60/155,299, filed September 21,1999. The aforementioned application, and the references cited therein, are incorporated herein by reference.

Field of the Invention The invention relates to devices and methods for high speed, low volume automated sample handling of biological samples, which are useful in the field of genomics for a variety of processes, including DNA sequencing, genetic analysis, and gene expression analysis. The invention further relates to devices and methods for setting up and executing assays for high throughput compound screening for pharmaceutical applications.

Background of the Invention Laboratory automation has played a key role in the advancement of genomics and drug discovery over the past decade. Early work in genomics focused on the automation of fingerprinting and STS mapping procedures through the adaptation of pipetting robots and image acquisition systems (Garcia et al., 1995, Kwok et al., 1992, Lamerdin and Carrano, 1993, MacMurray etal., 1991, Nizetic et al., 1994, Sloan et al, 1993). An interesting example was the"Genomatron"for STS/EST mapping, jointly developed by Intelligent Automation Systems and the Whitehead Institute Genome Center in Cambridge, MA (Dietrich et al., 1995, Hudson et al., 1995). This system performed all the necessary steps for high-speed PCR setup, thermocycling, sample processing for transfer of the reaction products onto nylon membranes, and hybridization with biotinylated probes for CCD based optical signal detection.

However, the machine was large, expensive to operate, and could not be easily adapted to performing other tasks. From 1990 onward, a large number and variety of laboratory

automation devices became available from an ever expanding set of instrumentation companies. Automated systems are now used in high-throughput sample preparation for DNA sequencing at many of the large sequencing centers.

The degree of automation of the various functions performed in sequencing centers varies widely, ranging from manually fed systems to fully integrated processes. Each configuration has demonstrated positive attributes that contribute to its successful implementation in the sequencing laboratory. However, with a manual setup, there are significant problems with human error resulting in misidentification of sequencing reads, while the fully integrated process is prone to system failure should one of the modules break.

The current approach in the automation community is to move away from large fully integrated systems to smaller workstations that fulfill specific independent functions in the sequencing process. This means that malfunction of one workstation does not result in a total system breakdown. In addition, this paradigm allows flexibility, which can accommodate changes in requirements as sequencing processes change and improve over time. Specifically, as it takes time to build an automation unit, having flexibility allows one to alter and modify different components as improvements become available. In high throughput sequencing facilities there are several functions which are tedious, inefficient and error prone. Examples of these are colony picking, template preparation, sequencing reaction setup, clone retrieval and gel loading.

Modern laboratories employ partially automated procedures for handling samples. In these procedures, reagents and templates are combined by manually feeding 96-channel pipettors with thermocycling plates. Other laboratories utilize pipetting robots, such as the Tecan Genesis (Ahmadi, 1997) to accomplish the same task.

Integrated systems that utilize a variety of pipetting robots, and plate-to-plate liquid transfers, plate sealing, and plate-based thermocycling with magnetic bead or filtration based purification procedures have been constructed. However, these systems are complicated, expensive to build, and suffer from sample evaporation problems and volume constraints.

Several groups have proposed using glass capillaries to handle large numbers of DNA sequencing samples. For example, the first protocols for chemical sequencing developed by Maxam and Gilbert (1977) utilized sealed glass capillaries to handle the samples. In one case, the capillaries are filled, mixed and handled individually as they are moved through several functional"stations"on a conveyor belt type of arrangement (Friedman and Meldrum, 1998). In another developmental project,

96 capillaries are attached to a Hydra dispenser (Robbins Scientific) so that the samples can be moved up and down past heating elements to perform PCR (Hunicke-Smith, 1997). In a revision of this device, copper heating elements were moved up and down with respect to the position of the samples (Stanford Technology Lab, 1998).

A significant drawback in standard 5-10 1ll sequencing reactions is that at least 50% of the sample is wasted, never being loaded on the gel.

Summary of the Invention The present invention addresses the foregoing by providing a capillary cassette having a frame defining an interior chamber, a plurality of capillaries, each having a first and second end, wherein at least one of the first end and the second end is mounted to the frame, such that each of the capillaries is fluidly coupled to an external surface of the frame.

According to another aspect of the invention, the present invention provides a sample handling cassette including a frame having a first end and a second end, with a passage through the frame extending from the first end to the second end, a first flat membrane layer disposed along opposed sides of the frame and defining, along with the frame a sample handling chamber.

According to another aspect of the invention, a docking port is provided including a guide cap having a concave end and, optionally, a fluid-tight seal.

According to another aspect of the invention, a method of performing dialysis on a biological sample is provided involving the use of a capillary cassette.

