EP2027251A2 - Thermal cycling system - Google Patents
Thermal cycling systemInfo
- Publication number
- EP2027251A2 EP2027251A2 EP07783905A EP07783905A EP2027251A2 EP 2027251 A2 EP2027251 A2 EP 2027251A2 EP 07783905 A EP07783905 A EP 07783905A EP 07783905 A EP07783905 A EP 07783905A EP 2027251 A2 EP2027251 A2 EP 2027251A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- sample
- pcr
- liquid composition
- temperature
- sample vessel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/168—Specific optical properties, e.g. reflective coatings
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1838—Means for temperature control using fluid heat transfer medium
- B01L2300/185—Means for temperature control using fluid heat transfer medium using a liquid as fluid
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/02—Water baths; Sand baths; Air baths
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
Definitions
- PCR is recognized as one of the most important scientific developments of the twentieth century. PCR has revolutionized molecular biology through vastly extending the capability to identify and reproduce genetic materials such as DNA.
- PCR is routinely practiced in medical and biological research laboratories for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.
- the method has been automated through the use of thermal stable DNA polymerases and a machine commonly referred to as "thermal cycler.”
- the conventional thermal cycler has several intrinsic limitations. Typically a conventional thermal cycler contains a metal heating block to carry out the thermal cycling of reaction samples.
- a desirable device would allow (a) rapid and uniform transfer of heat to effect a more specific amplification reaction of nucleic acids; and/or (b) real-time monitoring of the progress of the amplification reaction in real time.
- the present invention satisfies these needs and provides related advantages as well.
- this invention provides a method of performing PCR comprising: a) performing PCR in a sample vessel and generating a signal in the sample vessel indicating the course of the PCR, wherein substantially all said signal is reflected back into said sample vessel, wherein substantially all the signal is detected from said sample vessel from a discrete location; and b) measuring the signal emitted from the discrete location of the sample vessel.
- the method comprises measuring the signal over a plurality of PCR cycles in real time.
- the sample vessel comprises a wall transparent to the signal and at least part of the wall is immersed in a liquid composition that reflects substantially all the signal striking the liquid composition.
- the liquid composition comprises a metal or metal alloy.
- the signal is generated by a label or dye.
- said sample vessel comprises a material that reflects substantially all said signal in said sample.
- said sample vessel is comprised of a metal.
- said samp e vesse comprises a re ective materia . n anot er em o iment sai sample vessel is separate from said liquid composition by a receptacle.
- this invention provides a method of performing PCR comprising: a) performing PCR of a target nucleotide sequence in a reaction mixture under conditions wherem:(i) the rate of temperature change between primer extension and duplex dissociation and between primer annealing and primer extension is more than 10.5oC per second (ii) said PCR is conducted in a heat block comprising sample vessels, comprising temperature variance of less than 0.5 0 C within a sample vessel; and b) monitoring amplification of the target nucleotide sequence in real time.
- said heat block comprises a liquid composition.
- said method of performing PCR further comprises a temperature variance of less than 0.5 0 C as measured between two or more wells in said heat block.
- said temperature variance is 0.01 0 C as measured between two or more wells in said heat block.
- said heat block is a swap block.
- the rate of temperature change is effected by:(l) putting a sample vessel containing the reaction mixture in thermal contact with a liquid composition having a heat transfer coefficient of at least 0.1 W/M*K; wherein the temperature of the liquid composition controls the temperature of the reaction mixture.
- the temperature uniformity is maintained by inducing movement of the liquid composition.
- this invention provides a method of performing PCR comp ⁇ sing: conducting PCR in a thermal cycler which modulates sample temperature by more than 5 0 C per second; wherem said thermal cycler temperature is regulated by a liquid composition in heat block with a liquid tight seal; wherein said liquid composition comprises a heat transfer coefficient of at least 0.1 W/M*K.
- said thermal cycler provides ramp rates of at least about 10°C per second.
- said thermal cycler provides well-to-well and sample temperature uniformity of at least about 0.01 to 0.1° C.
