JPH0299855A - Dna pattern recording method and apparatus for executing the same - Google Patents

Abstract

PURPOSE:To enable direct and manual analysis from material recorded by recording an original data of a DNA sequence obtained with a light irradiation of a gel onto a recording medium as image as intact to be left as proof. CONSTITUTION:An electrophoretic device comprises an A/D conversion circuit 10 to digitize an electrical signal from an amplifier 9, a memory 41 for storing data thereof, a control section 44 and a film printer 46 for preserving data as film. In operation, data of the converter 10 is outputted to a data line synchronizing the falling of a clock and stored into the memory 41 at the rising of a stable strobe signal with a stable address and data made available. Likewise, in reading, a clock is applied to generate an address when the strobe signal reaches H, during the period, data are outputted and converted to analog from digital with an interface section 42. Based on a command of a control section 44, the data is outputted to a digital audio tape recorder 47 as intact, then, to a display 45 and further to an optical fiber printer to preserve.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a DNA pattern recording method and an apparatus for implementing the same, and in particular, to a method for recording a DNA pattern and an apparatus for implementing the same, and in particular, for acquiring detected raw data as is using a fluorescence detection type electrophoresis apparatus.

The present invention relates to an NA pattern recording technique suitable for keeping as evidence.

[Conventional technology]

Fluorescence detection electrophoresis devices have the advantage of not requiring dangerous and expensive radioisotopes.

Generally, when DNA sequencing (gene base sequence determination) is performed using a fluorescence method, electrophoresis is performed using DNA fragments labeled with a fluorescent substance.

These DNA fragments have various lengths and have specific bases at the cut end by controlling the reaction rate of a chemical reaction specific to each base using restriction enzymes on the DNA whose structure is to be determined. (any of the four types A, C, G, and T) can be created.

These fragments are separated by length by electrophoresis, and by exciting the fluorescent substance labeled on each fragment, the base sequence can be determined from the intensity distribution of the fluorescence.

An example of the distribution of DNA fragments subjected to electrophoresis is shown in Figure 8.
Since the electrophoretic distance differs depending on the length of the DNA fragment, DNA fragments of the same molecular weight gather as one hour passes, forming a pattern like band 31 as a whole. Here A, C, G.

The bands in lanes 32 to 35 for each base of T are always 1.
Because there is a molecular weight difference of more than a base, the electrophoresed distances are all different, and the bands in other lanes do not line up horizontally.

The distribution of DNA fragments can be determined by irradiating a gel with a laser beam or the like and detecting the fluorescence emitted in response with a photoelectric conversion element.

A conventional fluorescence detection type electrophoresis device of this type is disclosed in, for example, Japanese Patent Laid-Open No. 62843/1983.

As shown in FIG. 6, a conventional fluorescence detection type electrophoresis device includes a light source 1 for exciting fluorescence. A fiber 2 that guides light from a light source 1, an electrophoresis section 3 that performs electrophoresis, upper and lower electrodes 4 that apply voltage to the electrophoresis section 3, a power source 5 that applies voltage to the electrodes 4, and receives fluorescence. an optical amplifier 7 for amplifying the optical signal, a one-dimensional optical sensor 8 for converting the optically amplified image into an electrical signal, and -
an amplifier 9 for amplifying the electrical signal from the dimensional optical sensor 8; an analog-to-digital conversion circuit 10 for converting the signal from analog to digital; a processing unit 11 for determining the sequence of bases from the digitized signal string; It is comprised of an output device 12 and the like for outputting a symbol string of the obtained base sequence.

Next, the operation of the conventional fluorescence detection type electrophoresis device will be explained using FIGS. 6 and 7.

First, as shown in FIG. 7, the electrophoresis section 3 has a structure in which a gel 22 made of polyacrylamide or the like is sandwiched between glass 21 on both sides. Light from light source 1 is transmitted through optical fiber 2
, and the gel 22 in the electrophoresis section 3 is irradiated from the lower left side of the electrophoresis section 3 so as to form an optical path 13 . The phosphor present in the optical path 13 within the gel 22 is excited and generates fluorescence 24.

