JPS59126256A - 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 autoradiograph due to a 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+A, 2)T+C, 3)G and 4)C of a DNA specimen are transferred to a photographic photosensitive material 1. This photosensitive material 1 is mouned to a rotary drum 2 moving to the axial direction thereof and to 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 9. A reference line of 0)G+T+A+C is synthesized by logically adding the sampling point to the separated and developed lines 1), 2) and interpolation identification is performed according to a curve fitting method by arranging the reference line 0) in the arrangement line of the separated and developed line 1)-4) and the separated and developed lines formed by changing the arrangement order thereof to easily determine the base arrangement of the DNA specimen with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a signal processing method in autoradiography. More specifically, the present invention provides D
The present invention relates to a method for comparative identification of the separation and development position of a radiolabeled substance in digital signal processing for determining the base sequence of a partially degraded NA or 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 nucleic acid, the radiolabeled macromolecular substance, its derivative, or its decomposition product (hereinafter also referred to as the radiolabeled substance) is used. ) on a Kel-like support medium by electrophoresis or other lead section operations to perform separation and development to form a column of radiolabeled substances on the support medium, and then an autoradiograph of this column of column # development. is captured as a visible image on a radiation film, and position information of the radiolabeled substance is obtained from the visible image. In addition, methods for separating and identifying the polymeric substance, or evaluating the molecular weight, characteristics, etc. of the polymeric substance based on the obtained positional information of the radiolabeled substance have already been developed and have not been actually used. There is.

Particularly in recent years, autoradiography has been
It is effectively used to determine the base sequence of A (or partially degraded products such as DNA, hereinafter the same).

By using this autoradiography, DN
Methods for determining the base sequence of A include the Maxam-Gilbert method and the Sanger-Coulson method.
The law is known. In these methods, DNA has a double helical structure consisting of two chain molecules, and each of the two chain molecules contains four types of bases, namely adenine (A
), guanine CC, ), 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 fist Gilbert method is performed by the method described below.

First, phosphorus (P) is added to one end of the chain molecule of the DNA or degraded DNA whose base sequence is to be determined.
By bonding a group containing a radioactive isotope, the object becomes a radiolabeled substance, and then chemical means are used to cleave the bonds between each constituent unit of the chain molecule in a base-specific manner. Next, the DNA obtained by this operation or a mixture of many base-specific cleavage products of the DNA decomposition product is separated and developed by gel electrophoresis, and each of the many base-specific cleavage products forms a band. You get a separated separation expansion column (but you can't see it visually). Then, this separation and development array is visualized on an X-ray film to obtain an autoradiograph, and from the obtained autoradiograph and the cleavage means applied to the target base-specific cleavage product, the radioisotope bond is determined. The bases located in a certain positional relationship from the end of the chain molecule are sequentially determined, and in this way, the sequence of all the bases of the target object 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 radiolabeled substances on the support medium by visually observing the visualized autoradiograph.

In other words, 1. DNA base sequences are determined by visually determining the separation and development position of each radioactively labeled base-specific cleavage product or a mixture thereof, and then determining the separation and development sequence of these base-specific cleavage products. It is 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 is obtained by analyzing the autoradiograph, which is a visible image. There are problems such as differences in information between researchers, and there are limits to the accuracy of the information that can be obtained. In particular, if the autoradiograph visualized on radiographic film does not have good image quality (g-acuity, contrast), it is difficult to obtain satisfactory information, and its accuracy tends to decrease. .

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 use such measuring instruments.

Furthermore, 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 shift may occur in their overlapping, and in this case, the separation obtained as a visible image on the radiation film may Since the development row (for example, the migration row) is not parallel to the length of the film and is shifted, errors are likely to occur when visually determining the position information of the radiolabeled substance, and its accuracy may be affected. tends to decline. Furthermore, depending on the support medium and the separation and development position, the resulting separation and development rows are often 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 radiolabeled substance may not necessarily spread uniformly on the support medium. Similar non-uniformity in separation development occurs when the gel contains empty bubbles, or
It also occurs 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 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.