- said heat block has a temperature variance is 0.01 0 C as measured between two or more wells in said heat block.
- said liquid composition is contained within a heat block, In another embodiment said heat block is a swap block
- this invention provides a method of performing PCR comp ⁇ sing; conducting PCR in a thermal cycler which modulates sample temperature sufficiently to allow detecting amplification in real time at a signal index of at least 3, wherein said detection is via a non-specific nucleic acid label, wherein said thermal cycler modulates sample temperature by more than 1O 0 C per second.
- this invention provides a method for performing real-time PCR comprising: a) performing a PCR reaction of a target nucleotide sequence in a reaction mixture under conditions wherein the rate of temperature change between primer extension and duplex dissociation and between primer annealing and primer extension m the reaction mixture is at least 5°C per second, wherein said PCR reaction is in thermal contact with a liquid composition having heat transfer coefficient of at least 0.1 W/m*K; and b) monitoring the PCR in real time.
- said monitoring comprises, a) performing PCR m a sample vessel and generating signal in the sample vessel indicating the course of the PCR, wherein the signal is emitted from the sample vessel substantially from a discrete location; and b) measuring the signal emitted from the discrete location of the sample vessel.
- said rate of temperature increase in the reaction mixture is at least 40° C per second.
- said temperature change is regulated by a Peltier element.
- this invention provides a method for performing real-time PCR compnsing: a) cycling the temperature of the PCR reaction mixture between temperatures for duplex dissociation, primer annealing an p mer extens on or a p ura y o cyc es, w ere n eac cyc e s no more t an ve secon s; w ere n said temperatures are modulated by a liquid composition sealed in a thermal cycler; b) monitoring the course of PCR in the sample vessel in real time over a plurality of cycles.
- the method of claim 28 further comprising before step (a): 1) placing a closed end of a sample vessel containing the PCR reaction mixture into thermal contact with a liquid composition that is liquid above 60° C and that has a heat transfer coefficient of at least 0.1 watts/meter-degree Kelvin, whereby the temperature of the liquid composition controls the temperature of the PCR reaction mixture.30.
- said liquid composition reflects substantially all light inside the sample vessel.
- said monitoring is performed by measuring signal emitted from the top or bottom of the sample vessel.
- each cycle is no more than three seconds.
- said sample vessel comprises a reflective surface.
- said sample vessel is further covered with a cap.
- said cap is capable of absorbing heat from or being cooled by said liquid composition.
- this invention provides a method for conducting real time PCR comprising providing a thermal cycler comprising a liquid composition in thermal contact with PCR sample vessels; wherein said liquid composition modulates in temperature thus regulating reaction temperatures in the sample vessels, wherein a detectable signal is emitted from said sample vessels; and detecting said signal via an optical assembly comprising a light emitter and optical detector.
- said optical assembly comprises a pin photodiode CCD imager, a CMOS imager, a line scanner, a photodiode, a photorransistor, a photomultiplier or an avalanche photodiode.
- said sample vessel is further covered with a cap.
- this invention provides an apparatus comprising: a) a temperature control assembly comprising a container containing a liquid composition that is liquid above 60° C and that has a thermal conductivity of at least 0.1 watts/meter-degree Kelvin, said container having at least one aperture, each aperture adapted to receive a closed end of an sample vessel, wherein a sample vessel received into the aperture is placed in thermal contact with the liquid composition whereby the temperature of the liquid composition controls the temperature of a liquid sample in the sample vessel and wherein the liquid composition is capable of reflecting substantially all light inside the sample vessel; b) an optical assembly capable of detecting said light inside the sample vessel from a discrete location on said sample vessel; and c) a control assembly that controls the temperature of the liquid composition and the operation of the optical assembly.
- the liquid composition comprises gallium, a gallium-indium alloy or alloy comprising gallium, indium, rhodium, silver, zinc, tin or stannous.
- said temperature controller comprises a Peltier element.
- said temperature controller comprises resistive wire in thermal contact with the liquid composition.
