Tt liquid handling robot qiagen

Each well 12 is designed to accommodate a biological sample receptacle The receptacle 18 has substantially the same shape as the well, thereby maintaining the temperature of a biological sample in the receptacle during sample set up and prior to polymerase chain reaction.

Use of the metal block with an automated liquid handling device 20 and for genetic analysis of biological samples provides an improvement to liquid handling systems currently available. Here, the biological sample is inserted into the biological sample receptacle 18 as held by the wells 12 of the metal block 10 in the automated liquid handling device Subsequently, reverse transcriptase polymerase chain reaction is used to determine the presence of RNA or DNA in the sample via a nucleic acid amplification machine.

The handling device 20 controls dispensing, aspirating and transferring of liquid from a first microtiter plate well or other biological sample receptacle to a second microtiter plate well or other second biological sample receptacle. The automated liquid handling device is capable of functioning with test tubes, freezing vials, reservoirs and other wet chemistry containers.

The improvement to the liquid handling device comprises use ofthe metal block 10 comprising a plurality of wells 12 where each well 12 has an open cylindrical upper end 14 and a closed conical lower end Each well 12 accommodates a biological sample receptacle 18 having substantially the same shape as the well The biological sample and reagents are pipeted into the receptacle 18 and the temperature of a biological sample during sample set-up and prior to polymerase chain reaction analysis is maintained.

RNA laboratory is provided. Such method includes the steps of cl illing the metal block, inserting the biological sample receptacle into the metal block, positioning the metal block onto an automated liquid handling device and transferring the biological sample into biological sample receptacle in the metal block for polymerase chain reaction analysis.

Any material with having high thermal conductivity may be suitable for use in the present invention. Biological sample receptacles may be made of plastic or glass. Frequently, biological sample receptacles are plastic and are made of polypropylene or polycarbonate.

Thin- walled tubes and plates are preferred as they allow rapid and consistent heat transfer. Tube volume capacity may range from approximately 0. Volume capacities of individual microplate tubes vary from approximately 0.

The tissue from which the RNA originated may include, but are not limited to, epithelial, connective, muscular, and nerve tissues. It is important that RNA purification is done by a method that mimmizes degradation. The researcher analyzing the results of gene expression must collect and analyze animal tissues as quickly as possible, beginning at the time the animal is euthanized and the organs harvested. Other devices for purification include but are not limited to the Qiagen BioRobot or RNA may also be purified through precipitation reactions using phenol based products, isopropyl alcohol and lithium chloride.

Also, available is a product known as Nucleopin by BD Biosciences. As an alternative, some thermostable DNA polymerases such as Thermus thermophilus DNA polymerase have reverse transcriptase activity in the presence of manganese, allowing for the use of only one enzyme for reverse transcription and polymerase chain reaction.

If bicine buffer with manganese is used, intermediate additions between reverse transcription and amplification are not needed and stability at elevated temperatures is not a concern. However the presence of manganese may reduce the fidelity of nucleotide incorporation. Therefore, this method is not suitable for a high throughput RNA analysis. As described in more detail below, other reagents may include, but are not limited to, ohgonucleotide primers, a thermostable DNA polymerase and an appropriate reaction buffer such as mM KC1, mM Tris-HCL 0.

These devices are able to transfer reagents from one location to another according to a preprogrammed pattern. The refrigerated table designed to maintain sample temperature table is not satisfactory for maintaining the sample at a sufficient temperature to preserve the activity ofthe enzyme. In this liquid handling system, microtiter plates, tip support plates, and troughs are supported in a table attached to the laboratory workstation base.

Movement of the table is provided by a motor means causing the table to reciprocally move in at least one axis. A modular pod suspended above the table has an arm attached at one end for movement up and down a vertically extending tower rising from the base of the workstation. The pod is capable of motion along the arm in at least a second axis that is perpendicular to the first axis of movement of the support table. The arm moves up and down in a third direction perpendicular to both the first and second directions.

Fluid is dispensed using interchangeable modules of one or more nozzles. The nozzles have pipettor tips affixed to them that are automatically picked up and ejected by the pod. The table 24 acts as a surface for supporting the metal block, biological sample receptacles, reagent reservoirs and pipettor tips. The pod 28 is capable of movement horizontally and vertically. The temperature of the table 24 is controllable and is achieved through the use of one or more circulating water baths.

The device software allows the user to specify the location of the aspiration, dispensation and mixing, what type of labware the liquid is being aspirated from and into and the volume and height of the aspiration and dispensation. The Qiagen BioRobot is a nucleic acid purification and liquid handling workstation. It has robotic handling, automated vacuum and a buffer delivery system. Sample receptacles and reagent troughs are present on a platform and an 8 channel pipetting system performs high-speed dispensing.