According to another aspect of the invention, a method of performing temperature processing on a biological sample involving the use of a capillary cassette is provided.

According to another aspect of the invention, a biological sample handling system for use with a liquid handling machine is provided involving the use of a capillary cassette and a needle bed.

According to another aspect of the invention, a hotel for processing multiple biological samples is provided including a housing and a fluid management system.

Optionally, the hotel of the present invention can process a plurality of capillary cassettes.

According to another aspect of the invention, a template preparation module is provided involving an apparatus management device and a capillary cassette.

Brief Description of the Drawings The foregoing and other objects, features and advantages of the invention will be apparent from the following description and apparent from the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings illustrate principles of the invention and, although not to scale, show relative dimensions.

Figure 1 is a perspective view of a capillary cassette according to a variation of a first embodiment of the invention ; Figure 2 is a perspective view of a frame according to the first embodiment of the invention; Figure 3 is a close-up perspective cross-sectional view of a docking port according to the first embodiment of the invention; Figure 4 is a cross-sectional view of a docking port according to the first embodiment of the invention; Figure 5 is a cross-sectional view of a portion of a sample handling cassette according to a second embodiment of the invention; Figures 6 and 7 are perspective views of a capillary cassette used in conjunction with a liquid handling machine according to a third embodiment of the invention.

Figure 8 is a perspective cross-sectional view of the third embodiment of the invention; Figures 9 and 10 are a cross-sectional perspective view of a sample handling cassette according to the second embodiment of the invention used with a needle bed and microtiter plate according to the third embodiment of the invention; Figure 11 is a perspective view of a template preparation module in accordance with a fourth embodiment of the invention ;

Figures 12 and 13 are close-up perspective views of the template preparation module according to the fourth embodiment of the invention; Figure 14 is a perspective view of a hotel according to a fifth embodiment of the invention; Figure 15 is a perspective view of a hotel according to a sixth embodiment of the invention; and Figure 16 is a close-up perspective view of a capillary cassette and hotel according to the sixth embodiment of the invention.

Detailed Description of the Invention The present invention addresses a need in the art for small-volume DNA purification methods, preferably with re-usable components, that can be easily integrated with capillary-based sample handling, and that eliminate the need for centrifugation. The invention also performs capillary-based clean-up devices and sample handling alternatives to air-driven thermocycling, and methods for efficiently handling sample amounts that are just sufficient for each separation, in order to achieve significant cost savings.

Before further description of the invention, certain terms employed in the specification, examples and appended claims are, for convenience, collected here.

The term"biological sample"refers to a sample comprising one or more cellular or extracellular components of a biological organism. Such components include, but are not limited, to nucleotides (e. g., DNA, RNA, fragments thereof and plasmids), peptides (e. g., structural proteins and fragments thereof, enzymes, etc.), carbohydrates, etc. The biological samples described herein may also include transport media, biological buffers and other reagents well know in the art for carrying out the processes described above. Although the methods of the invention can be carried out with a biological sample of just about any volume, biological samples in accordance with the invention preferably have microliter (L) volumes and therefore can be referred to as microsamples, e. g., biological microsamples.

The term"cassette"refers to a structure or"module"capable of handling a plurality of samples, for example, 96 or more samples.

The term"dialysis"is art-recognized and is understood to refer to the separation of substances in solution by means of their unequal diffusion through a membrane. As used herein,"equilibrium dialysis"refers to dialysis which occurs without exchange or flow of dialysate."Flow dialysis"refers to dialysis which occurs with a flow (or counterflow) of dialysate."Exchange dialysis"refers to dialysis which includes at least one change of the dialysate surrounding the membrane.

The term"frame"refers to any suitable structure for providing mechanical support to a capillary.

The term"hotel"refers to a unit for housing one or more cassettes and provides a platform for sample processing. In one embodiment, the hotel is adapted for sample thermocycling by the inclusion of means for circulating a fluid, for example, water or air, to provide temperature control. The hotel also provides a platform for sample purification. For example, in one embodiment, the hotel is adapted for dialysis of the sample by the inclusion of means for circulating a dialysis fluid. In yet another embodiment, the hotel advantageously provides a washing platform by the inclusion of means for circulating a liquid (e. g., water or a chemical cleaning solution) such that cassettes can be washed to prevent sample carryover, or can be regenerated.

The terms"filter"and"membrane element"refer to a material which may used to separate substances in solution by means of unequal diffusion, for example., by size exclusion. Exemplary membrane elements and filters are semipermeable ; i. e., the membrane elements or filters are capable of permitting dialysis to take place.

