JP2003520582A - Integrated microfluidic disc - Google Patents

Abstract

Disclosed is a method for performing the steps of nucleic acid template purification, a thermocycling reaction and purification of the products of the thermocycling reaction characterised in that the steps take place sequentially in a microfluidic disc. Also disclosed is a microstructure for fluids comprising at least one inlet opening connected to a first chamber incorporating a means for purifying template nucleic acid which, in turn, is connected to a second chamber incorporating a means for a thermocycling reaction which, in turn, is connected to a third chamber incorporating a means for purifying products of the thermocycling reaction, and a microfluidic disc comprising a plurality of such microstructures.

Description

Detailed Description of the Invention

The present invention relates to a microfabricated device having a rotatable disc, in particular a microfluidic disc containing microstructures for fluids, wherein the steps required for nucleic acid sequencing are integrated and continuous. It is done in a traditional way.

[0002] The sequencing process has reached industrial scale by applying, among other things, automation in the form of robots and utilizing minute volumes of liquids in complex states. This process involves disrupting the genome, inserting the fragment of interest into a clone vector, isolating individual clones, purifying the vector containing the inserted fragment, and removing the inserted fragment. And as a template in a sequencing reaction. The sequencing data obtained is sequenced using software to obtain a continuous sequence from numerous fragments. This process is described in more detail below.

Depending on the size of the fragment, different cloning vectors can be used to clone the fragment. The purpose of cloning is the biological system (
Bacterium or virus) to replicate the insert sequence to give a vast number of replicates. Huge fragments are often called BAC (Bacterial Art).
ificial Chromosomes: cloned into a bacterial artificial chromosome) or cosmid.
The smaller fragment is generally cloned either in a bacterial plasmid such as pUC18 or in phage M13.

The usual process for de novo sequencing a genome with a fragment cloned in a plasmid has a number of steps that can occur sequentially. Specific examples of these stages are described extensively below.

1) Preparation of Bacterial Culture A fragment of the genome of interest is formed and inserted into a plasmid (eg pUC18) carried within a strain of E. coli bacteria. This process is called transformation.

Transformed bacteria (carrying a gene that confers antibiotic resistance to the host bacterium)
To select for these bacteria containing the plasmid, they are spread on agar plates containing growth medium and antibiotics. The agar plate may include not only clones containing "empty" or plasmids lacking the insert, but also indicators that clearly indicate the presence of bacteria carrying the plasmid containing the insert. The bacterial culture is diluted before the individual bacterial cells, and daughter colonies, are spread out on the plate sufficiently far apart from each other. This removes individual colonies containing clones consisting of only one sequence.

The plate is incubated overnight at 37 ° C. Individual bacterial cells form non-overlapping cell colonies on the plate.

Colonies are removed either manually or robotically and used to prepare plasmids directly, or more usually overnight liquid culture (usually 1-2 ml).
Are inoculated to obtain higher numbers of bacteria and higher numbers of insert sequence replicas.

2. Extraction of plasmids containing inserts The quality of the nucleic acid template is a key factor in the success of the sequencing reaction. This template may be a plasmid or a polymerase chain reaction (PCR) product obtained from the plasmid. There have been reports of direct sequencing of bacterial extracts (see, for example, Frothingham, R. and RL Allen (1
991), Chen Q., and C. Neville (1996) et al. “Rapid 16S ribosomal DNA seque.
ncing from a single colony without DNA extraction or purification ”, Bio
See technique 11 (1) et al. Most major sequencing machines pay great attention to extracting pure plasmids so that they can be sequenced successfully.

Numerous methods have been developed for extracting and purifying plasmids. One general method is (i) thawing bacterial cells using NaOH, (ii) precipitating protein and chromosomal DNA, (iii) in the presence of a chaotrope such as guanidine isothiocyanate or sodium iodide. Extract the plasmid in solution with a glass substrate (or other purification column that selectively retains the nucleic acid to be extracted by adsorption and absorption), wash with (iv) an ethanol solution to remove salts and residual contaminants, (v) Finally, extract the plasmid from the substrate with low ionic strength buffer or water.
The process also includes the step of exposing the RNA to RNA degradation.