In conventional autoradiography, which uses radiography, the four unreleased groups are detected by photoelectrically reading an autoradiograph containing positional information of a radioactive label imaged on a photosensitive material and converting it into a digital signal. The present invention has been achieved by realizing the simple and highly accurate determination of the base sequence of DNA or DNA partial decomposition products by performing suitable signal processing.

That is, the present invention is a signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products.
1) guanine-specific cleavage and decomposition products obtained by base-specific cleavage and decomposition of NA or DNA portions;
adenine-specific cleavage product, thymine-specific cleavage product,
and a set of reference array forming samples consisting of at least two types of base-specific cleavage products or a mixture of base-specific cleavage products arbitrarily combined to contain all of the cytosine-specific cleavage products as a whole. and 2) at least one type of base-specific cleavage degradation product or base-specific cleavage product mixture, respectively, on a supporting medium, each of the two sets of reference array forming samples of 1) above is placed on top of each other in duplicate. At least five rows in which the separation and development rows are one-dimensionally separated and developed in parallel with each other so as to sandwich at least one row of separation and development rows of the cleavage degradation product or base-specific cleavage product mixture of 2) above. is obtained by recording an autoradiograph having positional information of a group of radiolabeled substances constituting a separation and development array on a photographic light-sensitive material, and then photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material. For the digital signal corresponding to the autoradiograph of each separation column, l) detecting a sampling point for each of the separation columns; ii) detecting a sampling point on each duplicate of each reference column forming sample; 111) composing a plurality of straight lines, polygonal lines, or curves connecting corresponding reference sampling points between the plurality of reference columns; This method consists of a signal processing method in autoradiography, which includes the steps of: setting a line; and iv) comparing and identifying sampling points of each separation expansion series using the continuous &L lines.

In the present invention, the reference sequence refers to four types of bases of DNA,
In other words, it refers to a column corresponding to a separation and development column containing all of the base-specific cleavage products of guanine, adenine, thymine, and cytosine. The labeled salt serves as an internal standard for a separation and development column in which specific cleavage and decomposition products are separated and developed on a support medium.

In the present invention, "position information" refers to various information centering on the position of a radiolabeled substance or an aggregate thereof in a sample, such as the position and shape of an aggregate of radioactive substances present in a support medium, and the like. It means various types of information obtained as one or any combination of information such as the concentration of radioactive substances at a location, minute 45, etc.

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 automatic distortion of the radiolabeled substance that is separated and developed in the -dimensional direction to form a separated and developed chest. Even if the entire autoradiograph recorded on the radiographic film is distorted or scratched due to positional misalignment during the operation of forming the radiograph on the radiographic film, the base sequence of the DNA or DNA partial decomposition product can be determined with high precision. can be determined.

In particular, for distortion in the direction of separation development, by combining multiple reference columns that serve as internal standards from multiple separation development columns, the distortion can be measured on digital image data based on this reference column. After correcting the obtained distortion for each column, it becomes possible to identify the 0-separation development site for each column. Therefore, the target base sequence can be determined with high precision and rationality.

The sample to be measured used in the autoradiography of the present invention is a base-specific cleavage sample obtained by base-specific cleavage of radioactively labeled DNA or a DNA partial decomposition product. A support medium in which a substance or a mixture thereof is separated and developed in the -dimensional direction = As a method for separating and developing the above-mentioned radioactively labeled substance using a support medium, for example, a gel-like support medium (the shape is Typical methods include electrophoresis using various force supporting media such as layered, columnar, etc.), polymer moldings such as acetate, or various force supporting media such as filter paper, and thin layer chromatography using supporting media such as silica gel. Among these, electrophoresis using a gel-like support medium is a typical separation and development method and is preferred.

The basic structure of the photographic light-sensitive material used in the present invention is a support and a photographic emulsion layer.The photographic emulsion layer is made of a binder such as gelatin that contains 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. The following radiation films can be mentioned.