The Qiagen BioRobot is an automated liquid handling and sample processing workstation. It allows the integration of other hardware, such as cyclers or spectrophotometers.

It has fully automated plate processing by transferring labware to various positions on and off of the worktable, as well as temperature control, small volume liquid handling and customizable processing parameters. The Qiagen BioRobot is an automated workstation for nucleic acid purification, reaction set-up, PCR product cleanup, agarose-gel loading and sample rearray and has a worktable and programmable pipetting mechanism.

The Gilson Constellation Liquid Handler has a bed that can hold up to 12 microplates, a robotic gripper arm, capability to dispense nanoliter volumes and an optional heating and cooling recirculator.

The Robbins Scientific Tango Liquid Handling System comprises a worktable and automated aspiration and dispensing of liquid in a 96 or well format. These devices are able to transfer reagents from one location to another according to a pre-programmed pattern and may be suitable for use in connection with the present invention.

The metal block 10 having been previously refrigerated or frozen is fixed into position on an automated hquid handling device Biological sample receptacles 18 are then inserted into the metal block As the temperature of the liquefied biological sample is maintained, reagents are added to the liquid biological sample for polymerase chain reaction analysis.

Reagents are added into the biological sample receptacles 18 by the automated liquid handling device. The biological sample receptacles are then either moved by robot or manually to a sequence detection system where the reverse transcription, polymerase chain RT-PCR reaction amplification and analysis occur.

From the template, a pair of corresponding synthetic ohgonucleotide primers "primers" can be designed. The primers are designed to anneal to the separate complementary strands of template, one on each side of the region to be amplified, oriented with its 3' end toward the region between the primers. The mixture is then cooled to a lower temperature to allow the ohgonucleotide primers to anneal to the appropriate sequences on the separated strands ofthe template. Following annealing, the temperature ofthe reaction is adjusted to an efficient temperature for 5' to 3' DNA polymerase extension of each primer into the sequences present between the two primers.

This results in the formation of a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times to obtain a high concentration of the amplified target sequence. Each series of denaturation, annealing and extension constitutes one "cycle.

By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" hereinafter "PCR". With PCR, it is possible to amplify a single copy of a specific target DNA sequence to a level detectable by several different methodologies.

These methodologies include ethidium bromide staining, hybridization with a labeled probe, incorporation of biotinylated primers followed by avidin-enzyme conjugate detection, and incorporation of 32 P-labeled deoxynucleotide triphosphates such as Dctp or Datp into the amplified segment. In real-time PCR, cycle- by-cycle measurement of accumulated PCR product is made possible by combining thermal cycling and fluorescence detection of the amplified product in a single instrument.

Because the product is measured at each cycle, product accumulation can be plotted as a function of cycle number. The exponential phase of product amplification is readily determined and used to calculate the amount of template present in the original sample. A number of alternative methods are currently available for real-time PCR. Patent 5,,, hereby incorporated by reference used radioactive labels on the probes but further refinements of the method have focused on self-quenching fluorescent probes.

Originally, separation ofthe amplified products by electrophoresis or other methods was used to measure and calculate the amount of released label. This added time-consuming steps to the analysis. Furthermore, this end-stage analysis of the reactions cannot be readily applied to real-time PCR. The nonmatching nucleotides are linked to a fluorescence donor. A fluorescence quencher is positioned typically at the end of the probe. When the donor and quencher are in the same vicinity, the quencher prevents the fluorescence donor from emitting light.

Increased sensitivity in real-time detection can be achieved with dark quenchers such as dabcyl or the developed Eclipse Quencher from Epoch Biosciences, Inc. The dark quenchers absorb fluorescent energy but do not fluoresce themselves, thus reducing background fluorescence in the sample.

The dark quencher works effectively against a number of red-shifted fluoropores such as FAM, Cy3 and Ta ra due to its broader range of absorbance over dabcyl nm versus nm respectively and is thus better suited to multiplex assays. The minor groove binders can be attached to the 5' end, 3' end or an internal nucleotide of oligonucleotides to increase the ohgonucleotide' s temperature of melting, i.

The use of MGBs allows for the use of shorter ohgonucleotide probes as well as the placement of probes in AT-rich sequences without any loss in oligonucleotidal specificity, as well as better mismatch discrimination among closely related sequences.

Minor groove binders may be used in connection with dark quenchers or alone. An extension of both primers and the probe occurs until one ofthe amplification primers is extended to the probe. Taq polymerase then cleaves the nonpaired nucleotides from the 5' end of the probe, thereby releasing the fluorescence donor.

