JPS59182366A - Signal processing method of autoradiography - Google Patents

Abstract

PURPOSE:To perform arithmetic among signals from respective separated and developed samples obtain a desired signal at the time of a photoelectric read by visualizing the radiation energy from separate and developed sample to which a radioactive mark is added on a photosensitive material. CONSTITUTION:Three kinds of (1) a reference mixture containing all decomposed materials of specific cutting of bases (G, A, T, and C), (2) a group of a sample for reference combined with decomposed materials of specific cutting of G, A, T, and C on the whole, and (3) one kind of decomposed material of specific cutting of bases are used for the autoradiography of separate expansion series (migration series) wherein decomposed materials of specific cutting of respective bases (G, A, T, and C) of DNA having the added radioactive mark, and arithmetic is carried out on the basis of signals from them to detect the separated and developed position of the radioactive marked material as a sampling position.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a signal processing method in autoradiography. More specifically, the present invention relates to autoradiography using photographic materials, in which DNA
Alternatively, 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 DNA product.

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 in a support medium. In recent years.

Autoradiography is effectively used to determine the base sequence of DNA or partial DNA fragments.

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 (G), cytosine (C), and thymine (T), and there are hydrogen bonds between these four types of bases between these two chain molecules. By cleverly utilizing the characteristic structure of DNA, in which the hydrogen bonds between each structural unit are realized only in two types of combinations, G-C and A-T, This is a method for determining base sequences.

For example, the Maxam-Gilbert method is implemented by the method described below.

First, add phosphorus (P) to one end of the chain molecule of the DNA or DNA decomposition product whose base sequence is to be determined.
By bonding a group containing a radioactive isotope, the object becomes a radiolabeled substance, and then chemical means are used to cleave the bonds between each constituent unit of the chain molecule in a base-specific manner. Next, the DNA obtained by this operation or a mixture of 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. Separately expanded columns that are separated (but cannot be seen visually)
get. Then, the autoradiograph outside the separation stage is visualized on an X-ray film, and from the obtained visible image and each base-specific dino cleavage means, the chain molecules to which the radioactive diagonal elements are bound are determined. The bases in a certain positional relationship from the end are sequentially determined, and in this way the sequence of all the bases of the object is determined.

Conventionally, in the OI radiography described in 1., the analysis of the obtained autoradiograph is performed by human eyes, but as an alternative method, the present applicant
A method of obtaining one-dimensional positional information of a radiolabeled substance as a symbol and/or numerical value by photoelectrically reading a visualized autoradiograph, converting it into a digital signal, and subjecting the digital signal to appropriate signal processing. An application has already been filed for the invention (Japanese Patent Application No. 1336-1982)
issue).

According to the above signal processing method, the position information of radiolabeled substances, which conventionally relied on the visual judgment of researchers, can be obtained from
It is possible to obtain desired symbols and/or numerical values automatically and with high precision.

However, during the exposure operation in which the support medium on which the separation and development rows are formed and the photographic light-sensitive material are brought into close contact, a shift may occur in the overlay, and in this case, a visible image is not obtained on the photographic light-sensitive material. The separation and development array (e.g., electrophoresis array) that is used is not parallel to the length direction of the photosensitive material (e.g., radiation film), but is shifted, so it is difficult to visually determine the positional information of the radiolabeled substance. Errors tend to occur, and the accuracy tends to decrease.

In addition, when gel is used as a support medium, since this gel does not have self-supporting properties, it is usually separated and developed with both sides sandwiched between glass plates, but due to deformation of the covering, etc. 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, a so-called smiling effect often appears, in which, for example, the moving 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. Alternatively, in the case of minute-by-minute development by electrophoresis, the voltage may not be applied uniformly to the support medium, and even in such cases, the separation and development conditions will differ locally on the support medium.
Distortion tends to occur in the resulting separated and expanded array.

In the above cases, it becomes difficult to obtain positional information of the separated and developed radiolabeled substance, that is, the base sequence of DNA or DNA partial decomposition products, with sufficient accuracy.

Therefore, an object of the present invention is to provide a signal processing method for determining the base sequence with high precision in autoradiography for determining the base sequence of DNA or DNA partial decomposition products. be.

