DE60028819T2 - METHOD OF TREATING A MATRIX NUCLEIC ACID USING AN INTEGRATED MICROFLUIDIC PLATE - 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

The The present invention relates to a microfabricated apparatus comprising a rotatable plate (disc, disc), and especially a microfluidic Plate comprising microstructures for fluids, in which steps which are needed for sequencing nucleic acids on an integrated and sequential (manner) can.

The Method of sequencing has by the application of automation, especially in the form of robots and handling of small volumes of liquid in multiplex formats (bundled Formats, multiplexed formats), reaching an industrial scale. The process typically concludes fragmenting a genome, inserting fragments of interest in a cloning vector, isolating individual Cloning, cleaning the vector which inserted the (inserted) Contains fragment, and using this inserted Fragment as a template in a sequencing reaction. The obtained sequence data are then using computer programs (Software) matched to a contiguous sequence from the numerous To get fragments. This procedure will be detailed below described.

In dependence on the size of the fragment can Different cloning vectors are used to form fragments to clone. The purpose of cloning is to replicate of the insert to ensure large numbers of copies (copy numbers) over one to yield a biological system (bacterium or virus). Big fragments become common in BACs (Bacterial Artificial Chromosomes) or cosmids cloned. Smaller fragments are in the general either in bacterial plasmids, such as pUC18, or cloned into phage M13.

One typical method for de novo sequencing of a genome under Use of fragments which have been cloned into a plasmid, includes a number of possible ones Steps which are performed sequentially. Examples of these Steps are broadly described below:

1. Production of bacterial cultures

fragments of the candidate genome are generated and transformed into plasmids (for example pUC18) inserted, which are kept in a strain of the bacterium Escherichia coli become. This process is called transformation.

The transformed bacteria are spread on an agar plate, which contains growth medium and an antibiotic to the bacteria, which containing the plasmid (which carries a gene which is the host bacterium gives antibiotic resistance). The agar plate may also contain an indicator which specifically indicates the presence of bacteria containing plasmids containing the insert - that is not only one clone containing "empty" or insert-free Plasmid - indicating. The bacterial culture is prior to its distribution to such Diluted in degrees, that individual bacterial cells and thereby their daughter colonies with high probability well separated from each other on the plate are. This ensures that individual colonies are recorded (selected) which in turn contain clones of only a single sequence.

The Plates are over Night at 37 ° C incubated. Individual bacterial cells lead to the formation of cell colonies, which do not overlap on the plate should.

colonies be either by hand (manually) or by a robot recorded and can can be used directly to prepare plasmids or more commonly for an overnight liquid culture (typically 1 to 2 mL) to inoculate larger amounts of bacteria and thereby large copy numbers of the insert.

2. insulation insert-containing plasmid

The quality the template nucleic acid is a key factor for the Success in a sequencing reaction. The template can either a plasmid or a polymerase chain reaction product (PCR product), which has been prepared from a plasmid. While about one direct sequencing of bacterial extracts has been reported (See, for example, Frothingham, R., R.L. Allen, et al. (1991). "Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification. "Biotechniques 11 (1): 40-4 .; Chen, Q., C. Neville, et al. (1996)), most major sequencing facilities apply size Take care to isolate pure plasmid for sequencing success sure.

Many methods for plasmid isolation / purification have been developed. A common method is to (i) lyse bacterial cells using NaOH, (ii) precipitate protein and chromosomal DNA, (iii) the plasmid in solution on a glass matrix (or other purification column which selectively isolates the nucleic acid to be isolated by adsorption or absorption) in the presence of a chaotrope, such as guanidine isothiocyanate (see for example US 5,234,809 ) or sodium iodide, to isolate (iv) with egg to wash an ethanolic solution to remove salts and other residual contaminants, and finally (v) to elute the plasmid from the matrix with a buffer of low ionic strength or with water. The method also includes exposing RNase to degradation of RNA.

This method is the basis for a number of commercially available kits, such as the GFX Micro Plasmid Prep Kit ^™ (Amersham Pharmacia Biotech). These kits typically include a series of solutions, Solutions I, II and III, where Solution I comprises approximately 100 mM Tris-HCl, pH 7.5, 10 mM EDTA, 400 μg / mL RNase I, Solution II approximately 100 mM NaOH, 1% w / v SDS and solution III comprises a buffered solution containing acetate and a chaotrope.

Modifications to the surface of the glass in the glass matrix are for example in the US 5,606,046 been described. Alternative methods include isolation by reversible, non-specific binding to magnetic beads in the presence of PEG and salt, as described, for example, in Hawkins, TL, T. O'Connor-Morin, et al. (1994). "DNA purification and isolation using a solid phase." Nucleic Acids Res. 22 (21): 4543-4), or by triplex-mediated affinity capture ("triplex mediated affinity capture"). US 5,591,841 ) one. Suitable cleaning materials include gels, resins, membranes, glass, or any other surface which selectively retains nucleic acids.

The plasmid quality can then be determined using agarose gel electrophoresis and the quantity can be determined spectrophotometrically, using both techniques known to those of ordinary skill in the art. It is beneficial to plasmid in water or diluted Buffer which is compatible with the subsequent step (ie PCR or a direct sequencing reaction, such as cyclic sequencing).