This method is based on the GFX microplasmid preparation kit (Amersham Pharmacia Biotech
) Is the basis of many commercial kits such as. These kits usually have a series of solution I, solution II, solution III, which is approximately 100 mM Tris-HCl pH 7.5,
At 10 mM EDTA, 400 μg / mL RNase I, Solution II has approximately 100 mM NaOH, 1% w / v SDS and Solution III has a buffer solution containing acetate and chaotrope.

Modifying the glass surface within a glass substrate is described, for example, in US Pat. No. 5,606,
No. 046. In the presence of PEG and salt, magnetic beads (Magenet
ic beads) for reversible and unspecific binding, see, for example, Hawkins, TL and T. O'Conneor-Morin (1994), “DNA purification a.
nd isolation using a solid-phase ”, Nucleic Acids Res 22 (21): 4543-4, or triplex-mediated affinity capture (US Pat. No. 5,591,841). Suitable purification materials include gels, resins, membranes, glass, or other surfaces that selectively retain nucleic acids.

The plasmid quality can then be evaluated using agarose gel electrophoresis and the flow 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 that is compatible with the next step (ie direct sequencing reactions such as PCR or cycle sequencing).

If the yield (or perhaps quality) of the plasmid is insufficient for direct sequencing, the specific region of the plasmid that covers the insert and flanking sequences can be amplified by conventional polymerase chain reaction (PCR). , Get the sequencing template. This PCR product must be "cleaned up" before it can be used as a template for sequencing. Cleanup involves the removal of non-inserted nucleotides and primers that would otherwise interfere with the cycle sequencing reaction. One method involves exposure to exonuclease III and shrimp alkaline phosphatase, inactivation of these enzymes by heat denaturation and direct use in cycle sequencing of the reaction.

3) Cycle Sequencing The cycle sequencing reaction consists of a template nucleic acid, a sequencing primer, a thermostable DNA polymerase enzyme and a small amount of dideoxy (chain termination).
4 deoxynucleotides containing one base in the form (dATP, dCTP, dG
TP and dTT) mixing, followed by a heating and cooling cycle (ie, thermal cycling). The reaction is carried out in four different tubes-each containing a small amount of each dideoxynucleotide together with a fluorescently labeled primer, or in one tube through the use of a dideoxynucleotide with a different fluorescently labeled and unlabeled primer dideoxynucleotides. . The result is a fluorescently labeled ladder of nucleic acid strands that is complementary to the sequence of the template strand. Cycle sequencing reactions are generally performed on a 10-20 μl scale in microtiter plates (96 or 384 wells) in a thermocycler.

4) Cleanup If a labeled nucleotide terminator is used, the reaction mixture must then be "cleaned up" to remove non-inserted fluorescent nucleotides. These would otherwise be visible in the electrophoretic separation of the sequencing ladder,
Reduce the quality of results.

When using a capillary electrophoresis device, it is also necessary to remove salts from the sequencing ladder to facilitate electrokinetic injection. This clean-up is generally done by precipitation by addition of ethanol and salt or by gel filtration. For primer-labeling reactions, desalting is only necessary when using capillary electrophoresis.

5) Analysis of Sequencing Reaction The stopped sequencing reaction was then separated into slab gels (for slab analysis).
For example, as used in ABI PRISM 377 or ALFexpress), or a capillary column (e.g. MegaBACE (Amersham Pharmacia Biotech) or PE ABI.
Separation on a denaturing gel, which can be 3700 (PE Biosystems).

Recently, automation of these steps has been described with emphasis on microfabrication, ie performing these steps in as little as possible.

Most automation is aimed at the use of robots to perform the steps normally performed manually using pipettes and microtiter plates (96 and 384 wells). The individual steps are performed on separate plates and the transfer of liquid between the plate and the format is performed using a pipetting robot.
Recently, despite many robots, attempts have been made to integrate various stages into one device. One example is Trevor Hawkins (Whitehead Institute
) Was developed by Sequatron. This includes robots for purifying M13 and performing sequencing reactions with ultra-thin slab gels or preparations for capillaries. Methods are based on solid phase isolation of DNA (eg, Hawkins, TL, T
. O'Connor-Morin, et al. (1994). “DNA purification and isolation using
a solid-phase ”. See Nucleic Acids Res 22 (21): 4543-4), 2 per 24 hours.
Allows throughput of over 5,000 samples. In addition, Washington
The University group is also developing a robot for the collection of large robots and multititer plate-based M13 plaques and template preparations.

