KR20020087325A - Separation wall-integrated electrophoresis board and Nucleic acid sequencing appratus using the same - Google Patents

Abstract

PURPOSE: A separation wall integrated electrophoresis board and nucleic acid base sequence analysis apparatus is provided to ensure a wide dynamic range by minimizing interference of fluorescent signal, while obtaining a high light to dark ratio by widening the difference between the maximum signal and the minimum signal. CONSTITUTION: A nucleic acid base sequence analysis apparatus comprises a light source for generating excitation light; an electrophoresis board for electrophoresis of nucleic acid segments; an excitation light radiation unit for guiding the excitation light to the electrophoresis board; a light receiving unit for receiving fluorescence signal generated from a fluorescent material; and a detector unit for detecting the received fluorescence signal. The electrophoresis board is characterized in that a substrate and a separation wall for separating electrophoresis paths are formed integrally with each other. The separation wall is made of an optical absorption material which is selected from a group consisting of ferric oxide, cobalt oxide, crocus of copper, black pigment containing carbon or mixture thereof.

Description

Separation wall-integrated electrophoresis board and Nucleic acid sequencing appratus using the same

The present invention relates to a nucleic acid sequencing device equipped with an electrophoretic plate and an electrophoretic plate used in the electrophoresis method, more specifically, an electrophoretic plate formed integrally with a separation wall consisting of a light absorber and a nucleic acid base sequence using the same For the analysis device.

As the method for sequencing DNA known to date, electrophoresis is the most common method. These electrophoresis methods include mobile interface electrophoresis, paper electrophoresis, cellulose-acetate electrophoresis, gel electrophoresis, rod gel electrophoresis, starch-gel electrophoresis, agarose-gel electrophoresis, polyacrylamide-gel electrophoresis, disc Electrophoresis, SDS-polyacrylamide gel electrophoresis, isoelectric splicing, plate gel electrophoresis, immunoelectrophoresis and the like.

In performing the conventional electrophoresis method, the sample was analyzed by labeling it with radioactive isotopes, but this method has a problem that it takes a lot of time and labor. In addition, DNA sequencing work had to be carried out in a special facility for the safe management of radioactive material.

In order to solve these problems, a method of analyzing DNA sequencing by labeling a sample with a phosphor has recently been used. This method separates the fluorescently labeled DNA fragment mixtures from the polymer gel to which the potential difference is applied by using the difference in the moving speed according to the molecular weight size of the DNA fragments. Photo excitation and photodetection are formed at the end of the electrophoretic path through which the DNA fragments move, and the fluorescence generated in the DNA fragments passing through the portion is detected to recognize the terminal base of each DNA fragment. This is a method of analyzing the base sequence of DNA by determining the order of the bases.

In order to analyze the nucleotide sequence of a nucleic acid using a conventional electrophoretic apparatus, two glass plates are prepared, a spacer for maintaining a gap therebetween is prepared, and then a gel for electrophoresis is inserted. Thereafter, a separator comb wall having a comb shape is inserted into the gel layer between the glass plates to divide the moving plate into a plurality of moving paths.

As described above, in the case of electrophoresis of DNA fragments by using a conventional electrophoretic plate, the blades of the comb-shaped comb-shaped comb-shaped comb forming the isolation walls between the electrophoretic paths are not completely adhered to the electrophoretic plate, and thus each electrophoresis is performed. The DNA fragments in the furnace flow into the gap between the electrophoretic plate and the blade, which can mix with each other, thus making it difficult to read the nucleotide sequence.

A conventional DNA sequencing apparatus (S63-21556 (Japan), see FIG. 1) reflects and induces laser light 2 from the light source 1 to the mirror 3 to pass through the glass plate in the transverse direction of the gel layer of the electrophoretic plate. Investigate. When fluorescently labeled DNA fragments pass through the area irradiated with laser light 4 while being run in a gel, fluorescent signals are sequentially emitted from the DNA fragments. The fluorescence signal emitted from the DNA fragment passes through the light receiving lens 5 and forms an image in the light receiving portion 6. The formed signal is amplified and converted into an electrical signal, and the converted electrical signal 86 is processed by a computer to calculate a sequence of DNA fragments and to analyze the DNA sequence.

Between the glass plate 7 and the glass plate 8 of the conventional flat plate type | mold apparatus, the polymer gel layer swollen with electrolyte is inserted. Spacers 9 for supporting the gaps formed between the glass plates and for controlling the thickness of the gel layer are provided between the longitudinal ends of the two glass plates. Laser light 2 to excite the fluorescent label bound to the DNA fragment passes through the gel layer 11 through the condenser lens 10 and through the spacer 9 between the glass plates parallel to the glass plate. The laser beam excites fluorescent labels 14 and 15 bound to the DNA fragments while advancing the gel layer in which the DNA fragments are run. So the mold excited by laser light The fluorescence signals 18,19 generated from the photolabels are detected by a light receiving system to read the sequence of the base (FIG. 4).