If the plasmid yield (or possible quality) insufficient for the is direct sequencing, then a specific region of the Plasmids, which covers the insert and the flanking sequence, via a conventional Polymerase chain reaction (PCR) can be amplified to a sequencing template to surrender. This PCR product must be "purified" before being used as a template for sequencing is used. Clearing includes removing uninstalled ones Nucleotides and primers, which would otherwise be the cyclic sequencing reaction to disturb would. A procedure closes the exposure of exonuclease III and alkaline shrimp phosphatase, deactivating (killing) of these enzymes by heat denaturation and using the reaction directly in cyclic sequencing one.

3. Cyclic sequencing

A cyclic sequencing reaction involves mixing template nucleic acid with sequencing primers, a thermostable DNA polymerase enzyme and a mixture of the four Deoxynucleotides (dATP, dCTP, dGTP and dTTP), including one small proportion of a base in a chain-terminating (chain-terminating) dideoxy form, followed by periodic runs (cycles) of heating and cooling (this means Thermocycling). The reaction is either in four different Tubes (vessels, tubes) performed - respectively containing small amounts of each of the dideoxynucleotides, together with a fluorescently labeled primer - or in a single tube by the use of dideoxynucleotides with different fluorescent markers and unlabeled primer. The result is a fluorescently labeled Ladder of nucleic acid chains, which complements the sequence of the template strand. cyclic Sequencing reactions are usually on a scale of 10 to 20 μL in a microtiter plate (96-well or 384-well format) in one Thermocycler performed.

4. Purify

If labeled nucleotide terminators should be used, the reaction mixture subsequently "cleaned up" to non-built-in Remove fluorescent nucleotides. These would otherwise be in the electrophoretic Separation of the sequencing ladder will appear and the quality of the results Reduce.

If It is also necessary to use salts in capillary electrophoresis from the sequencing ladders to remove the electrokinetic To facilitate injection (injection). This purification will generally either by precipitation by addition of ethanol and salt or by gel filtration. In In the case of primer labeled reactions, desalting is only necessary if capillary electrophoresis is to be used.

5. Analysis the sequencing reaction

The stopped (stopped) sequencing reaction is then for subsequent analysis in a denatured gel which may be a slab gel (slab gel, slab gel, as used for example in the ABI PRISM 377 or ALFexpress), or in capillary columns (such as MegaBACE (Amersham Pharmacia Biotech) or PE ABI 3700 (PE Biosystems)).

Recently became the automation of these steps with a focus on the Microfabrication, that is, performing these steps in as possible small volumes, described.

Of the Main part of the automation efforts aimed at using robots to perform the steps which usually manually using pipetting equipment and microtiter plates (96 well and 384 well). The individual steps are performed in separate panels, and the liquid transfers between plates and formats are performed by pipetting robots. Recently Attempts have been made to take different steps in a single device although it included a number of robots. An example is the Sequatron, which was developed by Trevor Hawkins (Whitehead Institute) was developed. This consists of robots to M13 to clean, and leads Sequencing reactions in the preparation for separation in ultrathin slab gels or capillaries through. The technology is based on solid phase insulation DNA (see, for example, Hawkins, T.L., T. O'Connor-Morin, et al. (1994). "DNA purification and isolation using a solid phase. "Nucleic Acids Res. 22 (21): 4543-4) and allows throughputs of more than 25,000 samples in 24 hours. In addition, a group has at the Washington University Robot to record (select) from M13 plaques and templating in turn based on large robots and multi-well plates developed.

method for the isolation of DNA in the presence of chaotropes on micromachined Silicon structures have been published (Christel, L.A., 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 the manufacture and use of porous silicon structures, around the surface from heated Reaction chambers, electrophoresis devices and thermopneumatic Sensor actuators (sensor actuators), chemical preconcentrates and filter or control flow devices to increase. In particular, such silicon structures with a large surface or specific pore size in one significant improvement of adsorption, evaporation (evaporation), Desorption, condensation and flow of liquids and gases in applications, which use such methods on a miniaturized scale, useful be.

method for direct sequencing of plasmids from single bacterial Colonies in fused silica capillaries have been developed (Zhang, Y., H. Tan, et al. (1999). "Multiplexed automated DNA sequencing directly from single bacterial colonies. "Analytical Chemistry 71 (22): 5018-25) Close the procedure separation into glass chips, including detection methods, which are based on laser excitation confocal microscopy (laser-excited confocal microscopy) (see for example Kheterpal, I. and R.A. Mathies (1999). "Capillary array electrophoresis DNA sequencing. "Analytical Chemistry 71 (1): 31A-37A).

The US Patent 5,610,074 describes a centrifugal rotor for insulation, in a series of steps, a substance of a mixture of substances dissolved in a sample liquid suspended or distributed. Many samples are made simultaneously by a variety processed by fractionation cells, each one a number of each other connected (interconnected), sloping and ventilated compartments contains in which individual steps of the fractionation and isolation process occur. In this centrifugal rotor is the specific compartment, which through the sample fluid or one of their fractions at any step of the process is evidenced by a combination of both the speed as well as the direction of rotation of the rotor and gravity certainly. The interconnections, chambers and passages of each compartment are sized and angled to prevent predefined Quantities of sample and reagent liquids over the Overflowed the compartment. However, such a rotor is relatively bulky, requires relatively large volumes at solutions and is complicated in production.