The term"purification"is intended to encompass, in its various grammatical forms and synonyms (e. g., purification, purifying, clean up, etc.) any operation whereby an undesired component (s) is/are separated from a desired component (s). Such operations include, but are not limited to, filtration, ultrafiltration, dialysis/equilibrium dialysis, chromatography, etc. In certain embodiments, purification is achieved by molecular size discrimination among the components of the biological sample. Purification by molecular size discrimination can be achieved using any number of materials of varying porosity well known in the art including, but not limited to, filters, membranes, and semipermeable ultrafiltration fiber materials.

The terms"temperature processing,""temperature treating,"and "thermal processing"are used interchangeably herein to refer to the application of a variety of temperature conditions to the sample, depending on the particular process underway and include, but are not limited to, continuous and discontinuous heating

regimens, e. g., denaturation, annealing, incubation, precipitation, etc. For example, the terms broadly encompass thermocycling associated with PCR and similar processes.

The term"ultrafiltration"refers to any method of dialysis wherein the sample is under positive pressure relative to the dialysate.

According to the invention, purification of a biological sample may be achieved by a variety of methods, including dialysis, filtration, ultrafiltration and chromatography. The invention further provides various configurations to achieve purification, depending on the method of purification selected. For example, when equilibrium dialysis is the method of purification, the apparatus of the invention provides at least one capillary comprising a membrane element in operative contact with a dialysate, for example, water. When exchange dialysis is the method of purification method, the capillary may be exposed successively to at least two dialysates. When flow dialysis is employed as the method of purification, the capillary cassette 10 may further include one or more ports for inflow and/or outflow of dialysate.

The invention further includes microdialysis-based sample clean up and plasmid clean up.

As set forth herein, the present invention includes dialysis techniques, which may be used effectively to"clean up"polymerase chain reaction (PCR) and cycle sequencing reactions. Until now, one of the problems with conventional dialysis techniques has been one of scale. Typically, dialysis is carried out on relatively large sample volumes of at least 1 mL or more. The typical PCR or sequencing reaction, on the other hand, generally utilizes sample volumes of approximately 10 uL or less, significantly smaller than the sample volumes in conventional dialysis techniques.

The present invention addresses this disparity by optionally using a membrane element, such as one or more microfibers inserted within one of the capillaries. The microfiber performs the same separation functions as the much larger dialysis operations, but with much smaller sample volumes and without the use of centrifugation. The microfibers can be generated or manufactured by removing one or more hollow fibers from commercially available filtration cartridges. Typical cartridges contain many hundreds of fibers, since the cartridge is solely designed to perform dialysis on large sample volumes, e. g., 1 mL or more. Many types and sizes of hollow fiber filtration cartridges are available through such suppliers as Millipore Corp.

Bedford, MA or Spectrum Labs Laguna Hills, CA. Typically these cartridges are used as ultrafiltration devices, where the dialysis membrane acts as a filter, excluding the

desired products while allowing the undesired components to pass through when pressure or vacuum is applied to the system. The present invention achieves proper filtration or separation of components from small volumes of a biological sample by employing one fiber for each biological sample. In this way, dialysis on sample volumes of 10 to 0.05 L volumes is achieved.

According to one mode of operation, the present invention achieves appropriate purification of a sample by first performing a standard Big Dye Terminator Cycle Sequencing Ready Reaction Kit, part # 4303154 PE Applied Biosystems Foster City CA, on a reaction sample size of between 0.05-10 1ll. The sample volume is drawn up into a hollow fiber filter which has been cut out of a Spectrum cartridge cat # 132229 Spectrum Labs Laguna Hills, CA using a 10 zip syringe from Hamilton, Reno, Nevada (see FIG. 2). Purification is then achieved according to any of the various methods described herein.

The technique of dialysis, although well established, has heretofore been difficult to perform on small sample volumes without suffering loss of the sample.

Semi-permeable microfiber ultrafiltration materials are available in a variety of porosities, which allow small components to freely pass through while larger components are selectively retained. Although these are commonly used for ultrafiltration of proteins, only some of the materials are suitable for capillary-based dialysis. Because all of the reaction components to be removed from PCR and DNA sequencing reactions are much smaller than the desirable products, the process of the present invention is an optimal method to"clean up"these reactions.

The invention also relates to purifying and cleaning methods that remove contaminants quickly and efficiently from a DNA reaction mix. Current sequencing machines use electrophoresis through a gel to separate and detect different lengths of DNA that have been appropriately labeled. To make these machines provide results faster and more accurately, the shapes of the gel separation media have gone from thick gels to a gel captured by thin capillaries. A major drawback is the contaminants in the DNA being sequenced tend to physically plug the capillary and interfere with the accurate detection of the different DNA lengths. One major source of contaminants in the DNA sample is the result of by-products of the thermocycling reaction that generates the DNA sample. Both regular and dye-labeled nucleotides that are not incorporated into the DNA strings during the reaction become contaminants that degrade the DNA sequencer. Additionally, ionic components of the reaction reagents remaining in the reaction (e. g., salts) also degrade the machines.