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). “Ra
pid, automated nucleic acid probe assays using silicon microstructures f
or nucleic acid concentration. ”J Biomech Eng 121 (1): 22-7). US Pat. No. 5,882,496 describes a heated reaction chamber, an electrophoretic device and a thermopneumatic sensor-actuator, pre-chemistry. It describes the fabrication and use of concentrates and porous silicon structures to increase the surface area of bulk or controlled flow devices, in particular such high surface areas or specific pore size porous silicon structures are described. , In applications using such a process on a small scale, it is useful for adsorbing, evaporating, desorbing, condensing and for clearly increasing the flow of liquids and gases.

A direct sequencing method for plasmids from a single bacterial colony in a fused silica capillary has been developed (Zhang, Y., H. Tan, et al. (1999). “M.
ultiplexed automated DNA sequencing directly from single bacterial colon
ies. ”Analytical Chemistry 71 (22): 5018-25). Another method involves separation on glass chips, including detection methods based on laser-excited confocal microscopy (eg Kheterpal, I. and RA Mathies ( 1999). “Capillary array electrophores
is DNA sequencing. ”See Analytical Chemistry 71 (1): 31A-37A).

US Pat. No. 5,610,074 describes a centrifugal rotor for the isolation of substances in successive stages from a mixture of substances dissolved, suspended or dispersed in the same liquid. There is. Multiple samples are processed simultaneously by means of multiple fractionation cells, each containing a series of interconnected, chambered and vented compartments, each of which undergoes individual steps of the partitioning and isolation process. In this centrifugal rotor,
The specific compartment filled with the sample liquid or one of its fractions at any stage of the process is controlled by a combination of both the speed and direction of rotation of the rotor and gravity. The interconnects, chambers and passages in each compartment are sized and angled so that a predetermined amount of sample and reagent liquid does not overflow the compartments. However, such rotors are relatively bulky, require relatively large volumes of solution, and are complicated to manufacture.

Microchannel-based microanalysis systems formed from rotatable, usually plastic, disks often refer to "centrifugal rotors,""lab on a chip," or "CD devices." Often called. Such discs can be used to perform analysis and separation of small volumes of fluid. The principle of moving a liquid through the channels of a plastic disk for the purpose of performing an enzyme assay is described, for example, by Duffy, DC, HL Gillis, et al. (1999). “Microfabr
icated centrifugal microfluidic systems: characterizatoin and multiple e
nzymatic assays. ”Analytical Chemistry 71 (20): 4669-4678. One type of suitable plastic disc is the so-called compact disc or CD.

When such a disc is rotated, centrifugal force is directed toward the center of the disc. If there is fluid in the disc, this centrifugal force may be the result of surface tension, tension and capillary forces. Movement of the fluid outwards of the disc is usually accomplished by counteracting the centrifugal force by increasing the rotational speed of the disc.

To reduce costs, the disc should not be limited to use with one type of reagent or fluid, but should be able to work with different fluids. Furthermore,
During sample preparation, it is often desirable to be able to evacuate the exact volume of fluid or any combination of samples without modifying the disc. Due to the narrow width of the microchannel, the air bubbles present between the two fluid samples of the microchannel may act as a separation barrier, or block the microchannel, thereby permitting the fluid to enter the microchannel. Prevent you from entering. To solve this problem, US Pat. No. 5,591,643
The publication teaches the use of a centrifugal rotor containing microchannels having a cross-sectional area large enough to allow undesired air to be pumped out simultaneously as fluid enters the microchannels.

The present invention relates to an apparatus for template isolation, cycle sequencing and integration of cleanup steps into a CD device, and to the use of this apparatus. In particular, the present invention relates to a single closed device that allows handling of large numbers of samples, thus greatly simplifying automation, reducing reagent consumption and thus total cost, and allowing the required equipment size to be elemental. To date, through the acquisition of cleaned-up sequencing reactions within one encapsulated structure,
There are no reports of similar integrated level devices that allow isolation of DNA-templated bacterial colonies.