This fluorescence 24 reaches the lens system 6 and is condensed into the optical amplifier 7.
reach. The optical amplifier 7 can amplify the intensity of light by several thousand to several hundred times. The amplified light reaches the -dimensional optical sensor 8 and is converted into an electrical signal. here,
The -dimensional optical sensor 8 is a one-dimensional optical sensor for obtaining an image along the optical path 13. The electrical signal obtained by the -dimensional optical sensor 8 is amplified by an amplifier 9, digitized by an analog-to-digital (A/D) conversion circuit 10, and sent to a processing section 11.

In the processing unit 11, each lane 32 to 3 on the optical path 13
The presence or absence of a band is determined by capturing the change in fluorescence intensity at approximately the central position of 5, and determining the sequence of bases such as A, C, G, and T. The determined sequence of bases is output as a symbol such as a character code on a screen or printer, or recorded on a magnetic storage medium.

As described above, in the conventional fluorescence detection type electrophoresis apparatus, detected data is digitally processed and only specific data corresponding to each base is outputted as DNA sequence data to a display or the like.

[Problems to be Solved by the Invention] However, in the above-mentioned prior art, no consideration is given to preserving raw data of the DNA sequence that appears as a phosphor distribution image on the gel as evidence, and photoelectric conversion Because the data detected by the element is digitally processed and only specific data is output, it is impossible to confirm errors in the processing results, it is easy to falsify, and there are problems with the lack of scientific evidence. .

In addition, conversion to digital data, digital analysis, etc.
There was a problem that DNA sequencing equipment was large-scale and expensive.

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

The purpose of the present invention is to record the raw data of a DNA sequence obtained by irradiating a gel with light onto a recording medium as an image, and to preserve it as evidence for digital analysis. The purpose of this invention is to provide a technology that enables manual analysis directly from recorded materials without the use of .

The above and other objects and novel features of the present invention will become apparent from the description of this specification and the accompanying drawings.

[Means to solve the problem]

In order to achieve the above object, the invention according to claim (1) of the present application includes a step of performing electrophoresis using a DNA fragment labeled with a fluorescent substance, and a step of performing electrophoresis using a DNA fragment labeled with a fluorescent substance.

irradiating light or radiation one-dimensionally across the direction of electrophoresis; receiving the irradiated light with a light receiving section fixed in a direction perpendicular to the direction of electrophoresis; The most important feature is that it includes a step of recording the distribution of the received fluorescence intensity.

Further, the invention according to claim (2) acquires the distribution of fluorescence intensity received by the light receiving section according to claim 1 at predetermined time intervals,
The direction of the light receiving part is the first axis, and the direction of time is the second axis.
The main feature is that the distribution is two-dimensionally reconstructed as an axis of , and this distribution is recorded on a recording medium.

Further, the invention of claim (3) provides a light source for exciting fluorescence, an electrophoresis device that performs electrophoresis using DNA fragments labeled with a fluorescent substance, and an electrophoresis section of the electrophoresis device that includes a light source for exciting fluorescence; A light irradiation device that irradiates light from a light source or a radiation source so as to cross the electrophoresis direction in a one-dimensional manner; and a one-dimensional electrophoresis device fixed in a direction perpendicular to the electrophoresis direction of the electrophoresis section The main feature of the present invention is that it is equipped with a light receiving device that receives the fluorescence intensity of the area, and a recording device that records the distribution of the fluorescence intensity received by the light receiving device.

That is, the purpose is to sequentially memorize changes in fluorescence passing through a detection position with one axis being the scanning direction of the detector relative to the gel while labeling with a fluorescent substance and performing electrophoresis.

The stored data is achieved by making the passage of time correspond to an axis different from the scanning direction and recording it on a photosensitive film or the like.

[Effect]

According to the above-mentioned means, a fluorescent image on a gel containing DNA fragments labeled with a fluorescent substance and obtained while performing electrophoresis is
Since analog data or digital data is recorded as the density distribution of the image on the recording medium, it is the same format as conventional film exposed to gel labeled with radioactive isotopes. In this case, raw data can be stored as evidence for digital analysis, etc. Further, if sequencing is performed using a conventional machine using this film, expensive equipment such as a digital analysis device is not required.

〔Example〕

Hereinafter, one embodiment of the present invention will be specifically described using the drawings.

In addition, in all the times for explaining the example.

Components having the same function are given the same reference numerals, and repeated explanations thereof will be omitted.