Microanalysis systems, which on microchannels build in a rotatable, usually made of plastic Plate are made often a "centrifugal rotor", "laboratory on a chip" or "CD devices". Such plates can used to analyze and separate small quantities to perform on fluids. The principle of movement of liquids through channels in a plastic plate for the purpose of performing enzymatic assays is described, for example, in Duffy, D.C., H.L. Gillis, et al. (1999). "Microfabricated centrifugal microfluidic systems: characterization and multiple enzymatic assays. "Analytical Chemistry 71 (20): 4669-4678. One type of suitable plastic plate is one which be referred to as compact discs or compact discs.

When such plates are rotated, a centripetal force is directed toward the center of the plate. Where there is a fluid in the plate, this centripetal force can be the result of different Forces, including surface tension, tensile forces and capillary force. Movement of the fluids in the direction of the outer diameter of the plate is achieved by overcoming the centripetal force, usually by increasing the rotational speed of the plate.

Around To reduce costs, it is desirable that the plates not for use with just one type of reagent or fluid limited but can work with a variety of fluids. moreover it is often during the production of samples desirable, that the plate allows it to the user, exact volumes of any desired Combination on fluids or samples without modification of the plate leave. Due to the small width of the microchannels can any Bubbles present between two samples of fluids in the microchannels are, act as separation barriers or can block the microchannel and thereby prevent a fluid from entering a microchannel, which one should enter. To overcome this problem, the US patent teaches No. 5,591,643 disclose the use of a centrifugal rotor with microchannels having cross-sectional areas, enough are that big undesirable Air from the microchannel can be vented at the same time as the fluid Microchannel enters.

Either WO-A-91/21090 and US-A-5,863,708 relate to a process for nucleic acid processing, including the step of nucleic acid amplification, which on a plate with a variety of microstructures for the fluid flow takes place.

The The present invention relates to an apparatus for integrating the steps template isolation, cyclic sequencing, and purification in a CD device and methods for the Use of this apparatus. In particular, be the present meets Invention a single closed device, which in the Location is a big one Handle number of samples, greatly simplifying automation, Reagent consumption and thus the total cost and size of the required equipment to reduce. So far, there are no reports of an apparatus with a similar one Degree of integration, which is included within a single Structure the isolation of a DNA-templated bacterial colony by Obtain a purified sequencing reaction allowed.

• a) den Schritt der Nucleinsäuretemplat-Aufreinigung;
• b) den Schritt einer Thermocycling-Reaktion; und
• c) den Schritt der Reinigung der Produkte von Schritt b)

dadurch gekennzeichnet, dass die Schritte in Folge in einer mikrofluidischen Platte stattfinden.Accordingly, in a first aspect of the invention there is provided a method of performing a series of steps comprising:

• a) the step of nucleic acid template purification;
• b) the step of a thermocycling reaction; and
• c) the step of purifying the products of step b)

characterized in that the steps take place in sequence in a microfluidic plate.

suitably may be the nucleic acid template Be plasmid DNA, although genomic eukaryotic or prokaryotic derived template can also be used. Other nucleic acid templates can from bacterial artificial chromosomes (bacterial artificial chromosomes, BACs) or from phage M13 under Use of suitable extraction and purification protocols which are known to those of ordinary skill in the art.

In another embodiment The thermocycling reaction can be directly on a simple bacterial Extract without prior isolation of the plasmid can be performed.

• – der Position der Struktur auf der mikrofluidischen Platte (das heißt je weiter die Struktur von dem Zentrum der Platte entfernt ist, desto niedriger die benötigten Umdrehungen pro Minute, um die gleiche Zentrifugalkraft zu erreichen, wie in einer Struktur, welche näher am Zentrum der Platte liegt);
• – der physikalischen Dimensionen der Struktur, durch welche die Flüssigkeit passieren muss;
• – der Viskosität der Flüssigkeit; und
• – der chemischen und physikalischen Eigenschaften der Oberflächen in der Struktur.

In a preferred embodiment of the first aspect, fluid flow through the microfluidic plate may be effected by rotating the plate. The disk may be rotated (or spun) at various speeds ranging from about 250 revolutions per minute (low speed) to 15,000 revolutions per minute (high speed). The actual speed needed to get the right flow of fluids through the plate at a particular step in the process will depend on a number of factors, including:

• The position of the structure on the microfluidic plate (that is, the farther the structure is from the center of the plate, the lower the number of revolutions per minute required to achieve the same centrifugal force as in a structure closer to the center of the plate lies);
• The physical dimensions of the structure through which the fluid must pass;
• - the viscosity of the liquid; and
• - the chemical and physical properties of the surfaces in the structure.

In a particularly preferred embodiment of the first aspect is the thermocycling reaction, step b), a nucleic acid sequencing reaction or cyclic sequencing reaction. Other preferred thermocycling reactions shut down Polymerase Chain Reactions (PCR).