Therefore, according to a first aspect of the invention: a) a step of nucleic acid template purification: b) a step of a thermocycling reaction: and c) a step of the product purification of step b), wherein the step is a microfluidic Provided is a method of performing a series of steps, characterized in that they are performed sequentially on a disc.

Suitably the nucleic acid template is plasmid DNA, although genomic eukaryotic or prokaryotic derived templates may also be used. Other nucleic acid templates can be used with Bacterial Artificial Chromos using suitable extraction and purification protocols known to those of skill in the art.
It can be purified from omes (BAC) or M13.

In another embodiment, the thermocycling reaction is a direct simple bacterial extract,
It can be done without prior isolation of the plasmid.

In a preferred embodiment of the first aspect, the desired flow through the microfluidic disc can be achieved by rotation of the disc. About 250 rpm (
It spins (or spins) at various speeds ranging from low speed to 15,000 rpm (high speed). The actual velocities required to achieve an accurate flow of fluid through the disc at any particular stage of the method are: -the position of the structure on the microfluidic disc (ie the farther the structure is from the center of the disc) , Low rpm required to achieve the same centrifugal force as the structure is in the center of the disk);-the physical dimensions of the structure through which the liquid should pass; -the viscosity of the liquid; and-the surface chemistry in the structure. It depends on many factors, including physical and physical properties.

In a particularly preferred embodiment of the first aspect, the thermocycling reaction, step b), is a nucleic acid sequencing reaction or a cycle sequencing reaction. Other preferred thermocycling reactions include the polymerase chain reaction (PCR).

In another embodiment of the first aspect, the method further comprises: d) a step of separating the purified product obtained in step c); preferably step d) is the product of the sequencing reaction. Electrophoretic separation. Preferably, the separation step d) is also carried out on the same microfluidic disc as steps a) -c).

In a particularly preferred embodiment step a) is carried out by passing the nucleic acid template through a purification column in the microstructure contained in the microfluidic disc, step c).
) Is carried out by passing the product of step b) through a gel filtration column on the microstructure contained in the microfluidic disc.

In one embodiment of the first aspect: a) treating the culture of cells containing the template nucleic acid with a lysis reagent to thaw the cytoplasmic membrane; b) bringing the lysate of step a) into a microfluidic disc A structure, wherein each microstructure comprises a first chamber containing means for purifying the template nucleic acid, a second chamber containing means for the thermocycling reaction, and means for purifying the product of the thermocycling reaction. A third chamber comprising: c) providing a method for performing a nucleic acid sequencing reaction on a template nucleic acid, comprising recovering the purified product for analysis.

In one embodiment, the purified product obtained after purification in the third chamber is loaded onto a capillary electrophoresis DNA sequencer (such as MegaBACE® (Amersham Pharmacia Biotech) to obtain template sequence data. Can be transferred. In another embodiment, the purified products are analyzed in a circular device directly attached to a "sample preparation" microfluidic disc.

In another embodiment, the eluate from the first chamber is first directed to the volume-defining structure to ensure the correct transfer of the correct amount of liquid to the second chamber containing the means of the thermocycling reaction.

In a second aspect of the invention: a) at least one inlet; destination b) first chamber containing means for purifying the template nucleic acid; destination c) comprising means for thermocycling reaction. A second chamber; a connection d) a microstructure for a fluid, characterized in that it comprises a third chamber containing means for purifying the product of the thermocycling reaction.

In a preferred embodiment of the second aspect, the microstructure for fluid further comprises: e) a fourth chamber comprising means for applying an electric potential through the separation matrix connected to the third chamber.

In this aspect of the invention, the electrophoretic separation of the sequencing ladder is performed on the same microfluidic disc or CD. Separation proceeds from the outer end of the circular device to the inner center where one detector can detect the passed band (eg, Shi, Y., PC Simpson, et al. (1999). “Radial capillary array. ele
ctrophoresis microplate and scanner for high-performance nucleic acid an
alysis. ”Analytical Chemistry 71 (23): 5354-61), which allows for further reductions in the scale of sample preparation and a marked increase in overall process tightness and automation.