The purpose of item 1 is to provide a reference column containing all of the guanine-specific cleavage products, adenine-specific cleavage products, thymine-specific cleavage products, and cytosine-specific cleavage products during sample separation and development. This is achieved by virtually obtaining the separated expanded columns of the detection target by composing them on digital image data, and by correcting the separated expanded columns based on these reference columns.

That is, the present invention is a signal processing method in autoradiography for determining the base sequence of DNA or a DNA partial decomposition product, which
■) All of the guanine-specific cleavage products, adenine-specific cleavage products, thymine-specific cleavage products, and cytosine-specific cleavage products obtained by base-specific cleavage degradation of NA or DNA partial decomposition products. at least one set of reference mixtures containing 2) a reference mixture that is arbitrarily combined to collectively contain all of the guanine-specific cleavage product, the adenine-specific cleavage product, the thymine-specific cleavage product, and the cytosine-specific cleavage product; 3) at least one set of reference array forming samples consisting of at least two base-specific cleavage products or a mixture of base-specific cleavage products; and 3) a base-specific cleavage product containing at least one base-specific cleavage product. At least one set of cleavage decomposition products or a mixture of cleavage decomposition products, each of which is separated and developed on a support medium, and a separation and development row of the reference mixture of the set 1) above and two sets of the reference row forming sample of the set 2) above. The separation and development rows on the base are one-dimensionally separated and developed in a parallel relationship with each other so that at least one row of separation and development rows of the base-specific cleavage product or base-specific cleavage product mixture of 3) above is sandwiched therebetween. After recording on a photographic light-sensitive material an autoradiograph having positional information of a group of radiolabeled substances constituting at least the separation and development row of intaglios, the autoradiograph visualized on the photographic light-sensitive material is photoelectrically recorded. for the digital signal corresponding to the autoradiograph obtained by reading the reference mixture: i) detecting sampling points for each of the separation and development columns; ii) sampling points of the reference column consisting of the separation and development columns of the reference mixture; The sampling points of the reference rows synthesized from the separation and development rows on the two bases of the reference row forming sample are each taken as a reference sampling point, and a straight line, a polygonal line, or a line connecting the corresponding reference sampling points between the plurality of reference rows. The present invention provides a signal processing method in autoradiography that includes the steps of: setting a plurality of continuous lines consisting of curved lines; and 1 ii) comparing and identifying sampling points of each separation development sequence using the continuous lines.

In the present invention, the reference column refers to a separation and development column containing all of the base-specific cleavage products of four types of DNA salts and groups, namely guanine, adenine, thymine, and cytosine, or a separation and development column thereof. Separation development in which a base-specific cleavage product to which a radioactive label has been attached is separated and developed on a support medium in determining the base sequence of a target DNA or partial NA decomposition product. It serves as an internal standard for the column.

In addition, in the present invention, "position information" refers to various information centered on the position of a radioactively labeled substance or an aggregate of moss 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.

According to the present invention, in a signal processing method in autoradiography for determining the base sequence of DNA or DNA partial decomposition products, distortion in the direction of the separation and development force is detected based on the reference sampling points of the reference train. By measuring the distortion, correcting the distortion for each column, and identifying the separation and development conditions for each column, it is possible to detect the misalignment that occurs when overlapping the sample and photographic material during the exposure operation, or the radioactive label. When a substance is separated and developed on a support medium, the separation and development column may be distorted due to the separation and development conditions, and the entire autoradiograph transferred and recorded on the photographic light-sensitive material may be misaligned or distorted. This allows correction to be made on the digital image data obtained, making it possible to determine the target base sequence with high precision and rationally.

Furthermore, by using at least one set of reference mixtures as samples, it is possible to obtain reference sampling points of the reference series that serve as internal standards with high accuracy. On the other hand, by synthesizing the reference array from the separation and development array of the sample for forming the reference array, since the sample for forming the reference array is also the substance to be detected, the number of separation and deployment arrays may be increased unnecessarily. Without,
A reference sampling point of the reference column can be obtained. This leads to an improvement in the accuracy of the reference sequence that serves as an internal standard in DNA base sequencing (i.e., an improvement in the accuracy of the obtained position and information) and an increase in the amount of information obtained in - times of autoradiography. This means that they can be achieved at the same time.

In addition, when it is desired to obtain three or more reference rows, the reference rows can be obtained by using at least one set each of the above-mentioned reference mixture and sample for forming reference rows, and any combination thereof. be.

Since it is possible to detect the distribution position with high precision even when the area of each distribution site of the radiolabeled substance is small, the absolute amount of the radiolabeled substance used in -times of autoradiography can be can be reduced. Alternatively, the number of separation and development columns can be increased without expanding the width of the support medium, and more information can be obtained by performing autoradiography operations twice.

The sample to be measured used in the autoradiography of the present invention is radioactively labeled DNA or D
Each base-specific cleavage product or a mixture thereof obtained by base-specific cleavage and decomposition of an NA partial decomposition product is divided into 1! It is an expanded support medium.

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

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

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 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 (2) 271~
Page 289, “8. Autoradiography Town Sueyoshi Toru,
Akiyo Shigesue (1977, published by Tokyo Kagaku Kaijin) Next, in the present invention, an autoradiograph having one-dimensional positional information of a radiolabeled substance on a support medium recorded on a photographic light-sensitive material is read. The method for converting into a digital signal will be briefly described with reference to an example of a reading device shown in FIG. 1 of the accompanying drawings.

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

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

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

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

The digital signal corresponding to the autoradiograph of the radiolabeled substance thus obtained is then input to the signal processing circuit 10 shown in FIG. Signal processing circuit 10
Now, by converting the one-dimensional positional information of the radiolabeled substance into symbols and/or numerical values, the base sequence of the target DNA can be determined. In this embodiment, the DNA base sequencing method using the Maxam-Gilbert method described above is taken as an example, and the following five groups of base-specific base-specific combinations are used as typical base-specific cleavage and decomposition product combinations for base sequencing. A case will be described in which three different types of DNA are electrophoresed on the same support medium using a combination of target cleavage products.

l) Guanine-specific cleavage product + adenine-specific cleavage product + thymine-specific cleavage product + cytosine-specific cleavage product, 2) Guanine-specific cleavage product + adenine-specific cleavage product 3) Thymine-specific cleavage product Cleavage product + cytosine-specific cleavage product, 4) Guanine-specific cleavage product, 5) Cytosine-specific cleavage product, First, three types of DNs with radioactive signature (32P) added.
By cleaving each of A at each base unit by a conventional method, three sets each consisting of base-specific cleavage products of the five groups 1) to 5) above are obtained.

However, at least one set of the reference mixture 1) above may be prepared.

Next, each of the three sets of base-specific cleavage products of the five groups described above is separated and developed by electrophoresis on a gel support medium to obtain respective separation and development arrays. However, at least the separation and development column of the reference mixture of l) above and 2) and 3 above
) of the mixture of base-specific cleavage products (sample for forming reference rows) of 1 group of base-specific cleavage products (sample for forming reference rows).
Separation and development operations are performed by arranging the base-specific cleavage products of each group so as to sandwich the separation and development rows.

Next, this sample (gel-like support medium on which separation and development rows have been formed) and a photographic light-sensitive material are superimposed at a low temperature of -70 to -90°C for several days to carry out an exposure operation, and the autoradiograph of the sample is is recorded as an image on a photosensitive material.

Figure 2 shows three types of DNA labeled with radiolabeled substances.
An example of an autoradiograph of the above-mentioned five groups of electrophoretic arrays in which the base-specific cleavage and decomposition products of are separated and developed is shown. That is,
The 1st to 13th columns in FIG. C) Specific cleavage product (3) - (''G) Specific cleavage product (4) - (C) Specific cleavage product (5) - ('G) Specific cleavage product (6) - ( G) Specific cleavage degradation product + "(A) Specific cleavage degradation product (7) - (G) Specific cleavage degradation product + (A) Specific cleavage degradation product + (T) Specific cleavage degradation product + (C ) Specific cleavage product (8) - (T') Specific cleavage product + (C) @ Differential cleavage product (9) - (C) Specific cleavage product (10) - (G) Specific Cleavage product (11) - (C) Specific cleavage product (12) - (G) Specific cleavage product + (A) Specific cleavage product (13) - (T) Specific cleavage product + ( C) Shows each row (electrophoresis row) of specific cleavage products. The 1st to 4th columns, the 5th to 9th columns, and the 10th to 13th columns are different DNAs.
is a group of separate expansion sequences. Further, the seventh column is a reference mixture, and the first and second columns, and the twelfth and thirteenth columns are sets of samples for forming the reference column, respectively.