In mode of operation, the present invention provides for effective removal of contaminants from a thermocycling reaction. Once the reaction mixture is thermocycled, purification may be achieved by placing the mixture into a hollow membrane element, which is in contact with a solution having a lower concentration of ionic components. The difference in osmotic pressure across the membrane forces contaminants in the product to migrate across the membrane into the aqueous solution, effectively removing them from the product.

In another mode of operation, the invention provides an apparatus and method for purifying DNA molecules produced in host cells.

The invention involves processes including, but is not limited to, template purification, polymerase chain reaction (PCR), DNA sequencing, polynucleotide ligation, cloning, ligase chain reaction (LCR), single nucleotide extension reaction, exonuclease treatment, and oligonucleotide hybridization reactions.

Process steps associated with these processes include, for example, the aspiration, mixing, incubation, purification, temperature treating, such as heating or cooling, and delivery of the biological sample alone or in a biologically compatible carrier fluid in a selected manner.

As shown in Figure 1, a first embodiment of the present invention involves a capillary cassette 10 based on a standard microtiter plate configuration, such as an 8 x 12 array of elements on 9 mm centers, to allow immediate integration with existing laboratory automation devices and capillary electrophoresis (CE) sequencing instruments. Optionally, the footprint of this embodiment will be of the same dimensions as a 96-well plate, but the height may be increased to accommodate an appropriate length of capillary tubing. This embodiment enables the optional use of an existing 96 channel pipetting device, such as the Robbins Hydra, to perform the liquid handling aspects of the process. The capillary cassette 10 of the present invention may optionally be formed with any number of channel pipetting devices. Other devices may include 384 channels or more.

The capillary cassette 10 of the present invention includes a plurality of capillaries 12. Each capillary 12 has a first end 14 and a second end 15, each of which is securely mounted within a frame 16. Frame 16 may be formed so as to accommodate 96 capillaries 12, or may optionally be formed to accommodate a subset of the capillaries 12 within the capillary cassette 10, as shown in Figure 1. Open space 18 is preferably

provided between capillaries 12, so as to allow fluids, such as liquids or gasses, to pass within or through an interior of capillary cassette 10.

The assembled capillary cassette 10 thus defines an interior chamber 30, with capillaries located therein, and through which air or water can be introduced and circulated over the capillaries to achieve thermocycling or dialysis.

According to a preferred practice, the capillaries preferably have internal volumes that accommodate fluid sizes of less than about 1 microliter. The methods of the invention are advantageously practiced with biological samples having volumes ranging down to approximately 0.05 u. L, preferably 0.1 LL to 3 u. L.

An advantage of employing the novel submicroliter capillaries is that minimal amounts of expensive sequencing reagents and relatively small volumes of biological samples may be used in an automated sample handling format. The invention can be used, for example, to perform purification procedures on polymerase chain reaction (PCR) products, preparing sequencing ladders, and injecting the sequencing ladders into appropriate microtiter plates, or aspirating the biological products.

The capillary cassette 10 of the present invention is formed with a first and second end 20,22. Both the first and second end 20,22 of the capillary cassette 10 may be open or closed by an optional end plate 24. The end plate 24 may further optionally be provided with ports to facilitate the entry and exit of fluids, such as gasses or liquids, passed within an interior chamber 30 of capillary cassette 10. A sealing gasket 26 is preferably optionally provided between frame 16 and the optional end plate 24. An optional sealing gasket 26 is also provided between frames 16, if more than one frame 16 is used, as shown in Figure 1.

In a variation of the present invention involving a plurality of frames 16 forming a capillary cassette 10, holes are formed in the frames to accommodate one or more fasteners, such as pins 28, that are optionally provided to mount the plurality of frames 16 to one another. The pins 28 may also secure optional end plates 24 to the frames 16. Preferably, four pins 28, each having threads and associated nuts are used, spaced along a perimeter of frames 16, as shown in Figure 1. Optionally, screws, rivets or other compressive fasteners may be used in combination with or in place of the pins 28. The pins 28, or their alternative, are preferably formed of stainless steel.

Use of frames 16 accommodating subsets of the total number of capillaries 12 within capillary cassette 10 provides the ability to replace a portion of the capillaries 12 within the capillary cassette 10 in the event of a capillary failure.