• d) den Schritt der Trennung der gereinigten Produkte, welche in Schritt c) erhalten wurden;

und vorzugsweise ist der Schritt d) eine elektrophoretische Trennung der Produkte der Sequenzierungsreaktion. Vorzugsweise findet der Trennungsschritt d) in der gleichen mikrofluidischen Platte wie die Schritte a) bis c) statt.In another embodiment of the first aspect, the method further comprises:

• d) the step of separating the purified Pro products obtained in step c);

and preferably, step d) is an electrophoretic separation of the products of the sequencing reaction. Preferably, the separation step d) takes place in the same microfluidic plate as steps a) to c).

In particularly preferred embodiments Step a) is accomplished by passing the nucleic acid template through a purification column in a microstructure, which is contained in a microfluidic plate, and the step c) by passing the products of step b) through a gel filtration column in one in a microstructure, which in a microfluidic plate contained, performed.

• a) Behandeln einer Kultur von Zellen, enthaltend eine Templatnucleinsäure, mit einem Lyse-Reagenz, so dass die cytoplasmatischen Membranen lysiert werden;
• b) Einführen des Lysats von Schritt a) in Mikrostrukturen für Fluide auf einer mikrofluidischen Platte, wobei jede der Mikrostrukturen eine erste Kammer, welche ein Mittel für die Reinigung von Templatnucleinsäure einbaut, eine zweite Kammer, welche ein Mittel für eine Thermocycling-Reaktion einbaut, und eine dritte Kammer, welche ein Mittel für die Reinigung von Produkten der Thermocycling-Reaktion einbaut, umfasst; und
• c) Entfernen der gereinigten Produkte für die Analyse

umfasst.In one embodiment of the first aspect, there is provided a method of performing a nucleic acid sequencing reaction on a template nucleic acid, the method comprising:

• a) treating a culture of cells containing a template nucleic acid with a lysis reagent so that the cytoplasmic membranes are lysed;
• b) introducing the lysate of step a) into microstructures for fluids on a microfluidic plate, each of the microstructures incorporating a first chamber incorporating a means for purifying template nucleic acid, a second chamber incorporating a means for a thermocycling reaction, and a third chamber incorporating a means for purifying products of the thermocycling reaction; and
• c) removing the purified products for analysis

includes.

In one embodiment, the purified products obtained after purification in the third compartment may be transferred to a capillary electrophoresis DNA sequencer (such as MegaBACE ^™ (Amersham Pharmacia Biotech)) for analysis to obtain template sequence data. In another embodiment, the purified products may be analyzed in a circular (circular) device directly connected to the microfluidic "sample preparation" plate.

In a further embodiment For example, the eluate from the first chamber may initially be in a volume-defining Structure are routed to accurately transfer the correct volume on liquid in the second chamber, which is a means for the thermocycling reaction built-in, ensure.

• a) mindestens eine Einlassöffnung; verbunden mit
• b) einer ersten Kammer, welche ein Mittel für die Reinigung von Templat-Nucleinsäure einbaut; verbunden mit
• c) einer zweiten Kammer, welche ein Mittel für eine Thermocycling-Reaktion einbaut; verbunden mit
• d) einer dritten Kammer, welche eine Mittel für die Reinigung von Produkten der Thermocycling-Reaktion einbaut,

umfasst.In a second aspect of the invention there is provided a microstructure for fluids which is characterized in that it comprises:

• a) at least one inlet opening; attached to
• b) a first chamber which incorporates a means for the purification of template nucleic acid; attached to
• c) a second chamber which incorporates a means for a thermocycling reaction; attached to
• d) a third chamber which incorporates means for the purification of products of the thermocycling reaction,

includes.

• e) eine vierte Kammer, welche ein Mittel zum Anwenden eines Elektropotentials quer durch eine Trennmatrix einbaut, verbunden mit der dritten Kammer.

In a preferred embodiment of the second aspect, the microstructure for fluids further comprises:

• e) a fourth chamber which incorporates means for applying an electrical potential across a separation matrix connected to the third chamber.

In this embodiment The invention relates to the electrophoretic separation of the sequencing ladder performed in the same microfluidic plate or CD. The Separation would be from the outer periphery the circular one Device inwards to the center where a single detector detects the bands can as they happen (for example described in Shi, Y., P. C. Simpson, et al. (1999). "Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis. "Analytical Chemistry 71 (23): 5354-61). This would one further reduction in scale sample preparation and a significant increase in compactness and automation of the entire process.

suitable manner can the chambers and channels, which include the microstructure, depths in the range of about 10 to 500 microns have.

In another embodiment of the second aspect, the fluid microstructure further comprises a plurality at inlet openings and drain holes (Waste ports), which are arranged to allow the introduction of sample and reagents and allow the outlet of waste products.

In a particularly preferred embodiment can the drain holes with a vacuum pump for be connected to an effective outlet of the waste.

In a third aspect of the invention, there is provided an apparatus for performing a thermocycling reaction on a template nucleic acid, the apparatus comprising a microfluidic plate, the plate having a plurality of radial distributed microstructures for fluids according to the second aspect.