In the sequencing device, the laser light 4 excites the fluorescent label bound to the DNA fragment while passing through the gel layer as shown in FIG. 4, and the fluorescence signal 18 generated from the fluorescent label of any one DNA fragment. When the fluorescent signals 19 generated from the fluorescent labels of adjacent DNA fragments are mixed with each other, they act as noise 87 for the electrical signals detected by the light receiver 5.

Therefore, the width 82 of the dynamic range of the detection signal is narrowed, the contrast ratio 84 of each signal is small, making it difficult to analyze the base sequence of the target DNA, and there is a problem of reducing the noise of the electronic signal.

In addition, the conventional DNA sequencing apparatus (Pha 10-176992 (Japan), see Fig. 2) is a laser light 20 to excite the fluorescent label bound to the DNA fragments incidence 21 at a constant angle on the glass plate of the yeongdongro, A light receiver 23 excited with the fluorescent markers 12 and 13 bound to the DNA fragments that are run between two glass plates 7, 8 so that the fluorescent signal 22 generated by the fluorescent label of any one of the DNA fragments is placed perpendicular to the plate. It is a device for analyzing the nucleotide sequence by detecting in. The laser light 20 for exciting the fluorescent label bound to the DNA fragment is focused through the condensing lens group 24 and irradiated onto a polymer gel inserted between two glass plates to excite the fluorescent label 12 bound to the DNA fragment. . The fluorescence signal excited by laser light and generated in the fluorescent label is detected by the light receiving system 23 to read the sequence of the base.

However, as shown in FIG. 5, in the sequencing apparatus, the focused laser light 16 excites fluorescent label 12 bound to the DNA fragment passing through the gel layer 11, and the fluorescent label of any one DNA fragment. The fluorescence signal 17 generated by the fluorescence signal 26 is mixed with the fluorescence signal 25 generated by exciting the fluorescent label 13 which is reflected at the interface between the remaining laser light 26 and the glass plate and the polymer gel and bound to the adjacent DNA fragments. This acts as noise 87 for the electrical signal.

Therefore, the width 82 of the dynamic range of the detection signal is narrowed, the contrast ratio 84 of each signal is small, making it difficult to analyze the base sequence of the target DNA, and there is a problem of reducing the noise of the electronic signal.

In addition, when the DNA fragments were subjected to the conventional method, as shown in FIG. 6 due to the temperature difference of the plate gel layer 11, the direction of movement 27 of the DNA fragments was distorted during electrophoresis, thus making it difficult to read 28 nucleotide sequences.

Therefore, in the nucleic acid sequencing apparatus, the separation wall for dividing each raceway is separated from the electrophoresis plate and is not separately inserted, but by using a movable plate having a separation wall integrally formed with the movement plate. The electrophoretic plate and the electrophoretic device using the same are integrated with the isolation wall which prevents mixing from the fluorescent markers of the nucleic acid fragments of the fluorophore, and the electrophoretic device using the same. The noise of the electrical signal is reduced and the dynamic range of the detection signal is reduced. An object of the present invention is to provide an electrophoretic plate and a nucleic acid sequencing device capable of increasing the contrast ratio of a detection signal and analyzing a clear detection signal and a longer nucleotide sequence than in the prior art.

1 is a perspective view of a nucleic acid base analysis device having a light irradiation system in a direction parallel to the electrophoretic plate.

Fig. 2 is a plan view of a nucleic acid sequencing device having a light irradiation system in a direction not parallel to the yeongdong plate.

Fig. 3 is a perspective view of a plate type plate on a sequencing device.

FIG. 4 is a diagram showing fluorescence generated in a phosphor labeled on a fragment and light reflected from a glass plate in a nucleic acid sequencing device having a light irradiation system that is not parallel to the plate.

FIG. 5 is a diagram showing fluorescence generated in a phosphor labeled on a fragment in a sequencing device having a light irradiation angle and a light receiving system that are not parallel to the plate.

Fig. 6 is a diagram in which DNA fragments are run in a sequencing device having a plate-like plate in accordance with the prior art.

7 is a schematic diagram of an electrophoretic device according to the present invention.

8 is another embodiment of an electrophoretic device according to the present invention.

9 is a perspective view of an electrophoretic apparatus according to the present invention.