Therefore, all capillaries 12 within capillary cassette 10 need not be discarded. Only the capillaries 12 sharing a frame 16 with the failed capillary are discarded.

Figure 2 illustrates a frame 16 according to a variation of the first embodiment of the invention. Optionally, a docking port 40 may be provided according to the first embodiment of the invention. The docking port 40 may optionally be mounted to frames 16 so as to be fluidly connected to the first end 14 and the second end 15 of capillary 12. Optionally, a docking port 40 may only be provided to a single end of capillary 12, or may be omitted entirely.

As shown in Figures 3 and 4, the docking port 40 includes a guide cap 42, optionally configured with a concave surface facing away from the capillary 12 so as to guide a needle 44 to be co-axially aligned with the capillary 12. Preferably, the needle 44 will be a blunt syringe needle. The guide cap 42 is optionally securely mounted to the frame 16. The guide cap 42 may be press fit within a portion of the frame 16. However, an adhesive is preferably used for mounting the guide cap 42 to the frame 16 within a portion of the frame 16. Optionally, a sealing element 46 is provided within docking port 40. The sealing element 46 is preferably a rubber o-ring gland seal from Apple Rubber Corp., press fit within a cavity 47 of guide cap 42. The dimensions of the sealing element 46 and cavity 47 are formed so as to allow a blunt needle 44 to be inserted through the sealing element 46 without damaging the sealing element 46, while simultaneously providing a fluid-tight seal to the corresponding end of the capillary 12.

The use of a blunt needle 44 aids in reducing damage to the sealing element 46, and provides for extensive repetitive use of the docking port 40.

Different solutions (for instance, DNA and Big Dye Terminatos Cycle Sequencing Ready Reaction Mix (DT-mix)) can be aspirated in separate"slugs"with a small air gap in between (with minimal cross-contamination). In such a case, preferably, a docking ports 40 is mounted on each end of each capillary 12.

In preferred embodiments, the capillaries are made of glass, fused silica, polyimide coated fused silica, or TEFLON#. The frames and end plates are preferably fabricated as injection molded parts.

Assembly of individual capillaries in the apparatus of the present invention may be achieved in a variety of ways. In one embodiment, capillaries may be cut to size, assembled into cast grooves, and then secured in place. Capillaries may be secured using a waterproof and temperature resistant glue.

A significant advantage of employing multiple capillaries is that the sample volumes provided by each capillary tube allows the processing of significantly

smaller sample portions, since relatively small volumes of the overall carrier fluid disposed within the capillaries are subject to evaporation. This sample conservation advantage significantly reduces the sample volumes necessary to achieve selected processing of the sample, while concomitantly affording sample outputs that have sequencing ladders with improved signal strength and resolution. By way of example, the amount of fluorescently labeled DNA that can be detected on current sequencing machines is much lower than the amounts that are typically processed; 0.5-1 u. l samples are sufficient.

Figure 5 illustrates a second embodiment of the invention involving a variation of the construction of the sample handling chamber. The second embodiment of the invention uses a flat membrane 61 in place of capillaries. A flat membrane 61 is provided along both sides of a frame 62. In Figure 5, a sample handling chamber 64 is formed by a cutout of the frame 62 and two flat membranes 61 layers. A first flat membrane layer 66 is provided along a forward surface of the frame 62 as shown in Figure 5. A second flat membrane layer 68 is mounted along a back side of the frame 62 as illustrated in Figure 5.

Optionally, a hollow fiber 70 may be mounted within the first or second embodiment of the invention.

The second embodiment of the invention, as described above, provides additional durability and economy. By omitting capillaries, there is no glass to break during rough handling of the frame 62. Furthermore, by the use of the flat membrane 61 to form a plurality of sample handling chambers 64, each frame can be quickly and easily formed.

According to a third embodiment of the invention, the capillary cassette 10 is used in conjunction with a liquid handling machine 80. Hydra liquid handling machines, manufactured by Robbins Scientific, U. S. A., are convenient because of the ability to simultaneously, accurately, and coherently aspirate and deliver selected volumes from parallel channels. These systems offer ease of integration with physical plate-handling systems and PC-based programming systems through an RS232 port.

The Robbins Hydra is also preferred because of the Teflon seals on the syringe plungers, availability of 384-channel models, and lower overall cost.

According to the third embodiment of the invention, the liquid handling machine 80 is provided with needles 44 and optionally a needle alignment frame 82.

The needle alignment frame 82 is preferably configured so as to align the needles 44 axially with the capillaries 12 of the capillary cassette 10. Optionally, docking ports 40

are provided at a first end 14 of the capillaries 12 to assist in alignment of the needles 44 with the capillaries 12.