- said temperature controller comprises means to cycle the temperature of the liquid composition between temperatures for duplex dissociation, primer annealing and primer extension appropriate for PCR.
- said thermal cycler further comprises means for circulating current in the liquid composition.
- said optical assembly comprises a light emitter and optical detector.
- said apparatus further comprises a sample preparation station comprising means to add reagents to sample vessels.
- said apparatus further comprises a means for moving the sample vessels into the apertures.
- said apparatus further comprises a digital computer that controls the thermal cycler, the optical assembly and the sample preparation station.
- a not er aspect t is invention provi es a system or per orming rea time R comprising a) a t erma cycler comprising a liquid composition and means to engage a sample vessel and put said vessel m thermal contact with the composition, and b) an optical assembly comprising a light emitter and optical detector that directs light into an engaged sample vessel and detects light emitted from an engaged sample vessel
- said liquid composition comprises a metal or metal alloy
- said liquid composition comprises gallium
- said signals emitted are from a removable cap of said sample vessel
- said liquid composition is separated from said sample vessel by a receptacle
- said receptacle is transparent or translucent
- said system comprises a Peltier element
- said thermal cycler further comprises a motor operatively connected
- this invention provides an apparatus comprising a) a heat sink, b) a heating component in thermal contact with the heat sink, c) a barrier comprising a wall having a top and bottom surfaces, wherem the bottom surface is sealed to the said heating component wherein the sealed barrier and said heating component form a container containing a liquid composition, d) a first piece comprising a plurality of wells, wherein the first piece is sealed to the top surface of the barrier and the wells extend mto the container, e) a second piece comprising a plurality of sample vessels, each with an open end, wherein the sample vessels are removably inserted into the wells, f) a third piece comprising a plurality of extrusions, wherein the extrusions are removably inserted into the open ends of the sample vessels
- said liquid comprising a plurality of wells, wherein the first piece is sealed to the top surface of the barrier and the wells extend mto the container
- a second piece comprising a plurality
- anot er em o ment sai apparatus further comprises a second heating component, wherein said barrier is disposed between said heating component and said second heating component.
- said plurality of wells comprise 4, 8, 16, 32, 48, 96, 196, 384 or 1536 wells.
- said apparatus further comprises a fan and stir bar, wherein optionally said fan and stir bar are operatively connected to a single motor.
- said apparatus is powered by a battery.
- said fan is turned on and off automatically by a controller component operatively connected to said apparatus.
- said apparatus further comprises a metal heat spreader.
- said spreader comprises copper.
- said heating component is configured for gradient PCR.
- this invention provides a continuous flow PCR system comprising a sample preparation module; thermal cycler, wherein said thermal cycler comprises a liquid composition for modulating temperature in said sample; and an optical assembly for detecting an emission signal from said sample.
- said system further comprises a sampler and a waste collection.
- Figure 1 illustrates a thermal cycler body for use with a liquid composition swap heat block.
- thermal cycler body may comprise an optical assembly, control electronics, fans, and optionally a power supply.
- FIG. 2 illustrates a swap block embodiment comprising 48 sample vessel wells.
- the swap block comprises, from top to bottom: a single piece serving as 48 transparent caps; a single piece creating 48 sample vessel wells; a single receptacle piece with 48 reaction well, which forms the ceiling of liquid metal container; a plastic housing forming walls of liquid metal container; a metal plate forming bottom floor of liquid metal container; a Peltier device(s) for heating and cooling; and a metal heat sink for removing heat from Peltier device(s).
- FIG. 3 illustrates an exploded view of a swap block embodiment.
- the swap block comprises, from top to bottom: a single piece serving as 48 transparent lids, a single piece serving as 48 transparent caps; a single piece creating 48 sample vessel wells; a single receptacle piece with 48 reaction well; a rubber or plastic gasket or ring that forms a liquid tight seal to contain the liquid metal; a plastic housing forming walls of liquid metal container; a rubber or plastic gasket or ring that forms a liquid tight seal to contain the liquid metal; and optionally a metal plate forming bottom floor of liquid metal chamber; a Peltier device(s) for heating and cooling; and a metal heat sink for removing heat from Peltier device(s).
