JP2003520582A5 - - Google Patents

Description

[Claims]
1. a) Step of nucleic acid template purification:
b) a step of thermocycling reaction: and c) a step of purifying the product of step b), characterized in that the steps are carried out successively on a microfluidic disk, wherein
2. The method according to claim 1, wherein the flow of the fluid through the microfluidic disc can be performed by rotation of the disc.
3. The method according to claim 1, wherein the nucleic acid template is a plasmid.
4. The method according to claim 1, wherein the thermocycling reaction b) is a nucleic acid sequencing reaction.
5. Further:
d) a step of separating the purified product obtained in step c);
5. The method of claim 4, comprising:
6. The method according to claim 1, wherein step a) is carried out by passing the nucleic acid template through a purification column in a microstructure contained in a microfluidic disk.
7. The method according to claim 1, wherein step c) is carried out by passing through a gel filtration column in a microstructure contained in a microfluidic disk.
8. The method according to claim 5, wherein step d) is an electrophoretic separation of the products of a sequencing reaction.
9. a) treating the culture of cells containing the template nucleic acid with a lysis reagent to melt the cytoplasmic membrane;
b) placing the melt of step a) in microstructures of a microfluidic disc, wherein each microstructure comprises a first chamber containing means for purifying the template nucleic acid, a second chamber containing means for a thermocycling reaction And a third chamber comprising means for purifying the product of the thermocycling reaction: and c) a method of performing a nucleic acid sequencing reaction on a template nucleic acid, comprising recovering the purified product for analysis.
10. a) at least one inlet; a destination b) a first chamber containing means for purifying the template nucleic acid; a destination c) a second chamber containing means for a thermocycling reaction; a destination d. A) microstructure for a fluid, characterized in that it comprises a third chamber containing means for purifying the product of the thermocycling reaction.
11. Further:
11. The microstructure for a fluid of claim 10 , comprising e) a fourth chamber including means for applying an electrical potential through a separation matrix connected to the third chamber.
12. An apparatus for performing a thermocycling reaction of a template nucleic acid, comprising a microfluidic disc, wherein the disc comprises a plurality of radially extending microstructures for a fluid according to claim 10 or 11. .

Then, a plasmid quality was evaluated using agarose gel electrophoresis, the amount can be determined spectrophotometrically, both techniques well known to those skilled in the art. It is advantageous to obtain the plasmid in water or in a dilution buffer compatible with the next step (ie a direct sequencing reaction such as PCR or cycle sequencing).

A method for isolating DNA in the presence of a chaotrope on a silicon structure using a micromachine has been published (Christel, LA, K. Petersen, et al. (1999). “Rapid, automated nucleic acid probe assays using silicon microstructures”). for nucleic acid concentration. "J Biomech Eng 121 (1): 22-7). U.S. Patent No. 5,882,496 describes a heated reaction chamber, an electrophoresis device and a thermopneumatic sensor-actuator, a chemical pre-concentrate, and a porosity to increase the surface area of a filtration or controlled flow device. Describes the fabrication and use of silicon structures. In particular, such high surface areas or specific pore size porous silicon structures can significantly increase adsorption, evaporation, desorption, condensation and liquid and gas flow in applications using such processes on a small scale. Useful to let.

Microanalytical systems based on microchannels formed from rotatable, usually plastic discs, are often referred to as "centrifuge rotors", "lab on a chip" or "CD devices". Called. Such discs may be used to perform analysis and separation of small amounts of fluid. The principle of moving a liquid through the channels of a plastic disc for the purpose of performing an enzyme assay is described, for example, in Duffy, DC, HL Gillis, et al. (1999). “Microfabricated centrifugal microfluidic systems: characterizatoin and multiple enzymatic assays.” Analytical Chemistry 71 (20): 4669-4678. One type of suitable plastic disc is what is called a compact disc or CD.

The present invention relates to an apparatus for the integration of the steps of template isolation, cycle sequencing and cleanup into a CD device, and to the use of this apparatus. In particular, the present invention enables the handling of large numbers of samples, therefore, greatly simplifying the automation, consumption and thus reducing the overall cost of the reagents, makes reducing the size of required apparatus, and a sealing device. To date, there has been no report of a similar integrated level of equipment that allows the isolation of DNA-templated bacterial colonies via the acquisition of a cleaned-up sequencing reaction within one encapsulated structure.