Optionally, a needle bed 90 is provided below the capillary cassette 10 and is provided with a plurality of needles 92. Each needle 92 has a first end 94 oriented toward the capillary cassette 10 and a second end 96 oriented away from the capillary cassette 10. As shown in Figure 8, the first end 94 of needles 92 is axially aligned with the second end 15 of the capillaries 12 so as to be able to be inserted through docking port 40 located in fluid contact with the second end 15 of capillary 12. A second end 96 of the needles 96 may optionally be configured so as to be inserted within a well 102 of microtiter plate 100.

The liquid handling machine 80 is preferably configured so as to provide relative vertical movement between the needles 44, the needle alignment frame 82, the capillary cassette 10, the needle bed 90 and the microtiter plate 100. Preferably the liquid handling machine 80 is provided with a platen surface 84 to support the microtiter plate 100.

Figures 9 provides a cross-sectional perspective view of the non-capillary configuration of the second embodiment of the invention, used with a needle bed 90 and microtiter plate 100. Figure 10 provides a further perspective view showing an exemplary sample handling chamber 64 provided with a hollow fiber 70 and provided with guide caps 40. As described above, a first flat membrane layer 66 is provided along a forward side of frame 62 and a second flat membrane layer 68 is provided along a rear surface of frame 62.

For temperature control and thermocycling, a two-temperature fluid circulation system with appropriately placed valves may be used to enable a wide range of fluid temperatures to be quickly attained. For example, the heating source 40 can be employed to heat a sample disposed in the capillary 12. Various flow patterns can be created, such as front to back, side to side, and general circulation.

Optionally, the thermocycler may optionally use a combination of hot and cold fluid to change sample temperature. Simple blowers or fluid pumps or blowing ambient air and air heated by resistance heaters over the capillaries are another alternative to change the temperature. The temperature may be measured and controlled by standard Proportional Integral Differential (PID) controllers. The heating rate may be increased as desired by using, for example, superheated fluid for the first part of the heating cycle, then cooler fluid to avoid excessive overshoot of the temperature of the capillaries.

Optical sensors may optionally be employed in connection with the invention to detect liquid levels at one or more points, and provide open loop or feedback control to adjust, if necessary, the sample or fluid level volumes.

The present invention is capable of processing many samples in parallel, if desired, using standard micro-titer plates as reagent sources. The use of capillaries in connection with the present invention is beneficial in that only a small fraction of the liquid volume is exposed to the atmosphere, so that evaporation is minimized. This promotes the processing of the sample, while concomitantly eliminating or reducing sample loss. The capillaries of the system can be used to retrieve, mix and dispense fluids by integration with air or liquid-filled volumetric devices, such as piezoelectric elements, movable pistons or syringe-type plungers.

Typically, DNA sequencing products are purified to remove excess salt, nucleotides, primers, and templates from the biological sample. The illustrated microfiber 70 can be employed to perform the filtration process upon the DNA, to exclude the desired products, while concomitantly allowing undesired components to pass therethrough when the processing assembly is exposed to a pressure or vacuum condition at a proximal end. The DNA sample is cycled through the microfiber by the pressure formed within the system, thereby resulting in relatively small components being filtered out of the hollow fibers and hence the sample. The use of a capillary tube with one or more microfibers 70 disposed therein, provides for the ability to perform equilibrium dialysis upon very small volumes of between about 10 to 0.05 microliters.

A well plate, such as a microtiter plate, can be positioned, so that the pipettes attached to the top of the cassette can draw the samples from the microtiter plate into the capillaries for processing. The needles may then be withdrawn from the top and the bottom of the cassette, allowing the gland seals to close, thereby effectively sealing the capillaries in the cassette. The cassettes may be thermocycled or dialyzed in place, or moved to separate hotels for these operations, as described below.

In another embodiment of the invention, cassettes are hard-mounted to 96 channel pipettors, and a needle bed is hard mounted to the bottom of the cassette.

During processing of the samples, the top of the capillaries are sealed by syringes, and the bottom of the capillaries are sealed by driving the lower syringe tips against gasket material.

One variation of the invention involves a capillary cassette with ports to allow inflow and outflow of dialysate (e. g., water). Preferably the ports are located at opposite ends of the cassette.

Figure 14 illustrates one embodiment of a hotel. A hotel may be used while thermocycling and/or dialysis occurs, to provide a washing function, to prevent sample cross-contamination, and/or regenerate chemically cassettes that fail to perform adequately. In this embodiment, the system may also recirculate water, or some other solution (acid, base, or detergent), through the lumen of the thermocycling or dialysis capillaries. In another embodiment, the water or other solution is heated.