In an embodiment of the third aspect, a number of microstructures on a single built-in microfluidic plate. In a preferred embodiment The number of microstructures between 1 and 1000 is preferred 80 to 100 arranged radially on a single plate are. In a particularly preferred embodiment, there would be 96 microstructures arranged on a single plate to the plate apparatus with the 96-well assay formats compatible.

suitable manner the apparatus is formed from a 12 cm compact disc.

In another embodiment In the third aspect, the apparatus may further include a plurality of wells (Bulges) which are for a bacterial culture or initial Nucleic acid template production are suitable on the same plate. This would have the advantage of being the necessity a format change between microtiter plate and microfluidic Plate would be eliminated.

In a preferred embodiment are the inlet openings the microstructures on the microfluidic plate with a centralized Distribution channel connected, such as a common annular (Annular) Channel, allowing a centralized distribution of reagents in all Microstructures on a plate at the same time is made possible. Preferably, the drainage channels are through a common annular Drain channel connected, which in one embodiment in the direction of the outer periphery the plate can be oriented. Likewise, a variety of aeration in the microstructures, which is a liquid stream allow through the integrated structure.

Short description of Characters:

The The present invention is illustrated by non-limiting examples of embodiments illustrated by the following figures, wherein:

1a Figure 12 is a schematic plan view of a fluid microstructure according to the present invention showing its orientation on a microfluidic plate (partially shown).

1b FIG. 3 shows a plan view of a microstructure for fluids according to the present invention, FIG.

1c an enlarged view of the flow control structure ( 34 ) of the microchannel structure of 1b Fig. 2 shows where a shows the route (the route) of the liquids when the microfluidic plate is rotated at a low speed and b shows the path of the liquids when the microfluidic plate is rotated at a high speed;

1d an enlarged view of the area ( 36 ) between the sample preparation microchannel structure (ie parts ( 13 ) to ( 22 )) and the electrophoresis structure (parts ( 23 ) to ( 28 )) of the microstructure, which in the 1b shown shows;

1e an alternative embodiment of a microstructure for fluids according to the invention, which is arranged on a microfluidic plate (partially shown);

2 shows two possible constructions for wells on a microfluidic plate according to the present invention.

Detailed description of embodiments that illustrate the invention

An example of a microstructure for fluids ( 1 ) according to the present invention is in the 1a shown on a microfluidic plate ( 2 ), which is partially shown. The microstructure for fluids ( 1 ) is arranged radially on the plate with the inlet opening ( 5 ) closest to the central hole of the microfluidic plate ( 3 ). The outer edge of the microfluidic plate is shown ( 4 ). The microstructure for fluids consists of a series of connecting microchannels. The microfluidic plate ( 2 ) has a thickness which is much smaller than its diameter, and is provided to the central hole ( 3 ) to be rotated so that centrifugal force causes fluid, which is arranged in the microchannels in the plate, towards the outer edge or the periphery ( 4 ) of the plate to flow. The flow can be driven both by capillary action, pressure and centrifugal force, that is by rotating the plate. As described below, hydrophobic breaks (kinks, breaks, breaks) can be used to control the flow.

Conveniently, the microfluidic plate is of a one-piece or two-piece construction and is formed of an optionally transparent plastic or polymeric material by means of separate moldings which are assembled (e.g., by heating) to provide a closed structure with openings at defined positions to facilitate loading allow the device with liquids and a removal of liquid samples. Suitable plastics or polymeric materials may be selected to have hydrophobic properties. Preferred plastics or polymeric materia They preferably have low self-fluorescence and are selected from polystyrene and polycarbonate. Alternatively, the surface of the microchannels may be additionally selectively modified by chemical or physical means to alter the surface properties to produce localized areas of hydrophobicity or hydrophilicity within the microchannels to impart a desired property. Preferred plastics are selected from polymers having a charged surface, suitably chemically or ion-plasma treated polystyrene, polycarbonate or other rigid transparent polymers.

The microchannels can be formed by micro-machined methods, in which the microchannels into the surface of the Be micromachined plate and a cover plate, such as a plastic plate, glued (stuck) to the surface that will be the channels be included.

hydrophobic Interruptions can introduced into the microchannel structures, such as by marking with an overhead pen (permanent ink) (Snowman pen, Japan). The purpose of the hydrophobic breaks is to prevent the capillary action the fluid in undesired directions draws. Hydrophobic interruptions can be caused by centrifugal force, the is called by rotating the plate at a high speed, overcome become.

In the 1a The arrows indicate the direction of the flow of fluids and / or air from the inlet ports and the drain ports in the microstructure. These openings are described in more detail below.

The 1a also shows a well ( 12 ), which towards the outer edge ( 4 ) of the microfluidic plate is housed. This well may be used for sample preparation prior to adding the sample to the inlet port (FIG. 5 ) be used. For example, the bacterial colony may be first removed by, for example, a pipetting robot from the surface of a solid liquid medium by suspending it in about 10 μL of isotonic liquid, if the nucleic acid template to be used is derived from a bacterial colony. The suspension can then be placed in a well ( 12 ) are placed on a microfluidic plate. The bacterial cells can then be pelleted by rotating the microfluidic plate and the supernatant discarded. The pellet can be resuspended in Solution I, followed by rotation and resuspension in Solutions II and III in succession. The precipitated genomic DNA and the precipitated proteins are pelleted by rotation and the supernatant containing plasmid is processed further (see below).

The 1b shows a more detailed illustration of a microstructure for fluids ( 1 ). The microstructure comprises inlet openings ( 5 ) 8th ) and ( 9 ), which can each be used as an application area (application area) for reagents and samples, drain openings ( 6 ) and ( 11 ), a ventilation ( 10 ) and an opening ( 7 ), which can act both as an inlet opening and as ventilation. The vents open into the recirculating air over the upper surface of the plate and prevent any fluid in the microchannels from being sucked back into the structure.

suitably can the inlet openings and drain holes with an annular distribution channel be connected.

suitably can the outlet openings be connected to a vacuum, so that the removal of waste can be relieved.

The Microstructure further includes a series of chambers. The movement of liquids the microchannels and chambers when the microfluidic plate is in operation now described.

• 1. Nacktes Sephasil^TM (naked sephasil) in flüssiger Suspension wird in eine Kammer (13) durch die Einlassöffnung (5) eingeführt und die mikrofluidische Platte wird rotiert. Die Bewegung des Sephasil wird durch eine Änderung in der Tiefe von > 20 μm zu < 10 μm (gezeigt als schattierter Bereich (14)) zur Bildung einer Sephasil-Säule (15) angehalten. Andere geeignete Matrizes sollten vorzugsweise monodisperse Ku geln (spheres) sein, welche einfach zu packen sind, und einen Durchmesser in dem Bereich von 15 bis 50 μm aufweisen. Die 1c zeigt eine vergrößerte Ansicht einer Ablaufsteuerstruktur (34), gezeigt in der Mikrokanalstruktur der 1b, welche die Entfernung der Flüssigkeit aus der Sephasil-Suspension erlaubt. Beim Rotieren der Platte bei einer niedrigen Geschwindigkeit wird die Ablaufflüssigkeit von der Sephasil-Suspension der Wand der nächsten Öffnung folgen (das heißt der Richtung, welche durch den Pfeil a gekennzeichnet ist) und die Struktur durch eine Ablauföffnung (6) verlassen.
• 2. Sephadex^TM G-50 (DNA-Grad) in flüssiger Suspension wird in eine Kammer (16) durch die Einlassöffnung (9) eingeführt. Beim Rotieren der mikrofluidischen Platte wird die Bewegung des Sephadex G-50 aus der Kammer (16) durch eine Änderung in der Tiefe von > 20 μm zu < 10 μm angehalten (durch schattierten Bereich (17) gekennzeichnet), um ein Sephadex-G50-Bett (35) zu bilden.

Prior to sample addition, two cleaning columns are introduced into the microstructure as follows:

• 1. Naked Sephasil ^™ (naked sephasil) in liquid suspension is placed in a chamber ( 13 ) through the inlet opening ( 5 ) and the microfluidic plate is rotated. The motion of Sephasil is represented by a change in depth from> 20 μm to <10 μm (shown as a shaded area ( 14 )) to form a Sephasil column ( 15 ) stopped. Other suitable matrices should preferably be monodisperse spheres which are easy to pack and have a diameter in the range of 15 to 50 μm. The 1c shows an enlarged view of a flow control structure ( 34 ), shown in the microchannel structure of 1b , which allows the removal of the liquid from the Sephasil suspension. When rotating the plate at a low speed, the effluent from the Sephasil suspension will follow the wall of the next orifice (ie the direction indicated by the arrow a) and the structure through a drain orifice (FIG. 6 ) leave.
• 2. Sephadex ^™ G-50 (DNA grade) in liquid suspension is placed in a chamber ( 16 ) through the inlet opening ( 9 ) introduced. When rotating the microfluidic plate, the movement of the Sephadex G-50 out of the chamber ( 16 ) by a change in the depth of> 20 microns to <10 microns (by shaded area ( 17 ) draws a Sephadex G50 bed ( 35 ) to build.

The liquid from the suspension is removed by applying sufficient centrifugal force (by rotating the microfluidic plate) so that it passes the microstructure through the drainage port (FIG. 11 ) after passing through a control area ( 19 ) leaves. The area ( 19 ) controls the liquid flow by physically narrowing the channel and / or by increasing surface hydrophobicity so that the liquid breaks through the resulting fluidic barrier only when a certain centrifugal force is achieved by rotating the microfluidic plate.

The Microfluidic plate is now for the sample addition ready.

If the sample is bacterial plasmid nucleic acid, the bacterial lysis supernatant is added to the first chamber ( 13 ) via the inlet opening ( 5 ) added. By applying a centrifugal force, the sample is passed through the Sephasil ^™ column ( 15 ) happens. The plasmid is captured on the Sephasil ^™ and washed with a wash solution which enters the chamber ( 13 ) (first chamber) again through the inlet opening ( 5 is washed, and by rotating the microfluidic plate at low speed, the wash solution is caused to pass through the Sephasil ^™ into the flow control structure (FIG. 34 ), from where it passes the microstructure through the drainage opening ( 6 ) leaves.

