Capillary array electrophoresis apparatus

Details Capillary array electrophoresis apparatus Capillary array electrophoresis apparatus

1999-10-25

2003-03-11

Warden, Jill (Department: 1743)

Chemistry: electrical and wave energy

Apparatus

Electrophoretic or electro-osmotic apparatus

C204S452000, C356S344000

Reexamination Certificate

active

06531044

ABSTRACT:

BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to an apparatus for separating and analyzing DNA, RNA and proteins, and in particular relates to a capillary array electrophoresis apparatus for determining the base sequences of DNA and RNA, and measuring the polymorphism of the base sequences of DNA based on the diversity of individual base sequences.
The analysis of DNA and RNA is becoming increasingly important in genetic analysis and medicine including genetic diagnosis and biology. In recent years, in particular, high-speed, high throughput DNA analyzers are being developed in connection with genome analysis plans.
In DNA analysis, a fluorephore-labeled sample is separated according to molecular weight by gel electrophoresis, and the fluorescence of the fluorephore label is detected. In gel electrophoresis, a flat plate gel formed by polymerizing acrylamide between two glass plates separated by an interval of approximately 0.3 mm is widely used (Biotechnology 6, 816 (1988)). A sample injected at the upper end of the flat plate gel is caused to move by electrophoresis towards the lower end while undergoing molecular separation due to a voltage applied between the two ends of the flat plate gel. A position which is electrophoretically at a certain distance is then irradiated by a laser which irradiates the whole electrophoresis path from the side face of the flat plate gel, and the separated components of the fluorephore-labeled sample passing through the laser irradiation part are excited. The fluorescence from the fluorephore-labeled sample is continuously and periodically measured at a fixed time interval. The results are analyzed to determine a DNA base sequence.
Recently, instead of a flat plate gel, a capillary gel, i.e., a polymerized gel in a fused quartz glass capillary tube, has come to be used. Capillary gel electrophoresis is attracting attention as a larger electric field can be applied than in slab gel electrophoresis, thereby permitting high-speed analysis (Analytical Chemistry 62, 900 (1990)). Normally, an on-column fluorescence detection measurement is performed wherein one capillary tube is used, and the vicinity of the lower end of the capillary is irradiated by a laser. The entire outer surface of the capillary has a polyimide coating, and the coating is removed at the position where fluorescence is to be detected so that the glass is exposed to form a window (U.S. Pat. No. 5,312,535). When this window position is irradiated by the laser, separated components of a fluorephore-labeled sample subject to electrophoresis in the capillary tube are excited when they pass through the beam, the fluorescence from the fluorephore-labeled sample is measured, and is then analyzed to determine the DNA base sequence.
However, in the aforesaid on-line column measuring apparatus, there were disadvantages such as considerable scattering of the laser beam on the outer surface of the capillary, moreover the capillary can be used only once and throughput could not be increased. Recently, there have been several reports of high throughput capillary array electrophoresis devices wherein plural capillaries are disposed in an array, and a large number of samples are simultaneously analyzed at high speed.
The first report is a capillary array scanning method (Nature, 359, 167 (1992)). Plural capillaries are irradiated in sequence one at a time, and on-column fluorescence detection is performed. The fluorescence detection positions of the plural capillaries are disposed horizontally in a plane, a laser beam converged by a lens from a perpendicular direction to the plane is irradiated to one capillary in the array, and the fluorescence is detected by a light collecting lens on the side of the laser beam light source. The part of the capillary array in the plane is moved back and forth in a perpendicular direction to the axis of the capillaries, and laser irradiation and fluorescence detection are sequentially performed for each capillary. The laser irradiation system and fluorescence receiving optical system are fixed. A construction is adopted with a common focal point where the position at which the laser beam is most converged in the capillaries and the position of the light source incident on the fluorescence measuring instrument coincide, and each capillary is measured independently.
The second report is a multiple sheath-flow method (Nature, 361, 565-566 (1993), Japanese Patent Laid-Open Hei 06-138037 (Koho)). The sample elution end of a capillary array disposed in a plane is vertically immersed in a buffer solution, sample components separated by gel electrophoresis are eluted from the capillaries into the buffer solution, a part where there are no capillaries is irradiated by a laser, and fluorescence is detected. An arrangement is adopted wherein the buffer solution is made to flow gradually in the electrophoresis direction so that separate components eluted from different capillary gels are not mixed together in the buffer solution, or two components separated in one capillary gel are not mixed in the buffer solution. A buffer solution part where there are no capillaries, in the vicinity of the outlet of the capillary array, is irradiated by the laser. Hence, the problem of scattering of the laser beam on the surface of the capillaries is avoided, components eluted from plural capillaries are excited together, and fluorescence detection is performed simultaneously. The fluorescence from all the electrophoresis lanes is detected in one operation by a two-dimensional camera in a perpendicular direction to the plane of the capillary array.
The third report is a laser beam expansion method (Analytical Chemistry, 66, 1424-1431 (1994)). Fluorescence detection parts of plural capillaries are arranged horizontally in a plane, and a laser beam is irradiated at an angle of 45° relative to the axis of the capillaries. The laser beam is enlarged in the perpendicular direction to the capillary axes by a cylindrical lens, and all the capillaries are irradiated simultaneously. Fluorescence from all the capillaries is detected in one operation by a two-dimensional camera in a perpendicular direction to the plane of the capillary array.
The fourth report is a multiple laser focusing method (Analytical Chemistry, 68, 2699-2704 (1996)). The fluorescence detecting parts of plural capillaries are arranged in a plane, and a laser beam is irradiated from the side of the plane so as to pass through the center of each capillary. As the laser beam is repeatedly converged by the converging action of each capillary and is not dispersed by the capillary array, all the capillaries can be irradiated simultaneously. Fluorescence from all the capillaries is detected in one operation by a two-dimensional camera in a perpendicular direction to the plane of the capillary array.
SUMMARY OF THE INVENTION
In on-column measurement wherein a capillary is directly irradiated by a laser beam and the resulting fluorescence is detected, the reflection of the laser beam from the inner surface and outer surface of the capillary which enters the fluorescence detecting system gives rise to a high level of background light. If a sufficiently converged laser beam is incident perpendicularly to the axis of the capillary and a fluorescence measurement is performed in a perpendicular direction to the plane formed by the laser beam and capillary axis, the intensity of the laser reflection incident on the fluorescence detecting system is of the order of 10
−3
for a laser reflection intensity of 1. If fluorescence detection is performed in the same direction as that of laser incidence, the laser reflection intensity incident on the fluorescence detecting system is of the order of 10
−2
. On the other hand, the fraction of laser scattering intensity due to the separating medium packed in the interior of capillary which is incident on the fluorescence detecting system is of the order of 10
−5
. In other words, the laser reflection intensity is two to three orders of magnitude larger th

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