JP2701412B2 - Gel electrophoresis device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a gel electrophoresis apparatus used in a fluorescence-based base sequencer, and more specifically, to a method for gelling a nucleic acid fragment sample labeled with a fluorescent substance. The present invention relates to a gel electrophoresis apparatus provided with an excitation / detection system that is mechanically scanned in a direction perpendicular to a lane for an electrophoresis gel that is simultaneously electrophoresed in a plurality of lanes by electrophoresis, and that detects fluorescence during electrophoresis.

(Prior Art) An example of a fluorescence detection type gel electrophoresis apparatus is shown in FIG. 3 (see “High Technology”, December 1986, pages 46-50).

The sample is dyed and separated into four types of fluorescent dyes, and is electrophoresed simultaneously from the upper end of the electrophoresis gel 70 along a plurality of lanes in a mixed state. To detect DNA fragments during electrophoresis,
A multi-wavelength simultaneous oscillation laser 72 is used as an excitation light source, and a specific wavelength from the laser 72 is switched by a filter and applied to the electrophoresis gel 70. Fluorescence from each dye of the DNA fragment during the migration on the electrophoresis gel 70 is measured in a time division manner by one photomultiplier tube 76 by switching the interference filters 74 having different transmission wavelengths. Switching of the filter on the excitation light side and switching of the interference filter 74 on the light receiving side are performed in synchronization. Running multiple lanes
In order to detect DNA fragments, the irradiation position with excitation light
The beam is moved in a direction perpendicular to the lane by scanning the excitation light beam.

(Problems to be Solved by the Invention) In the apparatus shown in FIG. 3, the fluorescent substance detected at one time by one photomultiplier tube 76 is any one of four types. Since four types of fluorescent substances are used, the duty factor of each fluorescent substance is reduced to 1/4, the signal utilization is reduced, and the S / N per measurement time is deteriorated.

An object of the present invention is to increase the signal utilization rate and increase the S / N in an apparatus in which nucleic acid fragment samples labeled with four types of fluorescent substances are electrophoresed in a mixed state over a plurality of lanes. It is.

(Means for Solving the Problems) In the present invention, light from an excitation light source is spectrally separated by wavelength, and each of the separated light is irradiated as excitation light on a plurality of locations along a scanning direction on an electrophoretic gel, Fluorescence from each excitation light is received by an independent fluorescence photometer.

(Function) The light from the excitation light source is split, the light after the splitting is used as the excitation light, and the fluorescence generated from the nucleic acid fragment by each excitation light is received by an independent fluorescence photometer for each excitation light. Therefore, switching of the excitation light by the filter is avoided, and the utilization rate of the excitation light is increased.

Further, since a plurality of positions on the electrophoresis gel to which the excitation light is irradiated are aligned on a straight line in the scanning direction on the electrophoresis gel, the measurement location is constant regardless of the excitation wavelength.

(Embodiment) FIG. 1 is a view showing an optical system viewed from a direction orthogonal to a scanning direction of an excitation / detection system in one embodiment.

Reference numeral 2 denotes a polyacrylamide electrophoresis gel, which is cut in the scanning direction. The electrophoresis gel 2 is held between electrophoresis plates 2 and 6 made of Pyrex glass.

Reference numeral 8 denotes an argon laser as an excitation light source, and 12 denotes a mirror for directing the laser beam 10 in a direction perpendicular to the surface of the electrophoresis gel 2. The laser beam 10 includes 488 nm and 514 nm light that are simultaneously oscillated. Reference numeral 14 denotes a dichroic mirror which is inclined and placed on the optical path of the laser beam 10 reflected by the mirror 12, and has a characteristic of reflecting light having a wavelength of 500 nm or more and transmitting light having a wavelength less than 500 nm. Therefore, of the argon laser beam 10, the light of 488 nm is passed and made to travel straight, and the light of 514 nm is reflected.

The mirror 16, the lens 18, and the mirror 20 are an optical system that condenses the excitation light 10 a having a wavelength of 488 nm that has passed through the dichroic mirror 14 and irradiates the electrophoretic gel 2. Lens 22 and mirror 24
Is the transmitted wavelength 51 reflected by the dichroic mirror 14.
This is an optical system that collects the 4 nm excitation light 10b and irradiates the electrophoresis gel 2 with it. If the DNA fragment has been electrophoresed at the position on the electrophoresis gel irradiated with the excitation lights 10a and 10b, fluorescence is generated from the fluorescent substance labeling the DNA fragment.

The lens 26 emits the fluorescence 25 from the position irradiated with the excitation light 10a.
Is a lens that collects and collimates the light, and 28 is a mirror that makes the collimated fluorescence incident on the interference filter 30 or 32. The interference filter 30 has a characteristic of transmitting a wavelength of 520 nm, and the interference filter 32 has a characteristic of transmitting a wavelength of 550 nm. The interference filter 30 and the interference filter 32 are attached to a filter rotating plate 34, and are switched to the incident position of the fluorescent light 25 by the rotation of the filter rotating plate 34, and are arranged.
Reference numeral 36 denotes a photomultiplier tube, and reference numeral 38 denotes a lens for guiding the fluorescence passing through the interference filter 30 or 32 to the photomultiplier tube 36. Lens 42
Is a lens that collects the fluorescent light 40 from the position irradiated with the excitation light 10b and converts the collected fluorescent light into parallel light rays. The interference filter 46 has a characteristic of transmitting a wavelength of 580 nm, and the interference filter 48 has a characteristic of transmitting a wavelength of 610 nm. The interference filter 46 and the interference filter 48 are filter rotating plates.
Attach to 50, fluorescent light by rotation of filter rotating plate 50
Switched to 40 incident positions. Reference numeral 52 denotes a photomultiplier tube, and reference numeral 54 denotes a lens for guiding the fluorescence passing through the interference filter 46 or 48 to the photomultiplier tube 52.