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

The fluorescence detection type electrophoresis device with a recording device of this example has a sixth
The figure shows a light source 1 for exciting fluorescence, a fiber 2 for guiding light from the light source, and an electrophoresis section 3 for performing electrophoresis. Upper and lower electrodes 4 for applying voltage to the electrophoresis section 3 and their power source 5, a lens system 6 for receiving fluorescence, an optical amplifier 7 for amplifying the optical signal, converting the optically amplified image into an electrical signal. one-dimensional light sensor 8 for converting, the -dimensional light sensor 8
Up to the amplifier 9 that amplifies the output of is the same as before.
In FIG. 1, these are omitted.

The fluorescence detection type electrophoresis device with recording device of this example has a first
As shown in the figure, an analog-to-digital (A/D) conversion circuit 10 for digitizing an electrical signal from an amplifier 9;
A memory 41 for storing the data obtained by the above, an interface section 42 for connecting the memory 41 and an output device, an address generation circuit 43 for specifying an address for writing data into the memory 41, and a circuit for controlling the whole. control unit 44, display 45 for displaying images
, film printer 46 for saving as film
It consists of

The electrical signal from the -dimensional optical sensor 8 is amplified to an appropriate level by an amplifier 9 as in the conventional device, and is applied to an analog-to-digital conversion circuit 10. The quantization number in this embodiment is, for example, 8 bits. The timing of digitization is given by the control section 44. This timing signal is also applied to the address generation circuit 43 to synchronize the address generated from the address generation circuit 43 with data writing. In this way, the image data is stored in memory 41 in chronological order.

The detailed configuration of the memory 41 and address generation circuit 43 is shown in FIG. 2 (configuration block diagram).

As shown in FIG. 2, the address generation circuit 43 includes an address conversion circuit 431, an X counter 432, and an X counter 4.
It consists of 33.

The X counter 432 is, for example, from "0" to "2375"
This is a counter that repeatedly counts up to "2376"'. Further, when the count value is 2375, a carry (carry signal) is generated. This carry is X counter 43
3, and the X counter 433 is incremented by 1.

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

Normally, the capacity of the memory 41 is II 2 II to the N power, where N is the number of address input lines. Therefore, the address is from rr On to address "2N-1".

In this example, since the number of data in the
=4096) address is required. Therefore, simply
The output of the counter 433 is the upper address, and the X counter 43
If the output of 2 is added to the memory addressing as a lower address, it will change from "2376" to "40" for each Y address value.
95'' address is not used, resulting in poor memory usage efficiency. To avoid this, the address conversion circuit 431 performs conversion as shown in equation (1).

Memory address=YX2376+X (1) To perform the calculation of equation (1), an adder and a multiplier are basically required.

In this embodiment, the multiplication of the first term on the right side of equation (1) is calculated in advance, and the ROM or RAM with an address greater than or equal to the maximum value of Y is
etc. are written. Therefore, multiplication by 1 becomes a simple ROM access, which simplifies the circuit, makes it inexpensive and high-speed, and can accommodate changes in the number of pixels in the X direction. The address generation circuit is comprised of an adder that adds the outputs of this ROM and the X counter.

These operations will be explained using the timing chart shown in FIG.

=12 First, the case where the output from the analog-to-digital converter 10 is written to the memory 41 will be described (Fig. 3(a)
). The X counter 432 is incremented at the falling edge of the clock from the control section 44.

When reaching 2375, a carry is output as shown in FIG. 3, and the X counter 433 is incremented by one.

Data from the analog-to-digital converter 10 (the shaded area in the input data in Figure 3) is output to the data line in synchronization with the falling edge of the clock, and is output to the memory 4 at the rising edge of the stable strobe signal of the address and data.
Written to 1.

Next, the case of reading data from the memory to output data will be explained using FIG. 3(b). Similarly to writing, a clock is applied to read and an address is generated. Furthermore, when the strobe signal becomes true (high level), data is output during that period and sent to the interface section 42.

The interface section 42 has a function of directly outputting digital data from the memory and also has a digital-to-analog conversion function. Based on a command from the control unit 44, this data can be output as 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, if necessary, the image can be output to an optical film printer 46 and saved.