The invention provides"hotels"that accept a plurality of cassettes for parallel processing to process large numbers of samples efficiently during the dialysis and thermocycling steps. In one embodiment, a hotel can process up to 15-20 cassettes.

Each housing of the hotel accepts and processes a cassette independently of the others.

The hotel includes a fixture that accepts an individual cassette, and makes the appropriate fluid connections between the cassette and the dialysis, thermocycling or wash media. In a preferred embodiment, the end plates of the cassettes incorporate specialized fluid connectors that mate with similar fittings in the hotel. The cassette is optionally seated on a tray that includes means for grasping the cassette and holding it firmly against the housing of the hotel during processing.

In one embodiment, the dialysis hotel recirculates dialysis solution to the cassettes, and contains a reservoir and pump to perform this function. The pump constantly recirculates the solution through all the stations of the hotel. When a cassette is mounted, a valve opens to allow a stream of this recirculating dialysate to pass through the cassette and back to the reservoir. Figure 15 depicts one embodiment of a dialysis hotel according to the invention. A needle bed 390 may optionally be used to provide the dialysis solution.

In another embodiment, the thermocycling hotel recirculates hot and cold fluid, such as air or liquid, through a set of closed conduits. The hot air source is maintained at approximately 100 °C, for example, by electric resistance elements, and the cool air source may be ambient air. In other embodiments, the cool air source is a refrigeration unit. At each hotel housing, there is a proportional mixing valve that controls the temperature of the air circulating through the cassette by selectively mixing the hot and cold air sources. When a cassette is mounted, a valve may open to allow the air mixture to pass through the cassette. The invention also optionally provides a temperature sensor in the air-flow stream entering (or leaving) the cassette to provide positive feedback to a controller that operates the mixing valve to control the temperature and time profile during thermocycling. A control unit in each station of the

hotel causes the preset thermocycling protocol to initiate, and to preferably proceed until finished. In another embodiment, liquid, such as water, may be used instead of air as a heat transfer medium to achieve a uniform temperature profile across all of the capillaries within the cassette.

In yet another embodiment, a washing hotel serves to rinse out the inner surfaces of the capillaries after each use to minimize and/or eliminate cross contamination of samples, and also to retain the microporosity characteristics of the membrane element. In a preferred embodiment, the washing hotel circulates a wash fluid, followed by a water rinse whenever a cassette is introduced to a particular station.

The cassettes may be mounted differently in the washing station than in the thermocycling hotels to allow the circulating solutions to be directed to the insides of the capillaries rather than the outside. The circulation pumps are preferably capable of developing pressures in the range of about 15 psi to dislodge deposits from the capillary walls. Chemical regeneration may also be performed, by the use of appropriate regeneration chemicals.

As shown in Figures 11-13, one embodiment of the invention pertains to a system to prepare cleared lysates for plasmid template preparation or"template preparation module."The system is mounted on a Hydra, and includes heating and filtration functions to process cells from deep-well plates. The unit is based on the transfer Hydra design, but includes a specialized filtration manifold that utilizes a roll of filter material instead of multi-well filter plates. According to a preferred embodiment, a deep-well plate containing resuspended cells in lysis buffer is placed on the system and the samples are aspirated into a large-volume (100 uL) thermocycling cassette. The samples are heated at 95-100 °C for 1 minute, and the deep-well plate is removed. The filtration manifold is brought into place under the cassette, the filter material firmly clasped in place by the manifold (effectively sealing off each individual well), and the samples are drawn through the filter material into a receiving plate below the manifold by vacuum (about 3 min). Alternatively, pressure is applied to the samples to effect filtration. The filter material is then disengaged from the manifold, advanced, and the manifold and cassette is washed to prepare for the next set of samples (about 1 min).

The filtration apparatus moves laterally with respect to the cassette and needle bed, and the roll of filter material moves in and out to permit washing of the cassette, needles and manifold. Figure 16 depicts one embodiment of a template preparation module.

An apparatus management device 300 is preferably used to manipulate the above-noted components during processing.

According to the invention, automated processing may be provided by commercially available hardware and software. Custom automated systems to facilitate key aspects of the sequencing and finishing process have been developed and are known to the skilled artisan. Such systems preferably include: 1) a robust platform for silica- bead based template preparation, quantification, and sample reconfiguration utilizing a CRS systems robot, two TECAN liquid handling stations, a fluorometer, a Sagian 96 channel pipettor, and several plate stackers and sealers, 2) a custom platform for automated fabrication and spotting of"paper combs"using a Seiko D-Tran robot and plate handling system designed for microarray spotting and built for GTC by Intelligent Automation Systems, Inc., and 3) a finishing automation system built around a sophisticated storage and retrieval system (Gira), a Tecan liquid handling station, a 96 channel Quadra pipettor, two plate stackers and a plate sealer. In addition, the skilled artisan may use known databases and software tools to automate the apparatuses and methods of the invention.