The optical system in the area surrounded by the broken line is mechanically scanned in the X and -X directions as a unit. The scanning of the optical system and the rotation of the filter rotating plates 34 and 50 are performed in conjunction with each other. That is, when scanning is performed in the X direction, the interference filter 30
When the scanning is performed in the -X direction, the interference filter 32 and the interference filter 48 are set at the fluorescence receiving position.

 Next, the operation of the present embodiment will be described.

There are four types of fluorescent substances labeling the DNA fragments that migrate in the electrophoresis gel 2, FITC, NBD, TRITC, and Texas Red.
It is dyed by terminal base. FITC is excited by an argon laser beam at 488 nm and emits fluorescence with a peak at 520 nm. NBD is excited at 488 nm and emits fluorescence with a peak at 550 nm. TRITC is excited at 514 nm and emits fluorescence with a peak at 580 nm. Texas Red is excited at 514 nm, 61
It emits fluorescence with a peak at 0 nm.

The fluorescent substances excited by the irradiation of the excitation light 10a having a wavelength of 488 nm passing through the dichroic mirror 14 are FITC and NBD, and the wavelength of 514 nm reflected by the dichroic mirror 14
The fluorescent substance excited by the irradiation of the excitation light 10b is TRITC
And Texas Red. Of the fluorescence 25 generated by excitation with the excitation light 10a, the fluorescence detected by the photomultiplier tube 36 after passing through the interference filter 30 passing through 520 nm is the fluorescence from FITC and passing through the interference filter 32 passing through 550 nm. Then, the fluorescence detected by the photomultiplier tube 36 is the fluorescence from the NBD. Also,
Of the fluorescent light 40 excited by the excitation light 10b, the fluorescent light detected by the photomultiplier 52 after passing through the interference filter 46 passing through 580 nm is the fluorescent light from TRITC and passing through the interference filter 48 passing through 610 nm. And the fluorescence detected by the photomultiplier tube 52 is
Fluorescence from Texas Red.

When the optical system enclosed by the chain line is mechanically scanned in the X direction, the interference filters 30 and 580 nm are set at the fluorescence receiving positions because the interference filters 30 and 580 nm are set at the fluorescence receiving positions. If a DNA fragment labeled with FITC or TRITC is present at the irradiation position, the fluorescence from FITC is detected by the photomultiplier tube 36, and the fluorescence from TRITC is detected by the photomultiplier tube 52. When the optical system is mechanically scanned in the −X direction, the interference filter is located at the position irradiated with the excitation light 10a, 10b because the interference filter 32 of 550 nm and the interference filter 48 of 610 nm are set at the fluorescence receiving position. NBD
Or, if a DNA fragment labeled with Texas Red is present, the fluorescence from the NBD is detected by the photomultiplier tube 36, and the Texas
Fluorescence from Red is detected by the photomultiplier tube 52.

In the embodiment shown in FIG. 1, an interference filter is used to detect two types of fluorescent light excited by the respective excitation lights 10a and 10b with the respective photomultiplier tubes 36 and 52 which are independent for each excitation light. 30 and 32, and 46 and 48 are switched. Instead of switching the interference filters in this manner, as shown in FIG. 2, a dichroic mirror 56 is used, and the fluorescence separated by wavelength by the dichroic mirror 56 is used. Can be detected by separate photomultiplier tubes 58 and 60, respectively. For example, as an alternative to the switching mechanism of the interference filters 30 and 32, a dichroic mirror 56 having a characteristic of transmitting 520 nm light and reflecting 550 nm light can be used. 30 and 32 are the same interference filters as in FIG. 1, and 38 and 39 are condenser lenses. The switching mechanism of the interference filters 46 and 48 in FIG. 1 can be replaced with the same configuration as in FIG. However, the wavelength characteristics of the dichroic mirror are different.

When the fluorescence receiving mechanism as shown in FIG. 2 is used, the light utilization rate is further improved as compared with the embodiment shown in FIG.

(Effects of the Invention) In the present invention, light having a plurality of wavelengths from an excitation light source is simultaneously radiated to the electrophoresis gel as excitation light, and the fluorescence from each irradiation position is received by a separate fluorescence photometer. As compared with the case where only one wavelength of excitation light is irradiated at a time as described above, twice as many signals can be obtained, and the S / N per measurement time improves.

[Brief description of the drawings]

FIG. 1 is a plan view showing an optical system in one embodiment, FIG. 2 is a plan view showing a fluorescent light receiving section in another embodiment, and FIG. 3 is a perspective view showing a conventional gel electrophoresis apparatus. 2 ... electrophoresis gel, 8 ... argon laser, 10 ... laser beam, 10a, 10b ... excitation light, 14 ... dichroic mirror, 30, 32, 46, 48 ... interference filter, 36, 52 ... photoelectron Intensifier tube.

Claims (1)

(57) [Claims] 1. An excitation / detection system which is mechanically scanned in a direction perpendicular to lanes for an electrophoresis gel in which a nucleic acid fragment sample labeled with a fluorescent substance is simultaneously electrophoresed in a plurality of lanes by gel electrophoresis. In a gel electrophoresis apparatus that detects fluorescence during electrophoresis, the excitation / detection system disperses light from an excitation light source according to wavelength, and uses the separated light as excitation light along the scanning direction on the electrophoresis gel. A gel electrophoresis apparatus comprising: an excitation optical system for irradiating a plurality of locations; and a fluorescence photometric unit for receiving fluorescence by each excitation light.