The plasmid is separated from the Sephasil ^™ column by adding water to the first chamber ( 13 ) through the inlet opening ( 5 ) and applying a higher centripetal force such that the eluate enters the outer channel of the flow control structure (FIG. 34 ) (ie the direction indicated by the arrow b) and into a chamber ( 18 ), a U-bow structure, happens. The liquid flow is controlled, if necessary, by dimensional changes and / or changes in the surface hydrophobicity in the control region ( 20 ): the area ( 20 ) can control fluid flow by physically narrowing the channel and / or increasing surface hydrophobicity so that fluid only breaks through the resulting fluidic barrier when a certain centrifugal force is achieved by rotating the microfluidic plate.

The liquid in the chamber ( 18 ) is placed in a chamber ( 21 ), a double-U structure (second chamber), moved by centrifugal force. At the same time, reagents for carrying out the cyclic sequencing through the inlet opening ( 8th ) introduced. This will cause the plasmiduuate and the sequencing reagents in the second chamber ( 21 ) mixed.

A cyclic sequencing reaction is performed by periodically cycling the temperature of the chamber ( 21 ) between about 60 ° C and 95 ° C while the microfluidic plate is rotated, to reduce the evaporation in the chamber and also to reduce the risk of bubbling of the liquid column. Conveniently, the reaction volume for the cyclic sequencing reaction may be between 250 and 500 nL.

When the periodic cycling is complete, a liquid plug (liquid plug, liquid plug) is introduced into the chamber (FIG. 18 ) through the inlet opening ( 7 ) and the liquid plug is used to replace the liquid in the second chamber ( 21 ) and forces them through the Sephadex ^™ bed into a chamber ( 16 ) (third chamber) and further into a second U-arch structure, chamber ( 22 ). In this way, salt and unincorporated dye terminators are retained in the Sephadex ^™ and thus removed from the sequencing reaction by the gel filtration process. The purified reaction products (that is, the ladder of the sequencing products) are placed in the chamber ( 22 ) withheld. Suitably, the reaction products will be present in a volume of approximately less than 500 nanoliters.

A medium for electrophoresis, such as a high viscosity gel matrix, is placed in the channel ( 26 ) (fourth chamber), together with suitable buffers in the channels, which from the chambers ( 23 ) 24 ) 25 ) and ( 27 ) to lead.

Referring to the 1d becomes an electric potential between the chambers ( 23 ) and ( 24 ) is applied so that the plug of purified reaction products from the chamber ( 22 ) into the gel matrix in chamber ( 26 ) (fourth chamber) in the direction indicated by the arrow 1' is marked. A decrease in depth is indicated ( 37 ), which limits the movement of the high viscosity gel matrix and places it in the channel (FIG. 26 ) holds back.

An electric potential is between the chambers ( 25 ) and ( 27 ) are applied so that the reaction products (sequence ladder) through the gel matrix in chamber ( 26 ) (fourth chamber) in the direction indicated by the arrow 2 ' is marked, moved and so separated. The products can be detected when they reach the point ( 28 ) so that information is generated which results in the base sequence of the DNA in the plasmid.

In the alternative embodiment of the Mi The microstructure according to the invention, which in the 1e is missing, the electrophoretic structure, which in the 1b as chambers ( 23 ) to ( 25 ) 27 ) and ( 28 ) and the channel ( 26 ) (fourth chamber). In this embodiment, the purified products of the thermocycling reaction (that is, the eluate from chamber ( 16 )) for transfer to a separate electrophoretic device in a well ( 29 ) withheld.

In this embodiment the products are obtained in about submicroliter volume, which by the addition of a liquid (for example, formamide or water) for the transfer into a separate structure for dilute the further analysis can be.

The 2 FIG. 12 shows a side view of two possible constructions of wells, such as wells (FIG. 12 ) and ( 29 ). A suitable well is cylindrical in shape ( 31 ). The other is frustoconical in shape ( 32 ); the shape of both the top and bottom of the well is substantially circular and the bottom circle has a larger diameter than the diameter of the top circle. The direction of the centrifugal force is indicated by the arrow ( 33 ).

The Invention will be further understood by reference to the following non-limiting example described.

