated expansion sequence by comparing and collating the sampling point with the sampling point of the expansion sequence.

Next, regarding the first column, second column, fourth column, and fifth column,
The virtual standard 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, the Sk
Sampling point 33m of the reference column (third column) corresponding to m
is replaced with the matching sampling point Sk. Then, by tracing the reference columns in ascending order of n, the following diagram can be obtained, for example.

Sl j −S51 + S 45 + S 52 +
"2) '+S2□, S4□l S13, S5
21...-1-In the wiring formula, 5ln=G,
By replacing 5zn=A, San-T, 35n=c, the following diagram is obtained.

G-C-T-G-A-A-T-G-C-...In this way, the base sequence of a DNA chain molecule that has a complementary base sequence to the sample DNA is determined. 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 expanded DNA compound 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 two stranded molecules of DNA. In other words, by providing the information A + T, c + c, c - + c, T + A as combinations corresponding to each base in the diagram represented by the above symbols, DNA represented by the following diagram can be obtained. Obtain the base sequence of

G-C-T-G-A-A-T-G-C-...C
-G -A -C-T -T -A -C-G --・
----It should be noted that the signal processing method of the present invention allows the above (G
+A+T+C, G, A, T, C) (7) The DNA base sequencing method using the combination is an example of the DNA base sequencing method, and the signal processing method of the present invention uses the above combination. There is no limitation to these, and various combinations are possible, and the nucleotide sequence can be determined in a similar manner to the above-mentioned method using such combinations. However, in any combination, it is necessary to provide a column (reference column) of a mixture of all base-specific DNA compounds of G, A, T, and Cρ as an internal standard.

The separation 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 It is preferable to place the In addition, the standard sequence is not applied only to the combination of specific DNA compounds of the - set of DNA, but if the DNA is of the same type, it is separated and developed on the support medium and is applied to the combination of nine radioactive substances. Can be applied to each.

In the present invention, the sequence of a specific base is determined, for example, from a combination of at least one group of base-specific DNA compounds and an appropriate reference substance (for example, a mixture of each base-specific DNA compound). It is also possible.

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. In other words, the present invention is a method of performing identification while sequentially correcting deviations in the direction of separation development, centering on a reference column, so the present invention can be used more effectively as the number of separation development columns increases. Demo. Alternatively, it may be less than or equal to the number of balls. 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., electronic display devices such as CRTs (7), and those that record symbols and numbers displayed on CRTs, etc. by video. Recording devices based on various principles can be used, such as those that record on a printer or the like, and those that record on a heat-sensitive recording material using heat rays.

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. It is.

[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
2 light sources, C: light beam, 6: lens, 7 two photomultipliers, 8: amplifier, 9: A/D converter, IO: signal processing circuit. Figure 2 shows base-specific DNA synthesis of sample DNA. FIG. 2 is a diagram showing an example of an autoradiograph of a sample in which substances were separated and developed on a gel support medium. FIG. 3 is a partially enlarged view of the autoradiograph of FIG. 2. Patent applicant Fuji Photo Film Co., Ltd. Agent Patent attorney Yasuo Yanagawa Figure 1 Figure 2 Figure 3 Teitourine Ichisho Sho September 10, 1980

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. Labeled: 1) a base-specific DNA compound containing at least one type of base-specific DNA compound; and 2) a guanine-specific DNA compound, an adenine-specific DNA compound, and a thymine-specific DNA compound. and a DNA compound mixture consisting of a cytosine-specific DNA compound, each of at least two groups of base-specific DNA compounds and a DNA compound mixture comprising: After recording an autoradiograph having positional information of a group of radiolabeled substances separated and developed on a photographic light-sensitive material, each of the autoradiographs obtained by photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material Regarding the digital signal corresponding to the autoradiograph of the separation and development column, i) using the separation and development column of the DNA composite mixture as a reference column, detecting a reference sampling point for the reference column; ii) separation and development other than the reference column; iii) detecting the sampling points of the adjacent separation expansion column by comparing the sampling points of the separation expansion column adjacent to the reference column with reference to the reference sampling points of the reference column; A signal processing method in autoradiography, comprising: identifying a . 2. 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. Signal processing methods in autoradiography described. 3. A signal processing method in autoradiography for determining the base sequence of DNA or DNA fragments, which has a complementary base sequence to the DNA or DNA fragments and is tagged with a radioactive label. , 1) at least two groups of base-specific DNA compounds each comprising at least one type of base-specific DNA compound that is different from each other;
NA composite, and 2) guanine-specific DNA composite, adenine-specific DNA
A compound, a DNA compound mixture consisting of a thymine-specific DNA compound and a cytosine-specific DNA compound, each of at least three groups of base-specific DNA compounds and a DNA compound mixture 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 separation and development column obtained by reading the separation and development column, i) using the separation and development column of the DNA compound mixture as a reference column, detecting a reference sampling point for the reference column; ii) detecting sampling points for each separation and expansion column other than the reference column; iii) comparing and collating the sampling points of separation and expansion columns adjacent to the reference column using the reference sampling point of the reference column as a reference; A step of identifying sampling points of adjacent separation expansion columns and determining a new virtual standard sampling point based on the identified sampling points, iv) a standard sampling point newly determined in step iii) above. and a step of identifying a sampling point of an adjacent separation expansion sequence by comparing and matching the reference sampling point with a sampling point of an adjacent separation expansion sequence to the separation expansion sequence in which the standard sampling point is determined. signal processing methods in 4. Separation and expansion columns other than the reference column are arranged on both sides of the reference column, and the step iii) is performed for both of the two adjacent separation and expansion columns. Signal processing methods in autoradiography. 5. Claim 3, 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 development columns. Or the fourth
Signal processing methods in autoradiography as described in section. 6. 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, and 4) cytosine-specific DNA synthesis. 5) at least five groups of base-specific DNA compounds and DNA compound mixtures consisting of guanine-specific DNA compounds + adenine-specific DNA compounds + thymine-specific DNA compounds + cytosine-specific DNA compounds; A signal processing method in autoradiography according to any one of claims 3 to 5, characterized in that: