JPH0814571B2 - DNA pattern recording method and apparatus for implementing the same - Google Patents

Description

The present invention relates to a method for recording a DNA pattern and an apparatus for carrying out the method, and in particular, a DNA pattern detected by using a DNA sequencing device by a fluorescence detection type electrophoresis device. The present invention relates to a DNA pattern recording technique suitable for directly acquiring raw data of, and leaving it as evidence materials.

[Conventional technology]

A DNA sequencing device using a fluorescence detection type electrophoresis device has an advantage that a dangerous and expensive radioactive isotope is not required.

Generally, when DNA sequencing (determination of the nucleotide sequence of a gene) is performed by a fluorescence method, electrophoresis is performed using a DNA fragment labeled with a fluorescent substance.

This DNA fragment has various lengths and has a specific base at the cut end by controlling the reaction rate of a chemical reaction specific to each base part of the DNA whose structure is to be determined by a restriction enzyme. A fragment (fragment) having (any of four kinds of A, C, G, and T) can be created. These are separated by length by electrophoresis, and by exciting a fluorescent substance labeled on each fragment, the base sequence can be determined from the intensity distribution of fluorescence emitted from the fluorescent substance.

FIG. 8 shows an example of distribution of DNA fragments subjected to electrophoresis. D
Since the electrophoretic distance varies depending on the length of the NA fragment, the DNA fragments separated by band 31 form a distribution pattern as a whole by collecting DNA fragments having the same molecular weight over time. Here, lanes 32 to 35 for A, C, G, and T bases
Since the band 31 has a molecular weight difference of 1 base or more, the electrophoretic distances are all different, and the band 31 does not line up with the bands of other lanes.

The distribution of such DNA fragments can be known by irradiating the gel with a laser beam or the like and detecting the fluorescence emitted upon excitation with a photoelectric conversion element.

A conventional fluorescence detection type electrophoresis apparatus of this type is, for example,
It is disclosed in Japanese Patent Laid-Open No. 61-62843.

Next, DNA using such a fluorescence detection type electrophoresis apparatus
The sequencing device will be described. As shown in FIG. 6, the DNA sequencing apparatus applies a voltage to a light source 1 for exciting fluorescence, a fiber 2 for guiding light from the light source 1, an electrophoretic section 3 for electrophoresis, and an electrophoretic section 3. Upper and lower electrodes 4, a power source 5 for applying a voltage to the electrode 4, a lens system 6 for receiving fluorescent light, an optical amplifier 7 for amplifying an optical signal, and an optically amplified image converted into an electrical signal. For one-dimensional photosensor 8, an amplifier 9 for amplifying an electric signal from the one-dimensional photosensor 8, an analog-digital conversion circuit 10 for converting the signal from analog to digital, and a digitized signal train It is composed of a processing unit 11 that determines the arrangement of bases, and an output device 12 that outputs the symbol string of the obtained base sequence.

Next, the operation of the DNA sequencing device using this fluorescence detection type electrophoresis device will be described with reference to FIGS. 6 and 7.

As shown in FIG. 7, the electrophoretic section 3 has a configuration in which both sides of a gel 22 such as polyacrylamide are sandwiched by glass 21, and light from the light source 1 passes through the optical fiber 2 and the electrophoretic section 3 is provided. To the gel 22 from the lower left side of the electrophoresis unit 3
It is irradiated so that it becomes 13. The fluorescent substance labeled with DNA existing in the optical path 13 in the gel 22 is excited to generate fluorescence 24.

This fluorescent light 24 reaches the lens system 6 and is condensed to the optical amplifier 7.
Reach The optical amplifier 7 amplifies the light intensity several thousand to several ten thousand times. The amplified light reaches the one-dimensional photosensor 8 and is converted into an electric signal. Here, the one-dimensional optical sensor 8
Are arranged perpendicular to the electrophoretic direction in order to obtain an image along the optical path 13. The electric signal obtained by the one-dimensional optical sensor 8 is amplified by the amplifier 9, digitized by the analog / digital (A / D) conversion circuit 10, and processed by the processing unit 11.
Sent to.

The processing unit 11 captures the change in fluorescence intensity at approximately the central position of each lane 32 to 35 on the optical path 13, determines the presence or absence of a band, and determines the sequence of bases such as A, C, G, and T. The determined sequence of bases is converted into a symbol such as a character code on a screen, a printer, or the like, and is output or recorded in a magnetic storage medium.

As described above, in the DNA sequencing device using the fluorescence detection type electrophoretic device, the detected data is subjected to digital processing, and only specific data corresponding to each base is output as a DNA sequence data to a display or the like.

[Problems to be Solved by the Invention]

However, in such a conventional DNA sequencing device, the DN that appears as a distribution image of the fluorophore on the gel
No consideration is given to leaving the raw data of the A sequence as evidence data, and the data detected by the photoelectric conversion element is digitally processed and only specific data is output. Therefore, there is a problem that it is impossible to confirm the error of the processing result, the alteration is easy, and the scientific evidence ability is low.

The present invention has been made to solve the above problems.

An object of the present invention is to perform DNA sequencing,
Raw data of the DNA sequence obtained by irradiating the gel with light is recorded as it is on the recording medium, and it is left as the evidence data for the digital analysis together with the DNA sequencing result. It is to provide a technology that enables the analysis of.

[Means for solving the problem]

In order to achieve the above-mentioned object, the DNA pattern recording method of the present invention performs electrophoresis using a DNA fragment labeled with a fluorescent substance, and causes the electrophoresis section to cross the electrophoresis direction one-dimensionally. Irradiate light, receive the light in a direction perpendicular to the electrophoretic direction, digitally process the received fluorescence intensity distribution signal, and perform DNA sequencing determination, and at the same time, receive light. The signal of the distribution of the fluorescence intensity is acquired at a predetermined time interval, and the direction of the received light is used as the first axis, and the direction of the passage of time is used as the second axis to reconstruct the signal two-dimensionally and the signal of this distribution is recorded on the recording medium. Is recorded as a DNA pattern image of the grayscale image.

In addition, the recording device of the present invention, a light source for exciting fluorescence, an electrophoretic device for performing electrophoresis using a DNA fragment labeled with a fluorescent substance, and an electrophoretic unit of the electrophoretic device, from the light source. Light irradiation device that irradiates the light of the above-mentioned light so as to traverse the electrophoresis direction in a one-dimensional manner, and receives the fluorescence of the electrophoresis portion in a one-dimensional shape fixed in a direction perpendicular to the electrophoresis direction of the electrophoresis portion. A light-receiving device, a sequencing processing device for digitally processing the elapsed time from the fluorescence signal received by the light-receiving device to determine the DNA sequence, and a signal of the distribution of the fluorescence signal received by the light-receiving device as a grayscale image. DNA
A recording device for recording as a pattern image is provided.

That is, the purpose is to label with a fluorescent substance, and in the state of performing electrophoresis, one axis is the scanning direction for detecting the fluorescence of the DNA fragment in the gel, and the change in the fluorescence passing through the detection position is The data is sequentially stored, and the stored data is achieved by recording the result on the photosensitive film or the like along with the result of the sequencing judgment and the time lapse of the axis different from the scanning direction.

[Action]

According to the method described above, when DNA sequencing is performed, the DN obtained by labeling with a fluorescent substance and performing electrophoresis is used.
The fluorescence image from the gel containing the A fragment is subjected to DNA sequencing determination processing as analog data or digital data, but this analog data or digital data is recorded as it is on the recording medium as the concentration distribution of the image. , An image of the same type as a conventional film image exposed to a gel labeled with a radioactive isotope.

Therefore, when the recording medium is paper or film, raw data can be saved as evidence data such as digital analysis together with the analysis result of DNA sequencing. In addition, if the film or the like is used to perform sequencing using a conventional machine, the accuracy of the DNA sequencing result here can be confirmed.

〔Example〕

An embodiment of the present invention will be specifically described below with reference to the drawings.

In all the drawings for describing the embodiments, components having the same function are denoted by the same reference numerals, and their repeated description will be omitted.

FIG. 1 is a diagram showing a schematic configuration of a fluorescence detection type electrophoretic device with a recording device according to an embodiment of the present invention.

The fluorescence detection type electrophoresis apparatus with a recording apparatus of this embodiment is added to the DNA sequencing apparatus shown in FIG. 6, that is, a light source 1 for exciting fluorescence, and a fiber for guiding light from the light source. 2. Electrophoresis unit 3 for performing electrophoresis, upper and lower electrodes 4 for applying voltage to electrophoresis unit 3, power source 5 thereof, lens system 6 for receiving fluorescence, light for amplifying optical signal Since the signal path up to the amplifier 7 and the one-dimensional photosensor 8 for converting the optically amplified image into an electric signal is the same, they are omitted in FIG.

The fluorescence detection type electrophoretic device with a recording device of the present embodiment digitizes an electric signal from an amplifier 39 as shown in FIG. 1 in addition to the configuration for performing DNA sequencing shown in FIG. Analog / digital (A / D) conversion circuit 40, and the analog / digital (A / D) conversion circuit 40
A memory 41 for accumulating the data obtained by the
Interface part 4 for connecting the 41 to output devices
2, an address generation circuit 43 for designating an address for writing data in the memory 41, a control unit for controlling the whole
44, a display 45 for displaying an image and a film printer 46 for saving as a film.

The electrical signal from the one-dimensional optical sensor 8 is subjected to DNA sequencing by the signal path shown in FIG. 6 and is amplified to an appropriate level by the amplifier 39 shown in FIG. 1 and added to the analog / digital conversion circuit 40. To be The quantization number in this embodiment is, for example, 8 bits. The digitization timing is given by the control unit 44. The timing signal is also applied to the address generation circuit 43 to synchronize the writing of the address and the data generated by the address generation circuit 43. In this way, the image data is stored in the memory 41 in time series.

Detailed configurations of the memory 41 and the address generation circuit 43 are shown in FIG. 2 (configuration block diagram).

As shown in FIG. 2, the address generation circuit 43 is composed of an address conversion circuit 431, an X counter 432, and a Y counter 433.

The X counter 432 is, for example, "23" from "0" to "2375".
It is a counter that repeatedly counts 76 ". A carry (carry signal) is generated when the count value is 2375. This carry is added to the Y counter 433 and the Y counter 433 is incremented by one.

On the other hand, the address conversion circuit 431 performs address conversion in order to use the memory 41 efficiently.

Normally, the capacity of the memory 41 is "2" N powers, where N is the number of address input lines. Therefore, the address is from "0" to "2 ^N -1".

In this embodiment, since the number of data in the X direction is 2376, an address of at least 12 bits (2 ¹¹ = 2048 <2376 <2 ¹² = 4096) is required. Therefore, if the output of the Y counter 433 is simply used as the upper address and the output of the X counter 432 is used as the lower address for addressing the memory, the addresses from "2376" to "4095" will not be used for each Y address value. The efficiency of using will become poor. In order to avoid this, the address conversion circuit 431 performs the conversion represented by the expression (1).

Memory address = Y × 2376 + X (1) In order to carry out the calculation of this equation (1), an adder and a multiplier are basically required.

In the present embodiment, the multiplication of the first term on the right side of the equation (1) is calculated in advance and written in the ROM or RAM having an address equal to or larger than the maximum value of Y. Therefore, the multiplication is merely a ROM access, the circuit is simple, and the cost is low and the speed is high, or the number of pixels in the X direction can be changed. The address generation circuit is composed of an adder that adds the outputs of this ROM and the X counter.

These operations will be described with reference to the timing chart shown in FIG.

First, the case where the output from the analog / digital conversion circuit 40 is written in the memory 41 will be described (FIG. 3 (a)). X at the falling edge of the clock coming from the control unit 44
The counter 432 is incremented.

When it reaches 2375, a carry is output and the Y counter 433 is incremented by 1 as shown in FIG.

Data from the analog-digital conversion circuit 40 (3rd
The shaded portion in the input data in the figure) is output to the data line in synchronization with the falling edge of the clock, and the memory rises at the stable rising edge of the address and data strobe signal.
Written on 41.

Next, the case of reading from the memory to output data etc. will be described with reference to FIG. Similarly to writing, reading is also applied with a clock to generate an address. When the strobe signal becomes true (high level), data is output during that period and sent to the interface section 42.

The interface unit 42 has a digital-analog conversion function in addition to the function of directly outputting the digital data from the memory. Based on a command from the control unit 44, this data can be output as it is to the digital audio tape recorder 47, or can be output to the display 45 as a video signal converted into an analog signal. Further, it can be output to and stored in the optical film printer 46 as needed.

As the output result, a grayscale image of the DNA electrophoresis pattern as shown in FIGS. 4, 5A and 5B is obtained.
In this case, the horizontal axis represents the arrangement direction (main scanning direction) of the one-dimensional sensor along the optical path 13, and the vertical axis represents the passage direction of time. Unlike the arrangement shown in FIG. 8, the bands 51 are arranged such that the minimum pitch on the vertical axis is substantially evenly spaced as a whole. While this is an image of the entire migration surface in the middle of migration in FIG. 8,
FIG. 4 is due to the fluorescence distribution when passing through the position of the optical path 13. Further, in FIGS. 5A and 5B, the band 52
It is known that bending or inclination (called smileing) like the band 53 and the band 53 occurs due to various causes such as temperature distribution variation and gel state variation occurring during electrophoresis. Even in such a case, according to the present example, since the DNA sequence of the determination result is obtained together with the pattern image of the distribution of the DNA fragment, it is possible to reliably estimate the position of the band between the lanes and perform sequencing. The accuracy of the determination result can be determined.

Although the memory 41 is provided in the present embodiment, it can be eliminated by synchronizing the operation of the film printer 46, the display 45, the digital audio tape recorder 47, etc. with the operation of the analog-digital conversion. Is. In this embodiment, the image signal is once converted into a digital signal and then the image is output. However, an analog signal may be directly added to the electro-optical conversion element and printed on the film.

As can be seen from the above description, according to this example, a fluorescent image on a gel containing a DNA fragment obtained by labeling with a fluorescent substance and obtained by electrophoresis is used for DNA sequencing determination and analog data. Alternatively, since the digital data is recorded as a density distribution of the image on a recording medium, an image of the same type as that of a conventional film exposed to a gel labeled with a radioactive isotope can be obtained.

Therefore, when the recording medium is paper or film, raw data can be saved as evidence data such as digital analysis. In addition, if the film or the like is used to perform sequencing with a conventional machine or the like, the accuracy of the DNA sequencing determination result can be confirmed.

Further, the correctness of the sequencing determination result can be surely confirmed even when smileing or the like accompanying electrophoresis occurs.

As mentioned above, although the present invention was explained concretely based on an example, the present invention is not limited to the above-mentioned example.
It goes without saying that various modifications can be made without departing from the spirit of the invention.

〔The invention's effect〕

As described above, according to the present invention, in the DNA sequencing by the fluorescence method, the raw fluorescence image of the gel containing the DNA fragments is recorded as it is.
It can be used as evidence for sequencing.

It is possible to directly confirm the analysis result of the DNA sequence from the recorded result.

Further, the correctness of the sequencing determination result can be surely confirmed even when smileing or the like accompanying electrophoresis occurs.

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic configuration of a fluorescence detection type electrophoretic device with a recording device according to an embodiment of the present invention, and FIG. 2 is a block diagram showing a detailed configuration of a memory and an address generation circuit shown in FIG. 3, FIG. 3 is a timing chart for explaining the recording operation by the memory and the address generation circuit shown in FIG. 2, and FIGS. 4, 5A and 5B are diagrams showing the electrophoretic pattern of DNA, FIG. 6 to 8 are for explaining the problems of the conventional fluorescence detection type electrophoretic device. In the figure, 1 ... Light source, 2 ... Fiber, 3 ... Electrophoresis part, 4 ... Electrode, 5 ... Power supply, 6 ... Lens system, 7 ...
Optical amplifier, 8 ... One-dimensional optical sensor, 9 ... Amplifier, 10 ...
… Analog / digital conversion circuit, 11 …… Processing unit, 12…
… Output device, 13… Optical path, 21… Glass, 22… Gel,
24 …… fluorescence, 31 …… band, 41 …… memory, 42 …… interface section, 43 …… address generation circuit, 44 …… control section, 45 …… display, 46 …… film printer, 47
...... Digital audio tape recorder.

Claims (2)

[Claims] 1. A DNA fragment labeled with a fluorescent substance is electrophoresed, and the electrophoretic portion is irradiated with light so as to traverse the electrophoretic direction in a one-dimensional manner. Light is received in the direction perpendicular to the electrophoretic direction, and the received signal of the fluorescence intensity distribution is digitally processed to determine the DNA sequencing.At that time, the signal of the received fluorescence intensity distribution is measured at predetermined time intervals. Two-dimensional reconstruction is performed with the acquired and received light as the first axis and the time lapse direction as the second axis, and the signals of this distribution are recorded on the recording medium as the DNA pattern image of the grayscale image. Characteristic DNA pattern recording method. 2. A light source for exciting fluorescence, an electrophoretic device for performing electrophoresis using a DNA fragment labeled with a fluorescent substance, and an electrophoretic section of the electrophoretic device, which is provided with light from the light source. A light irradiation device that irradiates the electrophoretic direction so as to cross the electrophoretic direction in a one-dimensional manner, and a light receiving device that receives fluorescence of the electrophoretic portion in a one-dimensional manner fixed in a direction perpendicular to the electrophoretic direction of the electrophoretic portion. , A sequencing processing device for digitally processing the elapsed time from the fluorescence signal received by the light receiving device to determine the DNA sequence, and a signal of the distribution of the fluorescence signal received by the light receiving device as a DNA pattern image of a grayscale image A fluorescence detection type electrophoresis apparatus with a recording device, comprising a recording device for recording.