example 1

• 1. Transformed bacteria are on an agar plate containing LB medium and glucose containing 100 μg / mL ampicillin and even indicator. The Plate is over Night at 37 ° C incubated.
• 2. Colonies Derived from Individual Bacterial Cells (Clones) are detected (identified) by eye (or using a robot). The colony is passed through into a well in a microfluidic plate Resuspension in about 10 μL one transferred isotonic solution.
• 3. The bacterial cells are sedimented by centrifugation (spin down, spin down) and the supernatant is discarded.
• 4. Three microliters of solution I (100 mM Tris-HCl, pH 7.5, 10 mM EDTA, 400 μg / mL RNase I) are added and the bacterial cells are resuspended with a pipetting robot and incubated for three minutes (Note: all reagents for plasmid preparation is taken from the GFX Micro Plasmid Prep Kit ^™ , Amersham Pharmacia Biotech).
• 5. Three microliters of a solution II (190 mM NaOH, 1% w / v SDS) are mixed with a pipetting robot added, followed by incubation for three minutes.
• 6. Six microliters of a solution III (buffered solution, containing acetate and chaotrope) are mixed with the pipetting robot added.
• 7. The mixture is centrifuged and the supernatant transferred to a structure for plasmid isolation.
• 8. The supernatant is passed through a bed of bare Sephasil ^™ beads (previously prepared by adding Sephasil ^™ to the microstructure and rotating the microfluidic plate at about 1,000 rpm to remove the fluid) are trapped at the interface between the deep and flat sections in the structure. The plasmid is captured on the Sephasil ^™ column, and as the microfluidic plate rotates, unbound material passes out through a drainage channel.
• 9. The column is with washing solution (Tris EDTA buffer containing 80% ethanol) washed to contaminating Remove proteins. Again, the washings become the drain by rotating the microfluidic plate at about 1,000 Diverted revolutions per minute.
• 10. The plasmid is made by adding 1 to 2 μL of water eluted, followed by spinning at a higher speed. This Eluate is placed in a thermocycling chamber directed, where cyclic sequencing in a total volume of 250 to 500 μL carried out shall be.
• 11. Parallel to step 10, the cyclic sequencing reagents (enzyme, primers, buffers, nucleotides, and fluorescent terminators) (obtained from DYEnamic ET ^™ dye terminator kit (MegaBACE ^™ )) are introduced into the same thermocycling chamber.
• 12. Thermocycling is carried out by alternating application of heat and cold on the chamber to periodically cycling between 95 ° C and about 60 ° C for 25 to 35 passes To deliver (cycles).
• 13. The reaction mixture is removed from the thermocycling chamber ejected by centrifugal force and by a gel filtration chamber happens. The gel filtration chamber consists of monodisperse (sieved) Sephadex G-50 beads (beads, DNA grade), which at the interface trapped between deep and shallow sections in the microstructure are. Non-installed terminators and also salt are retained. The remaining one Sequencing ladder runs Continue into an end "intake" well (final "pickup" well), taking, if necessary, additional water is added to handle the liquid support and to reduce the evaporation.
• 14. The purified reaction is removed from the receiving well with a pipetting robot and placed in a microtiter plate and 5 to Dilute 10 μL for further processing by MegaBACE ^™ .

Claims (16)

A method of processing a nucleic acid template by conducting a thermocycling reaction based on the nucleic acid template, wherein the thermocycling reaction comprises a cyclic sequencing reaction or amplification of the template by polymerase chain reaction, characterized in that the method comprises the following sequence: a) Purification of the nucleic acid template ; b) thermocycling reaction based on the nucleic acid template purified in step a); and c) purifying the products of step b), wherein the steps are sequentially carried out in a microstructure ( 1 ) a microfluidic plate ( 2 ), which includes microstructures for the liquid flow. Method according to claim 1, characterized in that A) any microstructure ( 1 ) i) at least one inlet opening ( 5 ); associated with (ii) a first chamber ( 13 ) for purification of the nucleic acid template; connected to iii) a second chamber ( 21 ) for the thermocycling reaction; linked to (iv) a third compartment ( 16 ) for purifying the products of the thermocycling reaction; and B) step a) in the first chamber ( 13 ), Step b) in the second chamber ( 21 ) and step c) in the third chamber ( 16 ) is carried out. A method according to claim 2, characterized in that a cell culture containing the template nucleic acid is treated with a lysing agent so that the cytoplasmic membranes are lysed, after which the lysate in the microstructures ( 1 ) of the microfluidic plate ( 2 ) and steps a) -c) are carried out and the purified products are subsequently analyzed. Method according to one of claims 2-3, characterized in that the first chamber ( 13 ) a purification column ( 15 ) and that the template passes through the column ( 15 ) in step a). Method according to one of claims 2-4, characterized in that the third chamber ( 16 ) a gel filtration column ( 35 ) and that the products of step b) through the gel filtration column ( 35 ) in step c). Method according to one the claims 1-5, by characterized in that the thermocycling reaction, step b), a represents cyclic sequencing reaction. Method according to one the claims 1-6, by characterized in that the step sequence a) -c) is followed by a step d) which the separation of the purified products obtained in step c) represents. Method according to claim 7, characterized in that step d) an electrophoretic separation represents. Method according to claim 8, characterized in that A) the microstructure is a fourth chamber ( 26 ) associated with the third chamber ( 16 ), and which includes a means for applying an electric potential across a separation matrix contained in the fourth compartment, and C) step d) in the fourth compartment (Fig. 26 ) is performed. Method according to claim 7, characterized in that the purified product of step c) is transferred to a capillary electrophoresis DNA sequencer and step d) is performed in the sequencer. Method according to one of the claims 1-10, characterized in that each microstructure has a multiplicity of drainage openings ( 6 . 11 . 10 ) having. Method according to one of claims 2-11, characterized in that a drain opening ( 6 ) between the first and second chambers ( 13 . 21 ) is available. Method according to one of claims 9-12, characterized in that a drain opening ( 11 ) between the third and fourth chambers ( 16 . 26 ) is available. Method according to one the claims 1-13, by characterized in that the nucleic acid template is a plasmid. Method according to one of claims 1-14, characterized in that the liquid flow through the microfluidic plate ( 2 ) by rotation of the plate ( 2 ) is effected. Method according to claim 15, characterized in that the microstructures ( 1 ) are radially distributed.