In a preferred embodiment, the invention provides a method of plasmid isolation ("Automated Template Preparation"or"ATP") which is based on standard alkaline lysis chemistry coupled with reversible capture on silica beads (Engelstein, 1998). This method meets the design criteria for a filtration-based process that can produce templates yielding data of comparable or better quality than Qiagen-generated preparations, and the samples are stable upon storage at 4° C for 6 months. The ATP hardware preferably includes a Tecan Genesis 200 with Robotic Manipulator (RoMa), a CRS T475, a Sagian Multipette 96-channel pipettor, Tecan shakers, Scitec vacuum manifolds and a drying station.

The automated microplate heat sealer (Marsh BioMedical, NY) has RS232 capabilities and can function with plates manufactured from several different plastics. The material used to seal the plates is aluminum foil backed with a thin plastic film, which differs depending on the composition of the microplates to be sealed. The sealing process actually welds the seal to each well rim, with temperature and thickness of the plastic film controlling the strength of the seal. This permits both permanent and removable seals to be used in the process. Alternatively, thinner foils are also available which allow the seal to be pierced ("easy pierce seals") by the pipetting robot to gain access to samples.

The sample handling device may also be constructed as a series of independent modules that can be stacked together, or independently attached temporarily to a liquid handling device to effect the liquid handling steps. An advantage of this approach is that a single liquid handling device, which is the most expensive part of the system to build, could be more optimally utilized to process samples in a large number of dialysis and thermocycling modules. The latter modules can then be handled in a similar fashion as plates are handled on a typical integrated automation system, such that the independent units would be advantageously sealed automatically when detached from the liquid handling device, and that plate-to-plate transfers would be replaced by capillary-to-capillary transfers (avoiding contact with the atmosphere).

Visual examination (using a microscope) of 100 nanoliter samples being heated inside a capillary revealed rapid sample dispersal unless both ends of the capillary were sealed (presumably due to rapid outgassing or localized boiling). In experiments with larger sample volumes where only one end of the tubing was effectively sealed by a syringe, the sample slug was observed to rapidly migrate back and forth in the tubing during thermocycling. This was assumed to be a result of the changing vapor pressure in the closed air space within the capillary tubing. The application of slight pressure to the open end of the tubing eliminated the movement and allowed successful thermocycling to be achieved in long TEFLON capillaries.

Therefore, sealing means, valves, or a pressure control system are a desirable part of a robust flow-through capillary thermocycling system.

The invention relates to an integrated, capillary-based sample handling system for capillary-based aspiration, incubation, purification and delivery of a biological sample that is capable of processing many samples in parallel. The invention further provides integration of pipetting, mixing, temperature treatment, and sample purification that is easy to operate and can be re-used many times.

In another embodiment, the invention provides a capillary cassette system which allows integration with existing laboratory automation devices and sequencing instruments. The cassette includes open frames comprising a plurality of openings on the top and the bottom portions of the frame. In a preferred embodiment, there are twelve openings on the top and bottom portions of the frame. At each corner of the frame, slots are provided for means, e. g., pins for connecting the frames. In one embodiment, the frames are separated by sealing gaskets.

In another embodiment of the invention, the capillaries are designed to prevent leakage, e. g., by sealing, but at the same time to allow penetration, e. g., by a

syringe. This arrangement allows for the application of positive or negative pressure.

For example, this arrangement permits aspiration of samples from microtiter plates into the capillaries, and subsequent dispensing back into plates for further processing.

In a further embodiment of the invention, multiple samples are thermocycled in a single capillary at the same time by the use of air gaps and, preferably, sealing elements at each end of the capillary.

In another embodiment of the invention, DNA and sequencing reagents are metered and mixed in a single capillary.

In yet another embodiment, the invention provides a guide cap located above the seals at the top and bottom of each capillary tube. This arrangement serves to guide a syringe needle into the opening of the capillary.

In still another embodiment of the invention, the cassette includes open frames to allow for air or water flow through the capillaries for thermocycling and dialysis.

In an another embodiment of the invention, cassettes are processed in a hotel, e. g., an air or water temperature controlled station, for thermocycling or dialysis, or as a washing station to regenerate the cassettes.

Although not explicitly discussed herein, it will be appreciated that one or more fluid regulating elements could be positioned along the capillaries discussed herein.

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Equivalents Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.