Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
1999-11-19
2002-10-08
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S451000, C204S453000, C204S455000, C204S601000, C204S604000, C204S605000
Reexamination Certificate
active
06461492
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a capillary electrophoretic apparatus for separating and analyzing a biopolymer such as protein or nucleic acid.
Such a capillary electrophoretic apparatus is applied to sequence determination for DNA. The capillary electrophoretic apparatus for DNA sequence determination electrophoreses a DNA fragment sample prepared by labeling a primer or a terminator with a fluorochrome and detects fluorescence from the DNA fragment sample during electrophoresis for determining the base sequence.
2. Description of the Prior Art
A DNA sequencer having high sensitivity, a high speed and high throughput is necessary for sequence determination for DNA such as a human genome having long base sequence. As an example, capillary electrophoresis employing a capillary column charged with a gel in place of slab gel electrophoresis employing a flat plate type slab gel is proposed. With such a capillary column, a sample can not only be readily handled or injected but also electrophoresed at a high speed and detected in high sensitivity as compared with the slab gel. If a high voltage is applied to the slab gel, a band is spread or a temperature gradient is caused due to influence by Joulean heat. However, the capillary column hardly causes such a problem and can perform detection in high sensitivity with small band spreading even if performing high-speed electrophoresis with application of a high voltage.
A multi-capillary electrophoretic apparatus prepared by arranging a plurality of capillary columns is also proposed.
An automatic DNA sequencer utilizes a fluorochrome for identifying the four types of bases forming DNA. A Rhodamine derivative such as R6G, R-110 or ROX or a fluorescein derivative such as FAM is utilized as the fluorochrome. An argon ion laser unit having dominant wavelengths of 488.0 nm and 514.5 nm is utilized as a laser beam source.
However, both of the wavelengths of 488.0 nm and 514.5 nm are separate from the absorption maximum wavelengths of the fluorescein derivatives and the Rhodamine derivatives. While fluorescein derivatives having an absorption maximum wavelength of 493.5 nm are not much reduced in efficiency, the Rhodamine derivatives, which are excited at 514.5 nm although its absorption maximum wavelength is 550 nm, has inferior efficiency.
In order to solve this problem, there has been made an attempt (referred to as an energy transfer method) of introducing both of the fluorescein derivative and the Rhodamine derivative into the same molecule when using the Rhodamine derivative as a label thereby improving the efficiency of the Rhodamine derivative through the principle of energy transfer.
The energy transfer method is superior to general methods, but yet has the following problems:
1. It is technically difficult to introduce a plurality of fluorochromes into the same molecule, and the cost is increased following this difficulty.
2. Extreme influence is exerted by Raman scattering since the excitation wavelength is in the visible region. When excited at 488 nm, a Raman scattering line of water around 516 nm forms background noise of a channel detecting the fluorescein derivative having a fluorescence maximum at 510 nm to reduce an S-N (signal-to-noise) ratio.
3. Influence is exerted by Rayleigh scattering to readily reduce the S-N ratio.
Considering a multi-capillary electrophoretic apparatus in which a plurality of capillary columns are so arranged that a plurality of samples are injected into the capillary columns and simultaneously electrophoresed in all capillary columns, one ends of the plurality of capillary columns defining a sample injection side are two-dimensionally arranged and fixed by a sample injection side holder while the other ends defining a detection side are aligned with each other on a plane and fixed by a detection side holder for forming a capillary array. The detection side holder is provided with a slot along the arrangement of the capillary columns, and parts of the capillary columns exposed through the slot define a part to be detected. When separating and detecting a sample containing a DNA fragment labeled in four types with a fluorescent material, excitation light is applied to the part to be detected for detecting fluorescence generated from sample components electrophoresed to the part to be detected thereby identifying the sample components.
The prior art employs an epi-optical system comprising a condenser lens condensing and projecting excitation light onto each capillary column on the part to be detected and receiving the fluorescence generated from the sample electrophoresed in the capillary column as an objective lens for projecting the excitation light and receiving the fluorescence through the same lens as an excitation-light receiving optical system. The objective lens is scanned along a straight line parallel to the plane of arrangement of the capillary columns on the part to be detected and perpendicular to the electrophoresis direction, thereby detecting the fluorescence as to all capillary columns.
In such an optical system, the objective lens is preferably arranged in proximity to the part to be detected for collecting the maximum amount of fluorescence in consideration of detection sensitivity. Therefore, the condenser lens having a short focal length is employed as the objective lens.
When employing the condenser lens having a short focal length as the objective lens, the amount of collected fluorescence is reduced to reduce the detection sensitivity if the position of the part to be detected of the capillary array slightly deviates in the direction of application of the excitation light Therefore, high working accuracy is required when preparing the detection side holder and fixing the capillary columns to the detection side holder.
In the multi-capillary electrophoretic apparatus, the capillary columns are fixed to cassette holders on a sample introduction side and the detection side. The cassette holders two-dimensionally arrange the capillary columns on the sample introduction side and planarly align the same with each other on the detection side.
When charging each capillary column with a polymer, one end of the capillary column is stuck into and fixed to an elastic member such as a rubber stopper or fixed to a dedicated holder for polymer charging with an adhesive for filling up a clearance. The polymer is charged into the capillary column by fixing the elastic member or the dedicated holder to a vessel storing the polymer so that the end of the capillary column is dipped in the polymer, sealing the vessel and pressurizing the vessel with a pump for press-filling the polymer into the capillary column, or by connecting the elastic member or the dedicated holder to a pump, dipping another end of the capillary column into the polymer and decompressing the capillary column with the pump for inhaling the polymer into the capillary column.
When charging the polymer into the capillary column by press-filling or inhaling in the method of sticking and fixing the capillary column into and to the elastic member, pressure resistance of the elastic member may be so insufficient that the polymer cannot be smoothly charged into the capillary column. On the other hand, in the method of fixing the capillary column to the dedicated holder for polymer charging with an adhesive, it may be impossible to smoothly charge the polymer into the capillary column due to insufficient supply of the adhesive, to result in an inferior manufacturing yield.
SUMMARY OF THE INVENTION
A first objective of the present invention is to perform efficient detection in a capillary electrophoretic apparatus.
A second objective of the present invention is to provide a capillary cassette capable of reliably charging all capillary columns with polymers with a high yield in a multi-capillary electrophoretic apparatus.
A first aspect of the present invention for performing efficient detection comprises detection means exciting a fluorochrome bonded to a sample component as a lab
Fujiwake Hideshi
Hayashizaki Yoshihide
Nakamura Shin
Brown Jennine
Rader & Fishman & Grauer, PLLC
The Institute of Physical and Chemical Research
Warden Jill
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