Suitably, the chambers and channels containing the microstructures are deep in the range of about 10-500 microns.

In another embodiment of the second aspect, the fluid microstructure further comprises a plurality of inlets and waste outlets arranged to allow insertion of samples and reagents and exit of waste.

In a particularly preferred embodiment, the waste outlet can be connected to a vacuum pump for effective exit of the waste.

In a third aspect of the present invention there is provided a device for carrying out a thermocycling reaction of a template nucleic acid, said device comprising a microfluidic disc, said disc for fluid according to the second aspect. It includes a plurality of radially extending microstructures.

In one embodiment of the third aspect, many microstructures are included in one microfluidic disc. In a preferred embodiment, the number of microstructures is 1-1000, preferably 80-100, arranged radially on one disc. In a particularly preferred embodiment, the 96 microstructures are placed on one disc to make a disc device compatible with the 96-well assay format. Suitably, the device is in the form of a 12 cm compact disc.

In another embodiment of the third aspect, the device may further comprise a plurality of wells suitable for bacterial culture or initial nucleic acid template preparation on the same disc.
It has the advantage of avoiding the need for a format change between the microtiter plate and the microfluidic disc.

In a preferred embodiment, the entrances of the microstructures on the microfluidic disc are connected to centrally distributed channels, such as a common annular channel, while at the same time the center of the reagent to all microstructures on the disc. Enables a distribution that is concentrated in. Preferably,
The waste channel is connected to a common annular waste channel, which in one embodiment is toward the outer end of the disc. Also preferred are multiple vents in the microstructure that allow liquid flow through the integrated structure.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is illustrated by means of non-limiting examples of embodiments by the means of the accompanying drawings, in which: Figure 1a shows its orientation in a microfluidic disc. Fig. 1b shows a schematic diagram of a microstructure plan of (partially shown); Fig. 1b shows a schematic diagram of one microstructure plan for a fluid of the present invention; Shows an enlarged view of the control structure (34),
Where a shows the liquid path when the microfluidic disk rotates at low speed, and b shows the liquid path when the microfluidic disk rotates at high speed; FIG. 1d shows the microstructure shown in FIG. 1b. FIG. 1e shows an enlarged view of the region (36) between the sample preparation microchannel structure (ie the portion of (13)-(22)) and the electrophoretic structure (the portion of (23)-(28)); FIG. 2 shows another embodiment of a microstructure for a fluid of the invention arranged on a microfluidic disc; FIG. 2 shows two possible structures of wells on the microfluidic disc of the invention.

Detailed Description of the Embodiments Illustrating the Invention One example of a microstructure (1) for a fluid of the invention is shown in part in FIG. 1a, arranged on a microfluidic disc (2). Show. Microstructures for fluids (1) are microfluidic discs
Located radially on the disk with the inlet (5) closest to the central hole of (3). The outer edge of the microfluidic disc is shown (4). Microstructures for fluids include a series of dedicated microchannels. The microfluidic disc (2) has a thickness much smaller than its diameter and is considered to rotate around the central hole (3), whereby centrifugal forces are placed in the microchannels of the discner. Fluid is directed towards the outer edge or end (4) of the disc. The flow is driven by both capillary action, pressure and centrifugal force, ie rotation of the disc. Hydrophobic brakes can be used for flow control, as described below.

Suitably, the microfluidic disc is a one or two piece cast construct, optionally formed from a transparent or polymeric material, by means of separate moldings, which are assembled together (eg by heating). Providing a closed structure with an inlet at a defined location, allowing the device to be filled with liquid and the liquid sample removed. The preferred plastics or polymeric materials preferably have low autofluorescence and are selected from polystyrene and polycarbonate. Alternatively, the surface of the microchannels may be further modified selectively by chemical or physical means to alter the surface properties and create hydrophobic or hydrophilic local regions within the microchannels to achieve the desired properties. Give. Preferred plastics are selected from polymers with a charged surface, preferably chemically or ion-plasma treated polystyrene, polycarbonate or other rigid transparent polymers.

Microchannels are micromachined on the surface of a disk,
It may be formed by a micromachining method in which a cover plate, for example a plastic film, is attached to the surface and the channels are closed.

A hydrophobic brake may be used, for example, with an overhead pen (permanent ink) (Sno
wman pen, Japan) can be used to introduce the structure into myk channel. The purpose of the hydrophobic brakes is to prevent capillary action, which induces fluid in an undesired direction. The hydrophobic brake can be broken by centrifugal force, that is, by rotating the disc at high speed.

In FIG. 1 a, arrows indicate the flow of fluid and / or air from the inlet and outlet in the microstructure. These openings will be described in more detail below.

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

FIG. 1b is a more detailed view of the microstructure for fluid (1). The microstructures may be used as application areas for reagents and samples, respectively, inlets (5), (8) and (9), outlets (6).
And (11), including vents (10) and openings that can serve as both inlets and vents (7). The ventilation holes open to the outside air through the top surface of the disc,
Prevents fluid in the microchannels from being sucked back into the structure.

[0056]   Suitably, the inlet and outlet may be associated with an annular distribution channel.   Suitably, the waste outlet can be connected to a vacuum to facilitate removal of waste.

The microstructure further comprises a series of chambers. The movement of liquids through microchannels and chambers is described below using microfluidic disks.

Prior to sample addition, two purification columns are inserted into the microstructure as follows: 1) Naked Sephasil in suspension is introduced into the first chamber (13) through the inlet (5) and the microfluidic disc To rotate. Sephasil transfer from depth> 20μ to < 10
Stop by changing from μ (as shaded area (14)) to Sephasil column (15). Other suitable matrices are preferably easy to fill and
It must be a monodisperse sphere with a diameter in the range of 5-50 μm.

FIG. 1c shows a magnified view of the waste control structure (34) shown in the microchannel structure of FIG. 1b, which allows the removal of liquid from the Sephasil suspension. By rotating the disk at low speed, the waste liquid from the Sephasil suspension travels down the wall of the nearest outlet (ie in the direction indicated by arrow a) and out of the structure through the waste outlet (6).

2) Sephadex G-50 (DNA Great) in suspension into chamber (16), inlet (9)
Put through. The rotation of the microfluidic disk allows the Sephadex G-50 chamber (
The outward migration of 16) is stopped by changing the depth from> 20μ to < 10μ (indicated by the shaded area (17)) to the Sephadex G50 bed (35).

The liquid from the suspension is passed from the microstructure through the waste outlet (11) to the control area (1
It is removed by applying sufficient centrifugal force (by rotating the microfluidic disc) to pass through and exit 9). The region (19) allows liquid flow to pass through a fluid barrier where liquid is obtained by physical contraction of channels and / or increased surface hydrophobicity only when a certain centrifugal force is reached by rotation of the microfluidic disc. To control.

[0062]   The microfluidic disc is now ready for sample addition.

If the sample is bacterial plasmid nucleic acid, the supernatant from the bacterial lysate is added to the first chamber (13) through the inlet (5). The sample is passed through a Sephsil column (15) by applying centrifugal force. The plasmid was captured on Sephasil and washed with the wash solution inserted into the chamber (13) through the inlet (5) and the microfluidic disk was spun at low speed to remove the wash solution from the Sephasil control structure (34). ) And from there exits the microstructure through a waste outlet (6).

The plasmid was eluted from Sephasil by adding water to the chamber (13) through the inlet (5) and the eluate was discarded by applying a high speed centrifugal force to the waste control structure (3
4) outer channel (ie in the direction indicated by arrow b) and the second chamber (18
) And U-bend structure. The liquid flow can be changed, if necessary, by changing the dimension of the control area (20) and / or increasing the surface hydrophobicity, so that the liquid brings the resulting fluid barrier to a constant centrifugal force by the rotation of the microfluidic disc. Control to pass only when it reaches.

The liquid in the chamber (18) is moved to the third chamber, the double U structure by centrifugal force. At the same time, reagents for cycle sequencing are inserted through the inlet (8). Therefore, the plasmid eluate and sequencing reagent are mixed in chamber (21).