10 is an exploded perspective view of an electrophoretic plate having an integral separator wall according to the present invention.

11 is an exploded perspective view of a yeongdong plate having a separating wall and a light absorption window at the same time, according to the present invention.

12 is a perspective view of a yeongdong plate having a light absorption window according to the present invention.

FIG. 13 is a view showing fluorescence generated in the electrophoresis apparatus using the electrophoretic plate according to the present invention shown in FIG.

FIG. 14 is a view showing fluorescence generated in the electrophoresis apparatus using the electrophoretic plate according to the present invention shown in FIG.

FIG. 15 is a view showing fluorescence generated in the apparatus using the copper plate according to the present invention shown in FIG. 9 and the light absorption window of FIG.

Fig. 16 is a diagram showing a form in which DNA fragments are run in the DNA sequencing device using the yeongdong plate of the present invention shown in Figs.

17 is a view showing the output signal of the conventional sequencing device and the output signal of the sequencing device of the present invention.

<Explanation of symbols for the main parts of the drawings>

1: light source 2, 4: laser light

5: light receiving lens 6, 23: light receiving system

7, 8: Youngdong version 9, 71: spacer

10 condensing lens 11 polymer gel

12-15: fluorescent label 18, 19: fluorescent signal

22, 25: fluorescent signal 24: condensing lens group

26: residual excitation light 39,42: isolation wall

46: filter for separating the excitation light 47: photoelectric carrier

48: light receiving unit 52: dichroic beam spirit 56, 58: fluorescent light receiving element 55, 57: specific wavelength transmission filter 63: fluorescent detection sensor 82: dynamic range 85: contrast ratio of detection signal 74: fluorescent detection window

84: contrast ratio of the detection signal according to the prior art

86: electric signal according to the prior art 89: detection signal according to the present invention

88: background noise

The present invention relates to an electrophoretic plate and a nucleic acid sequencing device using the same, and more particularly, to an electrophoretic plate to which the isolation wall is integrally fixed and a nucleic acid sequencing device using the same.

The electrophoretic plate of the present invention is composed of a separation wall and a substrate formed between a plurality of fluorophores formed in the direction in which the DNA fragments are run. Separation walls formed between the dong-dong paths are those formed by fixing a new layer on the support plate, or they are dug from a plate of a certain thickness and consequently the iron remaining between them. It is composed of).

The separation wall formed on the electrophoretic plate of the present invention, unlike the comb of the hair comb-shaped inserted between the conventional electrophoretic plate, is fixed to the substrate or formed integrally with the substrate, so that the flow of nucleic acid fragments between each of the electrophoretic paths Block completely. In addition, since the width of the isolation wall can be sufficiently extended as necessary, it is possible to minimize the problem that the fluorescence generated in the adjacent yeongdongro mixed with each other by appropriately isolating each yeongdongro. Further, optionally, the isolation wall formed by permanently fixing to the substrate is formed by using a light absorber selected from the group containing black dye containing iron oxide, cobalt oxide, copper oxide, or carbon, so as to isolate the furnace. In addition to the effect of preventing the mixing of the fluorescence of each of the fluorescence path, and absorbs the residual excitation light or reflected light generated at the interface between the gel plate and the gel layer serves to prevent the generation of unnecessary noise signal.

Nucleic acid sequencing apparatus, which is another object of the present invention, includes a light source for generating excitation light, an electrophoretic plate on which nucleic acid fragments labeled with phosphors excited by the excitation light are moved, and inducing the excitation light to the electrophoretic plate. A nucleic acid sequencing device comprising an excitation light irradiation system, a light receiving unit for receiving fluorescence generated by the phosphor, and a detection unit for detecting the received fluorescence signal, wherein the electrophoretic plate separates the electrophoretic paths from the separation wall and the substrate. It is characterized by being integrally fixed.

In addition, a nucleic acid sequencing device according to another object of the present invention includes an electrophoretic plate on which a light source for generating excitation light, nucleic acid fragments labeled with phosphors excited by the excitation light are moved, and the excitation light is electrophoretic plate. A nucleic acid sequencing device comprising an excitation light irradiation system for inducing to, a light receiving unit for receiving fluorescence generated from the phosphor, and a detection unit for detecting the received fluorescence signal, wherein the electrophoretic plate includes a fluorescence detection window. do.

Still another object of the present invention is to provide a light source for generating excitation light, an electrophoretic plate on which nucleic acid fragments labeled with phosphors excited by the excitation light are moved, and an excitation light for inducing the excitation light to the electrophoretic plate. A nucleic acid sequencing device including an irradiation system, a light receiving unit for receiving fluorescence generated from the phosphor, and a detection unit for detecting the received fluorescent signal, the nucleic acid sequencing device comprising: a filter for separating the residual excitation light and the reflected excitation light; It is characterized by that.