Once it is physically separated from the quencher, the fluorescent donor can fluorescence in response to light stimulation. As more PCR product is formed, more fluorescent donors are released, allowing the formation of the PCR product to be measured and plotted as a function of cycle time.

The linear, exponential phase of the plot can be selected and used to calculate the amount of nucleotide in the sample. The development of these self-quenching fluorescent probes was a considerable advancement in quantitative PCR. Numerous improved self-quenching probes and methods for the use thereof have been subsequently reported in U. Patents 5,,, 6,, Kronick et al. This method also adds additional fiuorogenic probes to the PCR amplification.

When the primers are incorporated into a double stranded molecule, the hairpins are straightened, which separates the donor and quencher to cause an increase in fluorescence. Other applications use intercalating dyes, which only associate with double stranded DNA.

As more double stranded DNA is generated by the reaction, more fluorescence is observed as more dye becomes associated with DNA. Regardless of the method used, the end result is the same, a plot of fluorescence versus cycle number.

Further analysis of this data is then used to derive quantitative values for the RNA present in the samples. Hence, amplified segments created by the PCR process are efficient templates for subsequent PCR amplifications leading to a cascade of further amplification. The sequence detection system is able to vary reaction conditions to optimize amplification of a nucleic acid sequence. The system can analyze the amount of a given nucleic acid sequence present using any number of fluorescent probes, a fluorescence detection mechanism and system software.

Therefore, the cooling effect ofthe refrigerated table is dissipated and certain enzymes added to the sample receptacles lose activity. In addition, most available sample racks are not designed for use on an automated liquid handling device.

This type of device is simply a cooler and is not subject for use in an automated liquid handling device. Even with the cumbersome equipment set up, heat transfer is likely to occur through the well containing the sample receptacle.

Still other devices such as centrifuge rotors require a refrigeration system that maintains the entire chamber at a given temperature United States Patent No. Each well has an open cylindrical upper end and a closed conical lower end. Each well is design to accommodate a biological sample receptacle. The receptacle has substantially the same shape as the well, thereby maintaining the temperature of a biological sample in the receptacle during sample set up and prior to polymerase chain reaction.

Use of the metal block in an automated liquid handling device provides an improvement to liquid handling systems currently available. In a nucleic acid amplification device, the sample is then caused to undergo reverse transcriptase polymerase chain reaction to determine the presence of RNA or DNA. The metal block is first chilled and then fixed into position on an automated liquid handling device. The metal block and the liquefied biological sample temperature is. The typical handling device is adapted to transfer, dispense and aspirate liquid from one location to another automatically and is capable of a wide range of bioanalytical procedures including sample pipetting, serial dilution, reagent additions, mixing reaction timing and similar known manual procedures.

The typical handling device includes table for supporting microtiter plates and other biological sample receptacles, a pod for transferring fluid to a well located on the table and a means for moving the pod relative to the table between selected locations on said table.

The improvement to the liquid handling device is use of the metal block having a plurality of wells, each well having an open cylindrical upper end and a closed conical lower end. Each well accommodates a biological sample receptacle having substantially the same shape as the well and the temperature of a biological sample in the receptacle during sample set-up and prior to polymerase chain reaction analysis is maintained.

FIGURE 4 is a perspective view of a liquid handling device suitable for use in connection with the subject invention. Each well 12 has an open cylindrical upper end 14 and a closed conical lower end Each well 12 is designed to accommodate a biological sample receptacle The receptacle 18 has substantially the same shape as the well, thereby maintaining the temperature of a biological sample in the receptacle during sample set up and prior to polymerase chain reaction.

The automated liquid handling device is capable of functioning with test tubes, freezing vials, reservoirs and other wet chemistry containers.

Biological sample receptacles may be made of plastic or glass. Frequently, biological sample receptacles are plastic and are made of polypropylene or polycarbonate. Thin- walled tubes and plates are preferred as they allow rapid and consistent heat transfer.

Tube volume capacity may range from approximately 0. Volume capacities of individual microplate tubes vary from approximately 0. The tissue from which the RNA originated may include, but are not limited to, epithelial, connective, muscular, and nerve tissues. It is important that RNA purification is done by a method that mimmizes degradation. The researcher analyzing the results of gene expression must collect and analyze animal tissues as quickly as possible, beginning at the time the animal is euthanized and the organs harvested.

Other devices for purification include but are not limited to the Qiagen BioRobot or RNA may also be purified through precipitation reactions using phenol based products, isopropyl alcohol and lithium chloride. Also, available is a product known as Nucleopin by BD Biosciences.

