Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-12-11
2004-09-14
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06790651
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus and a method for performing a hybridization reaction between a DNA probe and a sample or for washing after the hybridization.
BACKGROUND ART
In the fields of molecular biology and biochemistry, biological polymers such as nucleic acids and proteins from organisms are identified and/or fractionated in order to search for useful genes or to diagnose diseases. A hybridization reaction is often used as a pretreatment for such processes, where a fluorescence-labeled target molecule in a sample is hybridized with a biological polymer (hereinafter, referred to as a “probe”) such as a nucleic acid or a protein having a known sequence. A DNA fragment or an RNA fragment is also used as a probe. According to a conventional hybridization reaction, probes are immobilized on a substrate such as a flat-surface rectangular glass plate, on which a hybridization reaction solution containing a target is dropped, which is then covered with a cover glass and left in a thermostat for a predetermined time. After the reaction, the substrate is taken out from the thermostat and left stand or shaken in a washing solution for an hour or longer to remove the non-reacting target from the surface of the substrate. The probes and the target are both biological substances, specifically DNA or RNA. Hybridization may take place between DNA and RNA. Alternatively, a target may be immobilized on a substrate to be subjected to a hybridization reaction with a fluorescence-labeled probe contained in a hybridization reaction solution.
According to a conventional hybridization reaction, the reaction requires very long time as over night (e.g., 15 hours or longer). In addition, the subsequent washing step needs to be performed after taking out the substrate from the thermostat, which renders the whole procedure troublesome and complicated.
In view of these facts relating to hybridization reaction, the present invention has an objective of providing a hybridization reaction apparatus which can shorten the reaction time by enhancing the reaction efficiency.
The present invention also has an objective of providing a hybridization reaction apparatus and a hybridization method, by which the hybridization reaction step and the subsequent washing step can be carried out continuously without the need of taking out the substrate from the apparatus, thereby simplifying the manipulation.
SUMMARY OF THE INVENTION
In order to achieve the above-mentioned objectives, the present invention uses an apparatus comprising: a case for accommodating a substrate on which biological substances such as DNAs are immobilized; a pump for injecting or discharging a reaction solution or a washing solution into or from the case; an agitating mechanism for the solution for enhancing the efficiency of the reaction; and a thermostat for controlling the temperature during the reaction. The substrate used for immobilizing the biological substances is a disc-shaped substrate having a throughhole at the center thereof. The reaction solution and the washing solution are agitated during hybridization reaction and subsequent washing, respectively, thereby shortening the reaction time and the washing time.
Thus, a hybridization reaction apparatus of the present invention is an apparatus for performing hybridization reaction between a biological substance immobilized on a substrate and a biological substance contained in a solution, the apparatus comprising: a case for accommodating the substrate immobilized with the biological substance; an injecting member for injecting a solution into the case; a discharging member for discharging the solution from the case; and an agitating member for agitating the solution injected into the case.
The substrate may be a circular substrate with a throughhole at the center, in which case, the agitating member may comprise a driver that rotates an agitator placed in the throughhole at the center of the substrate. The agitating member (as a shaker) may repeatedly tilt the case by moving the alternate ends of the case up and down. Alternatively, the agitating member may comprise an agitation fan attached to the case and a rotating member for rotating the agitation fan.
The case is detachable from the apparatus.
The injecting member for injecting the solution may comprise a solution selecting valve for selecting between the reaction solution and a washing solution, and an injection pump for sending the solution selected by the solution selecting valve to the case.
A hybridization method according to the present invention is a method for performing hybridization reaction between a biological substance immobilized on a substrate and a biological substance contained in a solution, the method comprising: injecting the solution containing the biological substance into a case accommodating the substrate immobilized with the biological substance; and allowing hybridization reaction while agitating the solution in the case.
Furthermore, a hybridization method according to the present invention is a method for performing hybridization reaction between a biological substance immobilized on a substrate and a biological substance contained in a solution, the method comprising: injecting the solution containing the biological substance and a liquid whose liquid density is lighter than the solution containing the biological substance and thus which does not mix with the solution into a case accommodating the substrate immobilized with the biological substance; and allowing hybridization reaction while agitating the solution containing the biological substance and the liquid in the case while keeping the solution phase and the liquid phase separated from each other. According to this method, the amount of the reaction solution injected in the case can be minimized.
REFERENCES:
patent: 4415270 (1983-11-01), Heinis et al.
patent: 5945334 (1999-08-01), Besemer et al.
Kijima Shigeru
Tachibana Mitsuhiro
Tanaka Toshiaki
A. Marquez, Esq. Juan Carlos
Fisher Esq. Stanley P.
Hitachi Software Engineering Co. Ltd.
Reed Smith LLP
Siew Jeffrey
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