The fluorescence detection window employed in the nucleic acid sequencing device of the present invention is attached to the electrophoretic plate of the present invention and serves to block the light generated from a portion other than the intended phoretic furnace into the light receiving portion.

Hereinafter, one preferred embodiment of the device of the present invention will be described in more detail with reference to the accompanying drawings. The sequencing device shown in the accompanying drawings is for explaining one embodiment of the present invention, and the scope of the present invention is not limited to the device shown in the drawings.

Example 1.

FIG. 10 is an exploded perspective view of the plate-type furnace of the present invention in which the isolation walls 39 and 42 are formed in the longitudinal direction in which the respective DNA fragments are moved on the surface or the rear surface of the electrophoretic plate. A plate-type yeongdong-ro was prepared by inserting a polymer gel swelled with electrolyte between the yeongdong-pan 37 and the other yeong-dong 38, which constituted the yeongdong-ro.

Example 2.

11 is an exploded perspective view of another embodiment of the flat type furnace of the present invention further comprising a light absorbing window in the flat type furnace having an isolation wall. A yeongdong-ro consisting of a yeongdong plate consisting of isolation walls 39 and 42 made of a light absorber and another yeongdong plate consisting of a light absorbing window 74 was prepared.

Example 3.

12 is a perspective view of the flat type electrophoretic furnace of the present invention in which the light receiving unit window 73 is formed in the flat type electrophoretic furnace. The copper furnace includes a spacer 71 for maintaining the gap between the glass plate and controlling the thickness of the polymer gel layer.

FIG. 13 is a diagram in which fluorescence is generated from a sample by using the electrophoretic apparatus using the flat electrophoretic furnace of the present invention shown in FIG. 10, and FIG. 14 is an electric furnace using the flat electrophoretic furnace of the present invention shown in FIG. Fluorescence was generated in the sample using the electrophoresis apparatus, and FIG. 15 is a figure in which the fluorescence signal was generated in the sample using the electrophoresis apparatus using the flat type electrophoretic furnace shown in FIG. 12.

Figure 16 is a photograph showing a DNA fragment electrophoresis of the sample using a plate-type electrophoretic furnace having a separation wall of the present invention.

Example 4.

7 is a diagram showing a sequencing device to which the plate-type yeongdongro of the present invention is applied. The nucleic acid sequencing apparatus of the present invention reflects one or more laser lights 29 and 30 to the mirrors 31 and 33 according to the number of samples to be analyzed as shown in FIG. It was composed of an excitation light irradiation system for exciting the labeled phosphor on the DNA fragment by incidence at an angle of 54. The excitation light 36 collected through the condenser lens 34 is irradiated to the fluorescent label 40 bound to the DNA fragments proceeding in each of the fluorescence paths and proceeds along each mobilization path, and is generated by the excitation by the irradiated excitation light. Is condensed by condensing lenses 43 and 47 through a filter 46 for separating the excitation light of the light receiving portion placed perpendicular to the plate. After the light was collected by the light collecting lenses 43 and 47, the fluorescence moved by the optical waveguide or the light transmission medium 49 was moved from the moving part to the area where the light receiving elements 56 and 58 were located. The fluorescence signal collected from the phosphor and the fluorescence signal generated by the excitation light configured to detect one or more samples are respectively separated by a dichroic beam splitter 52 and each separated fluorescence passes through a specific wavelength 55, 57. It was configured to be detected by the fluorescent light receiving elements 56, 58 through. The signal thus detected was converted to respective electrical signals to read DNA nucleotide sequences.

Example 5.

As shown in FIG. 8, the laser light 59 composed of a single color was reflected by the Dyck Ock beam splitter 61 to be condensed by a condenser lens 53, and then a fluorescent label coupled to the DNA fragment was excited to generate a fluorescent signal. The fluorescence signal generated by the excitation light is condensed with the condenser lens 53, separated from the primary excitation light by the dichroic beam splitter 61, and the excitation light is removed again by the wavelength transmission filter 62 for fluorescence, and then condensed by the fluorescence detection sensor 63. It was. In this embodiment, a flat type furnace (FIG. 10, FIG. 11) composed of the light absorption separator wall of the present invention was used.

Example 6.