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 material in close contact with each other and, for example, by keeping them in this state for several days at a low temperature below freezing. In the exposure operation, sensitization may be performed by using an intensifying screen or by applying pre-exposure such as flash exposure.

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

゛Biochemistry Experiment Course 6 Tracer Experiment Method (Part 1) 271
〜289 pages, "8. Autoradiograph E" Toru Sueyoshi, Akiyo Shigesue (1977, published by Tokyo Kagaku Dojin) Next, in the present invention, the radiolabeled substance on the support medium recorded on the photographic light-sensitive material. A method for reading an autoradiograph having one-dimensional 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 fitted with a photosensitive material l having a visible image on its outside. This rotating drum 2 is
It rotates at a constant speed and at the same time moves 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 a mirror 3, is attached to a transparent drum 2, passes through a photosensitive material 1, and enters a photoelectron multiplier v7.

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 an r 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 described above, but 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 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 using the signal processing method of the present invention will be explained using the above-mentioned DNA base sequencing method using the Maxam-Gilbert method as an example. A case will be described in which three different types of DNA are electrophoresed on the same support medium using the following combinations of four base-specific cleavage products.

1) Guanine-specific cleavage product + adenine-specific cleavage product 2) Thymine-specific cleavage product + cytosine-specific cleavage product, 3) Guanine-specific cleavage product, 4) Cytosine-specific cleavage product, First , three types of D labeled with radioactive label (32F)
By cleaving each of the NAs at each base unit using a conventional method, three sets each consisting of the four base-specific cleavage products described in 1) to 4) above are obtained.

Next, each of the three sets of base-specific cleavage products of 4-1 are separated and developed by electrophoresis on a gel support medium to obtain respective separation and development arrays. However, at least the two groups of base-specific cleavage and decomposition products in 1) and 2) above are arranged adjacent to each other at at least two locations on the support medium, and a set of adjacent separation and development rows is provided. The separation and development operation is performed by arranging the two groups of base-specific cleavage and decomposition products in 3) and 4) above so as to sandwich the separation and development columns.

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.
An autoradiograph of the unfolded train is recorded on radiographic film.

FIG. 2 shows an example of an autoradiograph of the four electrophoretic columns described above, in which base-specific cleavage products of radiolabeled DNA are separated and developed. In other words, the first
From column 1 to column 12, (1) - (G) specific cleavage product + (A) specific cleavage product (2) - (T) specific cleavage product + (C) specific cleavage product. Decomposition product (3) - (G) Specific cleavage product (4) - (C) Specific cleavage product (5) - (G) Specific cleavage product ('6) - (G) Specific Cleavage product + (A) specific cleavage product (7) - (T) specific cleavage product + (C) specific cleavage product (8) - (C) specific cleavage product (9) - ( G) Specific cleavage product (10) - (C) Specific cleavage product (11) - (G) Specific cleavage product + (A) Specific cleavage product ('12) - (T) Specific Each column (electrophoresis column) of the cleavage product + (C) specific cleavage product is shown. The 1st to 4th columns, the 5th to 8th columns, and the 1st to 4th columns are groups of separated and developed columns of different DNAs. Furthermore, the first and second columns, the sixth and seventh columns, and the eleventh and twelfth columns are sets of samples for forming reference columns, respectively.

The first autoradiograph recorded on radiographic film
By loading the digital signal into the reading device shown in the figure and reading it out, the digital signal input to the signal processing circuit 10 is transmitted to the address (x,
y) and the signal level (, Z) at that address, and the signal level corresponds to the amount of photostimulated light. That is, the digital signal corresponds to the autoradiograph of FIG. Therefore, in the signal processing circuit 10,
Digital image data having positional information of the radiolabeled substance is inputted.4 In the present invention, digital image data means a collection of digital signals corresponding to an autoradiograph of the radiolabeled substance.
- First, on the digital image data, the separated and developed positions of the radiolabeled substance are detected for each of the above 12 columns, and these are used as sampling points. There are 7 sampling points, which can be obtained, for example, as follows.