The cycle sequencing reaction was performed by changing the temperature of the chamber (21) from about 60 ° C to 9 ° C.
It is carried out at 5 ° C. during which the microfluidic disc is rotated to reduce evaporation in the chamber and to reduce the risk of liquid column breakage due to foam formation. Suitably, the reaction volume of the cycle sequencing reaction is 250-5.
It can be between 00nl.

When the cycling is complete, place a liquid plug into the chamber (18) through the inlet (7) and replace the liquid in the chamber (21) with the Sephaex bed in the fourth chamber (16) and further. , The second U-bend structure, through the chamber (22). In this way, salt and non-encapsulated dye terminators remain on Sephadex and are therefore removed from the sequencing reaction through the steps of gel filtration. The purified reaction product (ie, the ladder of sequencing products) is left in the chamber (22).
Suitably, the reaction sample has a volume of less than 500 nanoliters.

An electrophoresis medium such as a highly viscous gel matrix is placed in the chambers (23), (2
4), (25) and (27) channels with appropriate buffers for channel (
Put in 26).

With respect to FIG. 1d, an electric potential is applied between chambers (23) and (24) and a plug of purified reaction product is passed from the chamber (22) indicated by arrow 1 ′ through the gel matrix of channel (26). It shows a decrease in depth (37), which limits the migration of the highly viscous gel matrix and leaves it in the channel (26).

An electric potential is applied between chambers (25) and (27) to transfer the reaction product (sequence ladder) to the gel matrix of channel (26) indicated by arrow 2 ′. The product is a dot (2
It can be detected when it has passed 8), thus obtaining information that guides the base sequence of the DNA in the plasmid.

In another embodiment of the microstructure of the invention of FIG. 1e, the chambers (23)-(2
5), (27) and (28) lack the electrophoretic structure and channel (26) described in Figure 1b. 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 electrophoretic device. In this embodiment, the product is obtained in about submicroliter grade oil, which is a liquid (transferred to another structure for further analysis).
For example, it can be diluted by adding formamide or water).

FIG. 2 shows a side view of two possible structures of wells, such as wells (12) and (29). One suitable well is in the shape of a cylinder (31). The other is in the form of a frustroconical (32); both shapes have a substantially circular top and bottom shape of the well, the bottom circle having a diameter greater than the diameter of the top circle . The direction of centrifugal force is indicated by the arrow (33).

[0073]   The present invention is further described with reference to the following non-limiting examples.

Example 1 1. Transformed bacteria are spread on agar plates containing LB medium + glucose and 100 μg / ml ampicillin and even indicator. The plates are incubated overnight at 37 ° C. 2. Colonies from one bacterial cell (clone) are visually (or robotically used) identified. Colonies are transferred to the wells of a microfluidic disc by resuspending in about 10 μl of isotonic solution.

3. The bacterial cells are spun down by centrifugation and the supernatant is removed. 4.3 microliters of solution I (100 mM Tris-HCl, pH 7.5,
10 mM EDTA, 400 μg / ml RNase I) was added, and the bacterial cells were resuspended by a pipetting robot and incubated for 3 minutes (Note: all reagents for plasmid preparation are GFX Micro Plasmid Prep Kit, Amersham Pharmacia Bio
from teck).

5.3 microliters of Solution II (190 mM NaOH, 1% w / v SDS)
Is added with mixing with a pipetting robot and incubated for 3 minutes. 6.6 microliters of Solution III (buffer containing acetate and chaotrope)
Is added while mixing with a pipetting robot. 7. The mixture is centrifuged and the supernatant transferred to a structure for plasmid isolation.

8. The supernatant is passed through bare Sephasil beads (prepared by adding Sephasil to the microstructure, spinning the microfluidic disc at about 1000 rpm and removing the liquid) captured between the deep and shallow sections of the structure. The plasmid is captured on a Sephasil column and rotation of the microfluidic disc causes unbound material to exit through the waste channel.

9. The column is washed with a washing solution (Tris-EDTA buffer containing 80% ethanol) to remove contaminating proteins. Again, wash fluid to approximately 1000 microfluidic discs.
Direct to disposal by spinning at rpm. 10. The plasmid is eluted by adding 1-2 μl of water and then spinning at high speed. The eluate is directed to a thermocycling chamber where cycle sequencing is performed at 250-500 nl total volume.