By loading the autoradiograph recorded on the photographic light-sensitive material 2 into the reading device shown in Fig. 1 and reading it,
The digital signal input to the signal processing circuit IO is an address (x, y) expressed in a coordinate system fixed to the photosensitive material.
and the signal level (Z) at that address, and the signal level corresponds to the density of the image. That is, the digital signal obtained corresponds to the autoradiograph of FIG. Therefore, in the signal processing circuit 10,
Digital image data having position information of the radiolabeled substance will be input. In addition, in the present invention,
By digital image data is meant a collection of digital signals corresponding to an autoradiograph of a radiolabeled substance.

First, on the digital image data, the separated and developed positions of the radiolabeled substance are detected for each of the above 13 columns, and these are used as sampling points. The sampling points can be obtained, for example, as follows.

By scanning the above digital signal once in parallel at different positions on the digital image data so as to cross the one-dimensional distribution direction (separation and development direction) of the radiolabeled substance, each column on each scanning area is The scan to detect the distribution point of the radiolabeled substance is called a preliminary scan), connect the two distribution points for each column to obtain 13 straight lines, and connect the obtained straight lines to each column. is the scanning direction for sampling point detection at .

In addition, in the signal processing method of the present invention, the photographic light-sensitive material
The digital signal obtained by reading the
At O, it is temporarily stored in memory (that is, stored in a non-volatile memory such as a paperback memory or a magnetic disk).

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

Then, by scanning the digital image data along the scanning direction for sampling point detection obtained above,
The function f (w) [w
can obtain a value representing the position in the scanning direction. This function f(w) is then subjected to smoothing processing by convolution using, for example, an appropriate filter function to obtain the open 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 O by performing processing to set g(w)=0. The sampling point is g(w)
It is detected by setting each midpoint of the area of =1. Note that the threshold value (α0) in the above threshold processing is, for example,
For digital signals on the scanning area, the signal level and
It can be determined from the relationship with the frequency, that is, from the histogram.

In this way, for each column, the set of sampling points (S
ign (Xkn,!/kn+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. That is, the sampling point Skn means the nth sampling point in the first column.

Note that the method for detecting sampling points in the present invention is not limited to the above-mentioned method.

Next, by logically adding the sampling points in the first column and the sampling points in the second column, we newly create (G) specific cleavage decomposition product, (A) specific cleavage decomposition product, and (T) specific cleavage decomposition product. A set of sampling points containing all four types of base-specific cleavage decomposition products, including the decomposition products and (C) specific cleavage decomposition products;
That is, a set of reference sampling points (S14n) is obtained. This composition is expressed as an operation as follows.

(Scan) = (S t nl U (Szn) Here, the book represents a set of sampling points, and U represents a disjunction operator. This reference column is a virtual The reference sampling point 51
4 Set new coordinates of n.

By performing similar processing on the sampling points in the 12th column and the sampling points in the 13th column, a reference column having the reference sampling point (S I5n ) is synthesized and obtained.

Furthermore, since the seventh column is the portion # and development column of the reference mixture, that is, the reference column, its sampling point is the reference sampling point. In this way, the 1 obtained by separating and expanding
Three reference columns (including a virtual reference column) can be obtained on the digital image data at both ends and in the center of the three electrophoresis columns.

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. First, a polygonal line is obtained by connecting the reference sampling points S 141.371 and 5151, which are the farthest (n=1) from the separation development position, and further, when n=2.3. Similarly, by sequentially obtaining straight lines (or polygonal lines) for ......
A straight line (or broken line) with the same number as the minimum number of reference sampling points among the number of reference sampling points in each reference column.
get. Furthermore, the obtained polygonal line can also be approximated by an appropriate curve. A continuous line group consisting of these straight lines, polygonal lines, or curved lines is a contour line group (L
n). However, n matches the number of the reference sampling point.

FIG. 3 is a diagram showing a portion of the contour line approximated by a curved line as described below.

Based on these contour lines, comparative identification is performed for the sampling points in the remaining columns. For example, for sampling point S31 in the third column, sampling point S:5tf7) position a
It is determined which of the contour lines Ll and L2 (X3t + V3t') is closer to, and assuming that the closer contour line is Ll, the sampling point S31 can belong to the reference sampling point S141. In this way, all the sampling points in the third column are assigned to one of the reference sampling points in order. That is, the sampling points in the third column are a subset of the reference sampling points (S 1
4 n) 3 or a subset of contour lines (Ln)s.

By the process described in L, the sampling point Skn represented by the xy coordinate m (Xkn+Ykn) can be represented by a contour line Ln. Since these contour lines can also be called electrophoretic coordinates, this process can be performed from x-y coordinates to electrophoretic coordinates Ln.
It can be regarded as a coordinate transformation to .

The set of sampling points in the third column assigned to the reference sampling point is included in the set of sampling points in the first column (SIn) constituting this reference column, so (S+a,n) 3n (S I n) =
A virtual 16th column having a new set of sampling points (S Ill n ) expressed by a logical AND operation (Stsn) is obtained. The obtained 16th column is the 1st to 4th columns.
This corresponds to a separation and development column of adenine-specific cleavage products in a set of columns.

Similar processing is performed between the fourth column and the second 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 7 columns, reference sampling points 51 in order of decreasing n.
4n and the sampling point Skn are compared, and when they match, the reference sampling point 314n is replaced with the matched sampling point Skn. Then, by tracing the reference columns in ascending order of n, the following diagram can be obtained, for example.

331 ・S4t ・S 171 ・332・5161
・Arranged in the diagram shown in ゛°゛1-, 53n=G, 54n=C
.

By replacing S 16 n ” A and S 17 n ” T, the following diagram is obtained.

G-C-T-G-A-... In this way, the base sequence of one chain molecule of 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 fi49 and the set of columns 1O to 13, it is possible to determine the base sequence of one chain molecule of each DNA. can.

Information regarding the base sequence of the obtained DNA is available from I
The display format is not limited to the display formats listed above, and any display formats such as (a) and (a) are possible. For example, if desired, it is also possible to display the relative amount of each separated and expanded decomposition product by performing arbitrary arithmetic processing on the signals in the scanning direction of each column.

Or even 1. It is also possible to display the base sequences of both DNA chains. That is,
By providing information such as A+T, G+C, CG, and T+A as combinations corresponding to each base in the diagram represented by the above symbols, the DNA base sequence represented by the following diagram is obtained.

C, C-T-G-A -... C-G-A-C-T -... It should be noted that the above (G+A, T
The DNA base sequencing method using the combination of +C, G, C, G + T + C) is an example of the base sequencing method of DNA or DNA partial decomposition products, and the signal processing method of the present invention uses the above combination. Various combinations are possible without being limited to these combinations, and the base sequence can be determined in the same manner by a method similar to the above method using such combinations.

However, in any combination, G, A, T, C
A base mixture containing all of the base-specific cleavage products, and a reference array forming sample that is combined to contain all of the four types of base-specific 1) J cleavage products as a whole. is necessary. Furthermore, the S-developed rows on the two pairs of samples for forming reference rows of the negative set must be arranged close to each other. Also, the minute 1l11 to obtain this reference column
The set of expansion columns (including the MIN sequence of the base I! degree) should be arranged so that the length of the base-specific cleavage and decomposition product of the base sequence is determined. I feel safe.

Separation of this base 1/condition material and sample for forming reference rows Jj
In order to determine the base sequence of DNA with high precision, it is preferable that each set of #expanded positions between the four open columns be as far apart as possible. In other words, 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. preferable. If there are more than three sets, it is preferable to arrange them at appropriate intervals between the detection target minute # expansion columns. The installation of "3 groups or more" 2 is
This is particularly effective when the supporting medium in which the radioactive substance is separated and developed has a smiling effect, and the DN
The base sequence of A can be determined with high precision. The more accurate the contour lines obtained become as the number of sets of separation and development columns for the reference mixture and the sample for forming the reference column increases.
Therefore, it becomes possible to determine the base sequence of DNA with high precision.