The above digital signal is scanned twice in parallel at different positions on one digital image data so as to cross the one-dimensional distribution direction (separation development row direction) of the radiolabeled substance, so that each scanning area is The scanning to detect the distribution points of the radiolabeled substance in each row is called a preliminary scan), and the two distribution points for each row are connected to form a
Two straight lines are obtained, and each of the obtained straight lines is used as a scanning direction for detecting sampling points 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 temporarily stored in memory (that is, stored in buffer memory or non-volatile memory such as a magnetic disk).

In signal 1 processing, scanning digital image data means selectively extracting only the digital signal at this scanning location from the memory.

Then, by scanning the digital image data along the scanning direction for sampling point detection obtained above,
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, smoothing processing is performed on this function f (w) by, for example, convolution using an appropriate filter function to obtain a function g (w). Next, threshold processing is performed on this function g(w). That is,
For the threshold α0), when g (w) ≧ 00, g (w) = 1g (w:
)＜α. In this case, the function g (y) is converted into a continuous function of 1 or O by performing processing to set g(w)=0. The sampling points are detected by setting each midpoint of the region, g (w)=1. Note that the threshold value (α0) in the threshold processing described in “2” 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, for each column, the set of sampling points (S
kn (Xx n + Vkn + Zkn)). 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, by logically adding the sampling points in the first column and the sampling points in the second column, a new (G) specific,
Sampling containing all four types of base-specific cleavage decomposition products, (A) specific cleavage decomposition products, (T) specific cleavage decomposition products, and (C) specific cleavage decomposition products, a sequence of ξ , that is, a reference (internal standard) column (SL3n) is obtained. For example, this composition can be expressed as an operation as follows.

(SL3n)=(S ln) U (S2 n) Here, () represents a set of sampling points, and U represents a logical sum operator. This reference column has a reference sampling point S day so as to exist virtually midway between the first column and the second column.

Set new coordinates.

By performing similar processing on the sampling points of the 6th column and the sampling points of the 7th column, a reference column having the reference sampling point (S + a n ) is synthesized, and the sampling points of the 11th column and the 12th column are synthesized. A similar process is performed for the sampling points to synthesize and obtain a reference column having the reference and sampling points (S + s n ). In this way, reference rows of beads can be virtually provided on the digital image data at both ends and in the center of the double migration row obtained by separation and development.

A plurality of straight lines (or polygonal lines) are obtained by connecting corresponding reference sampling points in each reference column, that is, reference sampling points having the same sampling point number n. For example, a polygonal line is obtained by connecting the farthest (n-1) reference sampling points S'131.5141 and 5151 of the separation deployment position, and then n=2.3. By similarly obtaining straight lines (or broken lines) for . . . , as many straight lines (or broken lines) as there are reference sampling points in each reference column are obtained. Furthermore, the obtained polygonal line can also be approximated by a straight curve. A continuous line group consisting of these straight lines, polygonal lines, or curved lines can be regarded as a contour line group (Ln) regarding the separation development position.

However, n matches the reference sampling point number n.

FIG. 3 is a diagram showing a portion of the contour line approximated by the curve obtained as described above.

Based on this contour line, comparative identification is performed for the sampling points in the third column. For example, the third column sampling point S
31, the position of sampling point S31 (X3
It is determined which of the contour lines L1 and Ll t I y31) is closer to, and it is assumed that the closer contour line is Ll. , the sampling point S31 is the reference sampling point 51
31. In this way, the third
Assign all sampling points in the column to one of the reference sampling points. That is, the sampling points in the third column are represented by a set of reference sampling points (SL3.t13).

'' By the above operations, the sampling point Skn expressed by the y-coordinate (Yx n) can be expressed by the contour line Ln, and since this contour line can also be called the migration coordinate,
In other words, this operation can be regarded as coordinate transformation from xy coordinates to migration coordinates.

Furthermore, the set of sampling points in the third column is the set of - sampling points in the second column that constitute this reference column (S+n).
Since it is included in (SI3'l 't 3 /1'(S 1 n) =
(SI6n).