As an alternative, some thermostable DNA polymerases such as Thermus thermophilus DNA polymerase have reverse transcriptase activity in the presence of manganese, allowing for the use of only one enzyme for reverse transcription and polymerase chain reaction.

If bicine buffer with manganese is used, intermediate additions between reverse transcription and amplification are not needed and stability at elevated temperatures is not a concern.

However the presence of manganese may reduce the fidelity of nucleotide incorporation. Therefore, this method is not suitable for a high throughput RNA analysis. As described in more detail below, other reagents may include, but are not limited to, ohgonucleotide primers, a thermostable DNA polymerase and an appropriate reaction buffer such as mM KC1, mM Tris-HCL 0.

A modular pod suspended above the table has an arm attached at one end for movement up and down a vertically extending tower rising from the base of the workstation. The pod is capable of motion along the arm in at least a second axis that is perpendicular to the first axis of movement of the support table.

The arm moves up and down in a third direction perpendicular to both the first and second directions. Fluid is dispensed using interchangeable modules of one or more nozzles.

The nozzles have pipettor tips affixed to them that are automatically picked up and ejected by the pod. The table 24 acts as a surface for supporting the metal block, biological sample receptacles, reagent reservoirs and pipettor tips.

The pod 28 is capable of movement horizontally and vertically. The temperature of the table 24 is controllable and is achieved through the use of one or more circulating water baths. The device software allows the user to specify the location of the aspiration, dispensation and mixing, what type of labware the liquid is being aspirated from and into and the volume and height of the aspiration and dispensation.

The Qiagen BioRobot is a nucleic acid purification and liquid handling workstation. It has robotic handling, automated vacuum and a buffer delivery system. Sample receptacles and reagent troughs are present on a platform and an 8 channel pipetting system performs high-speed dispensing.

The Qiagen BioRobot is an automated liquid handling and sample processing workstation. It allows the integration of other hardware, such as cyclers or spectrophotometers. It has fully automated plate processing by transferring labware to various positions on and off of the worktable, as well as temperature control, small volume liquid handling and customizable processing parameters.

The Qiagen BioRobot is an automated workstation for nucleic acid purification, reaction set-up, PCR product cleanup, agarose-gel loading and sample rearray and has a worktable and programmable pipetting mechanism. The Gilson Constellation Liquid Handler has a bed that can hold up to 12 microplates, a robotic gripper arm, capability to dispense nanoliter volumes and an optional heating and cooling recirculator.

The Zymark Sciclone ALH Workstation has a 20 position deck, bulk dispensing capabilities to microplates by syringe or peristaltic pump and can pipet using a single channel, 8 channel, 12 channel or 96 channel head. The Robbins Scientific Tango Liquid Handling System comprises a worktable and automated aspiration and dispensing of liquid in a 96 or well format.

These devices are able to transfer reagents from one location to another according to a pre-programmed pattern and may be suitable for use in connection with the present invention. The metal block 10 having been previously refrigerated or frozen is fixed into position on an automated hquid handling device Biological sample receptacles 18 are then inserted into the metal block As the temperature of the liquefied biological sample is maintained, reagents are added to the liquid biological sample for polymerase chain reaction analysis.

The primers are designed to anneal to the separate complementary strands of template, one on each side of the region to be amplified, oriented with its 3' end toward the region between the primers. The mixture is then cooled to a lower temperature to allow the ohgonucleotide primers to anneal to the appropriate sequences on the separated strands ofthe template.

Following annealing, the temperature ofthe reaction is adjusted to an efficient temperature for 5' to 3' DNA polymerase extension of each primer into the sequences present between the two primers.

With PCR, it is possible to amplify a single copy of a specific target DNA sequence to a level detectable by several different methodologies. These methodologies include ethidium bromide staining, hybridization with a labeled probe, incorporation of biotinylated primers followed by avidin-enzyme conjugate detection, and incorporation of 32 P-labeled deoxynucleotide triphosphates such as Dctp or Datp into the amplified segment.

In real-time PCR, cycle- by-cycle measurement of accumulated PCR product is made possible by combining thermal cycling and fluorescence detection of the amplified product in a single instrument. Because the product is measured at each cycle, product accumulation can be plotted as a function of cycle number.

The exponential phase of product amplification is readily determined and used to calculate the amount of template present in the original sample. A number of alternative methods are currently available for real-time PCR. Patent 5,,, hereby incorporated by reference used radioactive labels on the probes but further refinements of the method have focused on self-quenching fluorescent probes.