Liquid purification or separation – With heater or heat exchanger
Reexamination Certificate
2000-09-05
2003-08-19
Reifsnyder, David A. (Department: 1723)
Liquid purification or separation
With heater or heat exchanger
C210S222000, C073S864130, C073S864160, C073S864170, C422S063000, C422S105000, C422S105000, C422S105000, C435S286400, C435S286700, C435S288200, C435S288400, C436S526000
Reexamination Certificate
active
06607662
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an apparatus for isolating and purifying nucleic acids such as DNA and RNA or proteins such as enzymes and antibodies from biological materials such as microorganisms, e.g., viruses and bacteria, animal and plant tissues.
BACKGROUND ART
In purification of nucleic acids and proteins, an objective substance alone is separated from contaminants by various methods. Among these methods, generally conducted in the art are insolubilizing contaminants other than the objective substance and utilizing the difference in molecular weights between the objective substance and other contaminants in this technical field. Specifically, the separation of the objective substance from other contaminants is carried out by centrifugation, filtration, gel chromatography, etc.
The devices which automate these operations are commercially available, but they are not totally capable of fast processing of a plurality of samples. One of the reasons for this is that the devices need to be equipped with a centrifuge and filter attaching and detaching mechanism, thereby increasing the size of the devices and the number of steps in purification.
To solve the above problems, a method has been proposed to separate nucleic acids from contaminants using particles which can be magnetically drawn. The details of this method and the composition of reagents are described in Japanese Patent No. 2680462. The method does not require centrifugation or filtration and allows purification of nucleic acids in a single container. The kits for purifying nucleic acids and proteins utilizing the principle of the above patent have already been put on the commercial market (“MagExtractor” produced by Toyobo co., Ltd., etc.).
The purification of nucleic acids or proteins using magnetically attractable particles can be generally carried out by repeating the procedure comprising the following steps.
(a) A solution containing an objective substance and contaminants, a reagent and magnetically attractable particles are placed into a specific container, followed by thorough mixing (stirring). For this step, the technique by which only the objective substance is specifically bonded to magnetically attractable particles has been established.
(b) A magnet is placed close to the outside of the container to make the magnetically attractable particles gather at a position inside the container and separate the particles from a supernatant (solid-liquid separation). In the course of purification, this supernatant is discarded and only the objective substance, which is bonded to magnetically attractable particles, is left in the container. Generally, the steps of adding another reagent, mixing the mixture, solid-liquid separation of the mixture, removing the supernatant are repeated for a few times.
(c) To composite of the objective substance and the magnetically attractable particles is added a reagent which detaches only the objective substance from the magnetically attractable particles, and the magnet is placed close to the container again to make the magnetically attractable particles gather at a position inside the container. Hence, only the supernatant in the container is recovered in a different container, whereby the objective substance can be purified.
The apparatuses for purifying nucleic acids or proteins which automate the above operations (a), (b) and (c) are commercially available (“MFX-2000” manufactured by TOYOBO Co., LTD., etc.). This MFX-2000 comprises a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end. A common piston pump transfers water with a piston moving back and forth in a cylinder. In the field of this technique, a minute amount of a reagent is dispensed (distributed) and mixed (stirred) together with samples by discharging and sucking up the liquid according to the principle similar to that of this pump. In the actual operation, a disposable tip is attached to the end of the piston pump, and the liquid is sucked up into and discharged from inside the tip only (see FIG.
1
). Such instrument is conventionally referred to as “pipetteman”. Further, the mixing or stirring by repeated sucking up a liquid into the tip and discharging the liquid using pipetteman is conventionally referred to as pipetting. Since pipetting is a piston action, it can be readily mechanized and applied to apparatuses for purifying nucleic acids or proteins.
In the above (a), mixing (stirring) is carried out by pipetting. Since the tip is disposable and attachable/detachable, it is convenient to avoid contamination between samples. In the above (b) and (c), solid-liquid separation is performed by discharging a liquid from inside the tip, stopping the piston pump, and placing the magnet close to the outside of the container. In this state, only the supernatant is sucked up. Thus, in the method for purifying nucleic acids or proteins using magnetically attractable particles, it is highly effective to use such mechanism equipped with a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end.
In this technical field, widely used are 96-hole plastic plates which comprises a series of holes in a grid pattern of 8 by 12 and are suitable for fast processing of a plurality of samples. Commercially available containers have the holes of a few &mgr;l to a few ml capacity. The 96-hole plate advantageously allows simultaneous handling of 96 samples in a container and can be carried with one hand. In this technical field, fast processing of a plurality of samples is substantially impossible without using the devices for sampling, extraction, purification and analysis compatible with the format of this container (mainly the intervals between the holes in lengthwise and widthwise directions). In general, the containers having holes with some hundred &mgr;l capacities are used for analysis. However, a container with holes of as great capacity as possible is ideal for extraction and purification in order to increase a recovery amount.
DISCLOSURE OF INVENTION
It is easily anticipated that the fast processing of a plurality of samples was enabled if the purification of nucleic acids or proteins using magnetically attractable particles can be performed using a 96 hole plate with the holes having as large capacity as possible. It is also considered that the automation of this processing could suitably use a mechanism comprising piston pumps equipped with nozzles capable of having disposable tips which are attachable/detachable at their ends. However, such automation entails the following problems.
I. Dispensing Method
As described in the above, the mixing (stirring) necessary for the purification of nucleic acids or proteins using magnetically attractable particles can be readily carried out by a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end. In conventional apparatuses, the operation of dispensing reagent necessary for purification is also conducted by a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end. This method is referred to as tip dispensing, and characteristically allows highly precise dispensing. In general, the purification of nucleic acids or proteins using magnetically attractable particles necessitates precise dispensing of reagents ranging from about several ten &mgr;l to about several hundred &mgr;l. For this reason, tip dispensing is considered suitable.
Reagents necessary for purification of nucleic acids or proteins using magnetically attractable particles are dispensed by tip dispensing comprising the following steps: placing a plurality of reagents necessary for purification in separate containers; moving a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end between a container, for example 96-hole plate used for purification and the reagent containers back and forth; and dispens
Ikeda Katsunori
Kawamura Yoshihisa
Kusumoto Masahiro
Nishiya Yoshiaki
Suzuki Masahiro
Kenyon & Kenyon
Reifsnyder David A.
Toyo Boseki Kabushiki Kaisha
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