A virtual 16th column with a new set of sampling points (S (6n)) expressed by the AND operation is obtained, and the obtained 16th column is adenine in the set of columns 1 to 4. This corresponds to a separation and development sequence of specific cleavage and decomposition products.

Perform the same operation between the 4th column and the 2nd column,
The 17th column corresponds to the separation and development column of thymine-specific cleavage products.
get column. Next, the 3rd column, 4th column, 16th column and the 1st column.
For the 7 columns, the reference sampling points Sl are arranged in descending order of n.
n and the sampling point Skn are compared, and when they match, the reference sampling point 5L3n is replaced with the matching sampling point Skn. If the reference sequence is traced to the four animals with small n, it is possible to obtain, for example, a first-order diagram.

S 3+, Sat, 5z7t, S32. S+6t
+In the above diagram, 53n=G, San=C151
By replacing 6n = A and S I7 n = T, we obtain the following diagram.

G-C-T-G-A-... In this way, the base sequence of one chain molecule of the DNA in the set of the first to fourth columns can be determined.

Furthermore, by performing the same process on the set of columns 5 to 8 and the set of columns 9 to 12, the base sequence of one chain molecule of each DNA can be determined. I can do it.

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 decomposed product 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 the information A+T, GC, C+G, and 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-... C-G-A-C-T-machi... By the signal processing method of the present invention, the above (G+A,
A DNA base sequencing method using a combination of T+C, G, C) is an example of a DNA base sequencing method,
The signal processing method of the present invention is not limited to the above-mentioned combinations, and the base sequence can be similarly determined by a method similar to the above-described method using various combinations.

However, in any combination, G, A, T,
It is necessary to have such a combination that a standard (internal standard) train consisting of all base-specific cleavage products of C can be synthesized and obtained. Furthermore, the separation and development arrays for the sample for forming reference arrays must be arranged closely; It is necessary to perform the separation and development by being arranged so as to sandwich the separation and development column of the cut and decomposed products.

The separation and development position of the separation and development row of this reference row forming sample is as follows:
In order to determine the DNA base sequence with high precision, it is preferable that each set be as far apart as possible. That is, when this set consists of two sets, it is preferable to arrange it at both ends of a plurality of separation deployment rows, and when it consists of three sets, it is preferable to arrange it at both ends and the center of a plurality of separation deployment rows. is preferred. If there are more than three sets, it is preferable to arrange them at appropriate intervals between the separated and deployed rows of detection targets. The installation of three sets or more is particularly effective when the above-mentioned smiling effect occurs in the support medium in which the radiolabeled substance is spread, and the DNA base sequence can be determined with high precision. . The more the number of sets of separation and development arrays of samples for forming reference arrays to be installed increases, the more accurate the contour lines obtained become, and therefore, the ability to determine DNA base sequences with high precision becomes n1 possible.

This sample for forming a reference array not only forms a reference array, but also participates in determining the base sequence of the DNA as a separate expanded array. Therefore, the greater the number of separation and expansion columns, the more reference columns can be synthesized.
Therefore, it becomes possible to obtain accurate contour lines. In other words, the base sequence can be determined with even higher accuracy.

In the example described above, 12 rows of radiolabeled substance groups 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 12 rows. Alternatively, it is also possible to determine the base sequences of several types of DNA or more at the same time.

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 are available, such as those that record on heat-sensitive recording materials using hot wire.

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

[Brief explanation of drawings]