As an output result, NA electrophoresis patterns as shown in FIGS. 4, 5A, and 5B are obtained. In this case, the horizontal axis is the direction of arrangement of the one-dimensional sensor along the optical path 13 (
(main scanning direction), and the vertical axis is the time elapsed direction. The arrangement of the bands 51 differs from that in FIG. 8 in that the minimum pitch on the vertical axis is approximately equal throughout. This is an image of the entire electrophoresis surface in the middle of electrophoresis in Fig. 8, whereas
The figure is for the fluorescence distribution when passing through the position of the optical path 13. In addition, in FIGS. 5A and 5B, the reason why bands 52 and 53 are bent or tilted (referred to as smiling) is due to variations in temperature distribution or variations in the state of the gel that occur during electrophoresis. It is known that this occurs due to various causes. Even in such a case, according to this embodiment, the position of the band between each lane can be reliably estimated.

In this embodiment, the memory 41 is provided, but it can be omitted by synchronizing the operation of the film printer 46, display 45, digital audio tape recorder 47, etc. with the analog-to-digital conversion operation. It is. Further, in this embodiment, the image signal is first converted into a digital signal and then the image is output. However, it is also possible to directly add an analog signal to the electro-optical conversion element and print it onto a film.

As can be seen from the above description, according to this example, a fluorescent image on a gel containing DNA fragments labeled with a fluorescent substance and obtained during electrophoresis is converted into analog data or digital data. Since it is recorded on the recording medium as a concentration distribution, an image is obtained in the same format as conventional film exposed to a gel labeled with a radioactive isotope.

Therefore, when the recording medium is paper or film, raw data can be stored as evidence for digital analysis and the like. Further, if sequencing is performed using a conventional machine using this film, expensive equipment such as a digital analysis device is not required.

Further, even if smiling or the like occurs due to electrophoresis, sequencing can be performed reliably.

The present invention has been specifically explained above based on examples, but
It goes without saying that the present invention is not limited to the embodiments described above, and can be modified in various ways without departing from the spirit thereof.

〔Effect of the invention〕

As explained above, according to the present invention, when performing DNA sequencing using the fluorescence method, raw fluorescence images of gels containing DNA fragments are recorded as they are.
It can be used as evidence for DNA sequencing.

Furthermore, it becomes possible to directly analyze the DNA sequence from the recorded results without necessarily using an expensive and complicated analysis device.

Further, even if smiling or the like occurs due to electrophoresis, sequencing can be performed reliably.

[Brief explanation of drawings]

FIG. 1 is a diagram showing a schematic configuration of a fluorescence detection type electrophoresis 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 the memory and address generation circuit shown in FIG. 1. , FIG. 3 is a timing chart for explaining the recording operation by the memory and address generation circuit shown in FIG. 2, FIGS. 4, 5A and 5B are diagrams showing electrophoretic patterns of DNA, 6 to 8 are for explaining the problems of the conventional fluorescence detection type electrophoresis device. In the figure, 1... light source, 2... fiber, 3... electrophoresis section, 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 unit, 45...Display, 46...Film printer, 47...Digital audio tape recorder.

Claims (3)

[Claims] (1) A step of performing electrophoresis using DNA fragments labeled with a fluorescent substance, a step of irradiating the electrophoresis area with light so as to cross the direction of electrophoresis in a one-dimensional manner, and a step of performing electrophoresis using a fragment of DNA labeled with a fluorescent substance; A DNA pattern recording method comprising the steps of: receiving light with a light receiving section fixed in a direction perpendicular to the direction of electrophoresis; and recording the distribution of fluorescence intensity received at the light receiving section. (2) Obtain the distribution of the fluorescence intensity received by the light receiving section according to claim 1 at predetermined time intervals, with the direction of the light receiving section being the first axis and the direction of time passing being the second axis. 2. The DNA pattern recording method according to claim 1, wherein the distribution is recorded on a recording medium by dimensional reconstruction. (3) A light source for exciting fluorescence, an electrophoresis device that performs electrophoresis using fragments of DNA labeled with a fluorescent substance, and an electrophoresis section of the electrophoresis device that receives light from the light source;
A light irradiation device that irradiates the electrophoresis direction in a one-dimensional manner, and a light receiving device that receives the fluorescence intensity of the electrophoresis region in a fixed one-dimensional direction perpendicular to the electrophoresis direction of the electrophoresis region. and a recording device for recording the distribution of fluorescence intensity received by the light receiving device.