As shown in FIG. 9, the laser light 65 emitted from the light source was reflected by the mirror 66 and irradiated horizontally through a side of a certain part of the gel of the electrophoretic plate. The plate-type yeongdong-ro used in this embodiment is composed of a polymer gel layer for electrophoresis interposed between the yeongdong plate 69 and the other yeongdong plate 70 and the spacer 71 to support the gap between the yeongdong plate and to control the thickness of the inserted gel layer Configured. The laser light 65 for exciting the fluorescent label bound to the DNA fragment was passed through the condensing lens group 67 and paralleled with the yeongdongro passage through the electrophoresis gel through the spacer 71. As shown in FIG. 15, the irradiated excitation light excites fluorescent labels 77 and 78 bound to the DNA fragments while traveling through the gel in which the DNA fragments are run. The fluorescence signal generated from the fluorescent label excited by the excitation light forms a light absorption layer 72 on the glass surface on the light receiving side so as not to be mixed with the adjacent fluorescence signal. The fluorescence signal separated from the adjacent fluorescence signal by the glass plate and the light absorbing layer is formed into the light receiving portion by an imaging lens, amplified and converted into an electrical signal. The converted electrical signal is processed by a computer to analyze an array of DNA fragments. DNA base sequences were determined.

Example 7.

Figure 17 is a diagram showing a base sequence read signal of the prior art and the base sequence read signal of the present invention. The result of electrophoresis using the DNA sequencing device composed of the plate-type electrophoresis furnace and the filter provided in the present invention is shown by the solid line 89 and the result of the electrophoresis using the sequencing device according to the prior art is a dotted line Shown as 86. Background noise 88 resulting from electrophoresis using the sequencing apparatus of the present invention is lower than background noise 87 according to the prior art, and the dynamic range range 83 by the apparatus of the present invention is wider than the dynamic range 82 according to the prior art. And the contrast ratio 85 of the detection signal by the apparatus of the present invention was larger than the contrast ratio 84 of the detection signal according to the prior art.

In the electrophoretic apparatus of the present invention, the DNA fragments to be analyzed are selected alone or selectively by a filter which separates the residual excitation light and the reflected excitation light from the isolation wall, the light absorption window, and / or the fluorescent signal made of the light absorber. By minimizing the interference of fluorescence signals generated in the yeongdong-ro and the adjacent yeongdong-ro, the wider dynamic range is achieved compared to the prior art, and by increasing the maximum signal and the minimum signal difference, high contrast ratio is achieved, resulting in a clearer detection signal. As a result, longer nucleotide sequences can be analyzed by one run.

Claims (10)

An electrophoretic plate for use in an electrophoretic device, the electrophoretic plate fixed to the separator wall, characterized in that the separator wall and the substrate separating the electrophoretic paths from each other are formed integrally. The electrophoretic plate according to claim 1, wherein the separating wall is made of a light absorber. The electrophoretic plate according to claim 1, wherein the light absorber is selected from iron oxide, cobalt oxide, copper oxide, black pigment containing carbon, or a mixture thereof. A light source for generating excitation light, an electrophoretic plate on which nucleic acid fragments labeled with phosphors excited by the excitation light are moved, an excitation light irradiation system for inducing the excitation light to the electrophoretic plate, and receiving fluorescence generated from the phosphor A nucleic acid sequencing device comprising a light receiving unit and a detection unit detecting a received fluorescent signal, wherein the electrophoretic plate is a nucleic acid sequencing characterized in that the separation wall and the substrate separating the electrophoretic paths from each other are integrally fixed. Device. The nucleic acid sequencing device according to claim 4, wherein the electrophoretic plate further comprises a fluorescent detection window. The nucleic acid sequencing device according to claim 5, wherein the light receiving unit further comprises a filter for separating residual excitation light or reflected excitation light. The nucleic acid sequencing device according to claim 4, wherein the light receiving unit further comprises a filter for separating residual excitation light or reflected excitation light. A light source for generating excitation light, an electrophoretic plate on which nucleic acid fragments labeled with phosphors excited by the excitation light are moved, an excitation light irradiation system for inducing the excitation light to the electrophoretic plate, and receiving fluorescence generated from the phosphor A nucleic acid sequencing device comprising a light receiving unit and a detecting unit detecting a received fluorescent signal, wherein the electrophoretic plate comprises a fluorescence detection window. The nucleic acid sequencing device according to claim 8, wherein the light receiving unit further comprises a filter for separating residual excitation light or reflected excitation light. A light source for generating excitation light, an electrophoretic plate on which nucleic acid fragments labeled with phosphors excited by the excitation light are moved, an excitation light irradiation system for inducing the excitation light to the electrophoretic plate, and receiving fluorescence generated from the phosphor A nucleic acid sequencing device comprising a light receiving unit and a detection unit detecting a received fluorescence signal, wherein the light receiving unit comprises a filter for separating residual excitation light or reflected excitation light. .