FIG. 1 shows an example of a reading device for reading an autoradiograph having positional information of a radiolabeled substance in a sample recorded on a photosensitive material in the present invention. ■=Photosensitive 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 FIG. 2 is a diagram showing an example of an autoradiograph of a separation and development sequence of DNA base-specific cleavage products. FIG. 3 is a diagram showing a portion of a contour line that fills the reference sampling point approximated by a curve. Patent applicant Fuji Photo Film Co., Ltd. Agent Patent attorney Engraving of 100 illustrations of Yasuo Yanagawa (no changes to the content) Figure 1 Figure 2 1 2 34 5 67.8 9101112 Teito Mitsune Minamishosho January 1982 Kazuo Wakasugi, Commissioner of the Japan Patent Office on the 25th Patent application filed on January 8, 1982 (22) 2゜Name of the invention Signal processing method in autoradiography 3゜Relationship with the amended case Patent applicant address (520) ) Fuji Photo Film Co., Ltd. Name Representative Minoru Ohnishi 4゜Written Amendment to Agent Proceedings January 1920 // Date゛President of the Japan Patent Office Mr. Kazuo Wakasugi 1, Indication of the Case 1988 Patent Essay R342 No. 2 Invention Name Signal processing method in autoradiography 3 Person making the amendment Relationship with the case Patent applicant 4, agent 8 Contents of the amendment As shown in the attached sheet, the "Detailed Description of the Invention" column of the specification has been amended as follows. Masu. ki 12 jitankiichi - 2 choku old E- (1) Page 7, line 16 Previous →-Delete (
'2) Page 23, line 8 Reading device □ → Listening device (3) Page 23, line 8 Reading → Reading Hiki A (4) Page 23, line 13 Stimulating light →
Transmission X (5) Page 25, line 15 g (y)
→ Present ■ Return Σ (e) Page 29, line 1 Gathering (1st on the 8th)
3 → Part Raikai” Godan 111 (, 7) 2! Page 3, line 10, 2nd column -→ 1st Sword (8), page 29, line 12
(Sol) 3/1 (S1n)= (S+6n)□
↓ (S 5=S (, 9) Page 30, line 1 Sin + Dandan 1 (lO) Drawing The corrected drawing of Figure 3 is attached.

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.
1) All of the guanine-specific cleavage product, adenine-specific cleavage product, thymine-specific cleavage product, and tocin-specific cleavage product obtained by base-specific cleavage degradation of the A partial decomposition product as a whole. at least two sets of samples for forming a reference array consisting of at least two kinds of base-specific cleavage decomposition products or mixtures of base-specific cleavage decomposition products arbitrarily combined to contain base-specific cleavage decomposition products; and 2) base-specific cleavage decomposition products. Object or base specific! ;Ij
At least one of the cut fragment mixtures or the cut decomposition product of 2) above, in which at least one spread column a is formed on each of the two sets of samples for forming reference rows of 1) above on a support medium. Or, it has position information of a group of radiolabeled substances constituting at least five separation and development rows that are one-dimensionally separated and developed in parallel with each other so as to sandwich the separation and development rows of the base-specific cleavage product mixture. After recording an autoradiograph on a photographic light-sensitive material, the digital signal corresponding to the autoradiograph of each separated development row obtained by photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material, i) Detecting sampling points for each of the separation and development columns; 11) composing each reference column from the separation and development columns on each two bases of each reference column forming sample, and detecting a reference sampling point for each reference column; 1ii) setting a plurality of continuous lines consisting of straight lines, polygonal lines, or curves connecting corresponding reference sampling points between the plurality of reference columns, and iv) detecting each separation expansion column by the continuous lines. A signal processing method in autoradiography comprising the step of comparing and identifying sampling points of. Two or more separation and development rows or at least three sets of each of the reference row forming samples of 2°-21) are arranged, and at least one separation and development row of the two adjacent sets of reference row formation samples is arranged. -22) The signal processing method in autoradiography according to claim 1, wherein the signal processing method is arranged so as to sandwich the separation and development column of the cleavage decomposition product or the cleavage decomposition product mixture of item 22). Claim 1 outside the scope of claim 1, characterized in that the 3° sampling point is detected by performing smoothing and/or threshold processing on digital image data in the scanning direction of each of a plurality of separation expansion columns. Or the second
Signal processing methods in autoradiography as described in section. The 4°-set of samples for forming reference rows consists of two types: 1) guanine-specific cleavage product + adenine-specific cleavage product, and 2) thymine-specific cleavage product + cytosine-specific cleavage product. The signal processing method in autoradiography according to any one of claims 1 to 3, characterized in that the signal processing method is comprised of a mixture of base-specific cleavage products.