11. In parallel with step 10, cycle sequencing reagents (enzymes, primers, buffers, nucleotides and fluorescent terminators) (DYEnamic ET dye terminat)
Place the or kit (obtained from MegaBACE®) in the same thermocycling chamber. 12. Thermocycling is performed by varying the application of heat and cooling to the chamber, cycling 25-35 cycles between 95 ° C and about 60 ° C.

13. The reaction mixture is discharged from the thermocycling chamber by centrifugal force and through the gel filtration chamber. The gel filtration chamber contains monodisperse (sieved) Sephadex G-50 DNA trapped between deep and shallow sections in the microstructure.
Includes grade beads. Non-inclusion terminators and also salts are retained. No k
A logical array ladder follows the final "pick-up" well, adding more water as needed to aid liquid handling and reduce evaporation. 14. The clean-up reaction is removed from the pick-up well by the pipetting robot, placed in a microtiter plate and diluted to 5-10 μl for further processing with MegaBACE.

[Brief description of drawings]

FIG. 1a shows a schematic diagram of a plan for the microstructure of a tonnage-on fluid (partially shown) showing its orientation in a microfluidic disk;

FIG. 1b shows a schematic diagram of one microstructure plan for the fluid of the present invention;

1c shows a close-up view of the microchannel discard control structure (34) of FIG. 1b, where a shows the path of liquid when the microfluidic disk rotates at low speed,
b shows the path of the liquid when the microfluidic disk rotates at high speed;

1d is a microstructured sample preparation microchannel structure shown in FIG. 1b.
Shows an enlarged view of the region (36) between (ie, the portion of (13)-(22)) and the electrophoretic structure (the portion of (23)-(28));

FIG. 1e shows another embodiment of a microstructure for a fluid of the invention arranged on a microfluidic disc;

FIG. 2 shows two possible configurations of wells on a microfluidic disc of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 30/88 G01N 37/00 101 35/08 C12N 15/00 A 35/10 G01N 35/06 A 37 / 00 101 27/26 331E (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ) , SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM , HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN , YU, ZA, ZW F terms (reference) 2G058 AA09 BA08 DA07 EA14 4B024 AA11 CA01 EA04 HA19 4B029 AA07 AA23 FA15 4B063 QA13 QQ43 QR32 QS12 QS16 QX05 4G075 AA39 BB03 BB05 BB07 ED01 [Summary]

Claims (12)

[Claims] 1. A step of purifying a nucleic acid template: b) a step of thermocycling reaction: and c) a step of purifying the product of step b), wherein the steps are carried out sequentially in a microfluidic disc. A characteristic method of performing a series of steps. 2. The method of claim 1, wherein the flow of liquid through the microfluidic disk can be accomplished by rotating the disk. 3. The method according to claim 1, wherein the nucleic acid template is a plasmid. 4. The method according to any one of claims 1 to 3, 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); The method of claim 4, comprising: 6. The method according to claim 1, wherein step a) is performed by passing the nucleic acid template through a purification column in the microstructure contained in the microfluidic disc. 7. The method according to claim 1, wherein step c) is carried out by passing through gel filtration through the microstructure contained in the microfluidic disc. 8. The method according to claim 5, wherein step d) is an electrophoretic separation of the products of a sequencing reaction. 9. A) treating a culture of cells containing template nucleic acid with a lysis reagent to thaw the cytoplasmic membrane; b) placing the lysate of step a) in the microstructures of a microfluidic disc, where each microstructure. Includes a first chamber containing means for purifying the template nucleic acid, a second chamber containing means for the thermocycling reaction, and a third chamber containing 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 connection destination b) a first chamber containing means for purifying a template nucleic acid; a connection destination c) a second chamber containing means for a thermocycling reaction; a connection destination d. ) A microstructure for fluids, characterized in that it comprises a third chamber containing means for purifying the product of the thermocycling reaction. 11. The microstructure for a fluid of claim 11, further 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 carrying out a thermocycling reaction of template nucleic acids, comprising a microfluidic disc, said disc being the claim 10 or 11.
A device comprising a plurality of radially extending microstructures for a fluid according to.