In a preferred embodiment of the first aspect, the flow of the fluid through the microfluidic disc can be effected by rotation of the disc. The disc from about 250 rpm (low speed) ∎ You can at 15,000rpm rotated at various speeds in the range of (Fast) (or rotation). The actual speed required to achieve accurate flow of fluid through the disc at any particular stage of the method is:
The position of the structure on the microfluidic disk (ie, the further the structure is from the center of the disk, the lower the rpm required to achieve the same centrifugal force as the structure is at the center of the disk);
The physical dimensions of the structure through which the liquid must pass;
It depends on a number of factors, including the viscosity of the liquid; and the chemical and physical properties of the surface in the structure.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described by way of non-limiting examples of embodiments by means of the accompanying drawings, in which:
FIG. 1a shows a schematic diagram (partially shown) of a fluid microstructure plan of the invention showing its orientation in a microfluidic disc;
FIG. 1b shows a schematic diagram of one microstructure plan for the fluid of the present invention;
FIG. 1c shows an enlarged view of the microchannel discard control structure (34) of FIG. 1b, where a shows the path of the liquid when the microfluidic disc rotates at a low speed and b shows the microfluidic disc at a high speed. Shows the path of the liquid when rotated at;
FIG. 1d shows the region (36) between the sample preparation microchannel structure of the microstructure shown in FIG. 1b (i.e., (13)-(22)) and the electrophoretic structure ((23)-(28)). ) Shows an enlarged view;
FIG. 1e shows another embodiment of a microstructure for a fluid of the invention disposed on a microfluidic disk;
FIG. 2 shows two possible configurations of a well on a microfluidic disk of the present invention.

Hydrophobic brake, for example, by marking with an overhead pen (permanent ink) (Snowman pen, Japan), can be introduced into the microphone Rochaneru structure. The purpose of the hydrophobic brake is to prevent capillary action, which directs fluid in undesirable directions. The hydrophobic brake can be broken by centrifugal force, that is, by rotating the disc at high speed.

FIG. 1a also shows the well (12) positioned towards the outer edge (4) of the microfluidic disc. This well can be used for sample preparation before addition to the sample inlet (5). For example, if the nucleic acid template used is derived from a bacterial colony, the bacterial colony is first removed, for example, by a pipetting robot, and suspended from the surface of the solid liquid medium in about 10 μl of isotonic solution. The suspension is then placed in the wells (12) of a microfluidic disk. Bacterial cells can then be pelleted by spinning the microfluidic disc and the supernatant decanted. The pellet is then resuspended in solution I, followed by rotation and successive resuspension in solutions II and III. The precipitated genomic DNA and protein are pelleted by spinning, and the plasmid-containing supernatant is further processed (see below).

In another embodiment of the inventive microstructure of FIG. 1e, the electrophoretic structures and channels (26) described in FIG. 1b as chambers (23)-(25), (27) and (28) are missing. In this embodiment, the purified product of the thermocycling reaction (ie, the eluate from chamber (16)) is retained in well (29) for transfer to another electrophoresis device. In this embodiment, the product is obtained in about a sub-microliter volume , which can be diluted by the addition of a liquid (eg, formamide or water) to transfer to another structure for further analysis.

13. The reaction mixture is drained from the thermocycling chamber by centrifugal force and through a gel filtration chamber. The gel filtration chamber contains monodisperse (sieved) Sephadex G-50 DNA grade beads captured between deep and shallow sections in the microstructure. Non-inclusive terminators and also salts are retained. The remaining sequence ladder follows the final "pick-up" well, adding more water, if necessary, to aid liquid handling and reduce evaporation.
14． The clean-up reaction is removed from the pickup well by a pipetting robot, placed in a microtiter plate, and diluted to 5-10 μl for further processing by MegaBACE.

[Brief description of the drawings]
FIG. 1a shows a schematic representation (partially shown) of a plan of the fluid microstructure of the invention showing its orientation in the microfluidic disc;
FIG. 1b shows a schematic diagram of one microstructure plan for the fluid of the present invention;
FIG. 1c shows an enlarged view of the microchannel discard control structure (34) of FIG. 1b, where a shows the path of the liquid when the microfluidic disc rotates at low speed and b shows the microfluidic disc at high speed. Shows the path of the liquid when rotated at;
FIG. 1d shows the region (36) between the sample preparation microchannel structure of the microstructure shown in FIG. 1b (ie, part (13)-(22)) and the electrophoretic structure (part (23)-(28)). ) Shows an enlarged view;
FIG. 1e illustrates another embodiment of a microstructure for a fluid of the present invention disposed on a microfluidic disk;
FIG. 2 shows two possible configurations of a well on a microfluidic disk of the present invention.