Further, since the reference row consisting of the separation and development row of the reference mixture is provided as a single row on the support medium, it is more accurate than the virtual reference row that is synthesized, and the accuracy of the contour line can be improved. That is, by providing a reference column in the sample, the base sequence can be determined with higher accuracy.

Moreover, 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 one-minute #expansion array.

Therefore, the greater the number of separation and expansion columns, the more reference columns can be synthesized, and thus it becomes possible to obtain accurate contour lines. In other words, the base sequence can be determined with even higher accuracy. Therefore, in the above example, 13 rows of radiolabeled substance groups are separated and developed in one-dimensional direction on the support medium, but the number of separated and developed rows is not limited to 13 rows.

Information about the DNA base sequence determined by the signal processing method described above is output from the signal processing circuit 1O, 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 numerical values displayed on a CRT, etc., on a video printer, etc. Recording apparatuses based on various principles can be used, such as those that record on a heat-sensitive recording material using heat rays.

In addition, the information obtained as described above 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: Amplifier, 9: A/D converter, 10: Signal processing circuit FIG. 2 is a diagram showing an example of an autoradiograph of a separation and expansion sequence of DNA base-specific cleavage products. FIG. 3 is a diagram showing a portion of contour lines connecting reference sampling points approximated by curves. Patent applicant Fuji Photo Film Co., Ltd. Agent Patent attorney Yasuo Yanagawa

Claims (1)

[Claims] The key to determining the base sequence of 1oDNA or DNA partial decomposition products in autoradiography is a processing method in which radioactively labeled DNA or DNA
l) All of the guanine-specific cleavage product, adenine-specific cleavage product, thymine-specific cleavage product, and cytosine-specific J cleavage product obtained by base-specific cleavage degradation of the A partial decomposition product. at least one set of reference mixtures containing 2) a reference mixture that is arbitrarily combined to collectively contain all of the guanine-specific cleavage product, the adenine-specific cleavage product, the thymine-specific cleavage product, and the cytosine-specific cleavage product; 3) at least one set of reference array forming samples consisting of at least two base-specific cleavage products or a mixture of base-specific cleavage products; and 3) a base-specific cleavage product containing at least one base-specific cleavage product. At least one set of cleavage decomposition products or a mixture of cleavage decomposition products, each of which is placed on a support medium between giK of the reference mixture of the set of 1) above and 1.2.
) so that the two sides of the sample for forming reference rows of ``2'' separation and development rows sandwich at least one row of base-specific cleavage degradation products or base-specific cleavage product mixtures of 3) above. After recording on a photosensitive material an autoradiograph having positional information of a group of radiolabeled substances constituting at least an external segment a development array which is one-dimensionally separated and developed in a parallel relationship with each other, With regard to the digital signal corresponding to the autoradiograph obtained by photoelectrically reading the autoradiograph visualized on the photographic light-sensitive material, i) detecting sampling points for each of the intervals outside the interval #Il; The sampling points of a reference column consisting of a separation and development column of the AA reference mixture and the sampling points of a reference column synthesized from two or more separation and development columns of the sample for forming the reference column are each referred to as reference sampling points, and the plurality of a step of setting a plurality of continuous lines consisting of straight lines, polygonal lines, or curves connecting corresponding reference sampling points between the reference columns, and 111) comparing and identifying the sampling points of each separation expansion column using the continuous lines; 2. A total of at least three sets of separation and development columns for the reference mixture in 1) above and two or more separation and development columns for the reference column forming sample in 2) above are arranged, and Claims characterized in that two sets of separation and development rows that are close to each other are arranged so as to sandwich at least one row of separation and development rows for base-specific cleavage and decomposition products or mixtures of cleavage and decomposition products as described in 3) above. 1st
Signal processing methods in autoradiography as described in section. Claim 1, characterized in that the 3° sampling point is 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゜The samples for forming reference rows in the above 2) are (1) guanine-specific cleavage product + azocone-specific cleavage product, and (2) thymine-specific cleavage product + cytosine-specific The signal in autoradiography according to any one of claims 1 to 3, characterized in that the signal is composed of a mixture of two types of base-specific cleavage products: Processing method.