- FIG. 5 illustrates a view of a sandwich embodiment of a thermal cycler component comprising a heat block comprising a liquid metal (A) This view shows a liquid metal chamber is formed between two Peltier devices (two large walls) and the
- the liquid metal chamber comprises holes for capillary samples tubes Further, each Peltier device is thermally coupled to a heat sink, which is in turn connected to a fan (B) A close up of the central portion of the heat block sandwiched between the Peltier devices (C) the liquid metal chamber comprises holes for capillary samples tubes Capillary tubes are slid into holes, and immersed directly in liquid metal composition for thermal cycling Peltier devices are located on both sides of liquid metal reservoir to allow rapid heating and cooling [0023]
- Figure 6 illustrates an embodiment for detecting a signal from a sample vessel cycled in a heat block comprising a liquid metal composition An LED is used to excite the sample contained within the sample vessel and any resulting signal is detected by a PIN photodiode
- Figure 7 illustrates PCR amplification using primers specific for the HBV virus and a patients blood sample, wherem the PCR was run on a Roche Lightcycler The melting curve's positive peak (green) indicates an HBV+ test, but the negative control peak (brown) is also prominent [00
- FIG. 13 illustrates a method of manufacture for metal sample vessels. This method can rapidly and inexpensively produce sample vessels by cutting and crimping them from a pulled metal stock in one movement. The groove is used to prevent the sample vessel walls from flaring out.
- This invention provides an apparatus comprising a thermal cycler for cycling the temperature of a sample vessel containing a reaction mixture, an optical assembly for detecting signal from the sample vessel and control means for controlling the operation of the thermal cycler and the optical assembly.
- the thermal cycler employs a heat block comprising a liquid composition (such as a liquid metal or a thermally conductive fluid) with high thermal conductivity to rapidly cycle the temperatures in the sample vessel.
- a liquid composition such as a liquid metal or a thermally conductive fluid
- the use of a liquid metal provides two main advantages. First, metal has high thermal conductivity, providing rapid heat transfer. Second, liquid provides tighter contact between the thermally conductive material and the sample vessel, providing more uniform heat transfer. As a result, the temperatures within the sample vessels are remarkably uniform.
- the sample vessel alone or in combination with the thermal cycler, emits substantially all of a signal generated therein out through a discrete portion of the sample vessel, for example, the top of the vessel, whereby the emitted light can be collected by the optical assembly.
- a light detector detects substantially all of the light emitted from a sample vessel.
- the liquid metal or sample vessel is highly reflective and reflects light transmitted through the walls of a transparent sample vessel back into the sample vessel.
- the apparatus of this invention is particularly adapted for performing PCR (polymerase chain reaction), reverse transcription PCR and real time PCR.
- Thermal cyclers comprising the liquid metal heat block will perform PCR faster and more cheaply than devices presently available on the market.
- a thermal cycler comprising a heat block comprising a liquid composition is powered by a battery.
- a t erma cyc er comprising a eat oc comprising a iqui composition is powered y a AC or DC current
- the liquid metal heating blocks of the present invention can be used widely in the field of biotechnology and chemistry Examples include but are not limited to incubations of enzymatic reactions such as restriction enzymes, biochemical assays and polymerase reactions, cell culturing and transformation, hybridization, and any treatment requiring precise temperature control Based on the present disclosure, one of ordinary skill in the art can readily adapt the liquid metal technology to va ⁇ ous analyses of biological/chemical samples which require accurate temperature control
- a liquid composition such as a liquid metal or a thermally conductive fluid
- a liquid metal heat block is used as a thermal cycler
- the faster heat ramping, and superior thermal uniformity lead to lower error rates by DNA polymerases than when used in conventional thermal cyclers ( Figures 7-9) This is due to the decreased time in which the
- a liquid metal or a thermally conductive fluid is used as a heating and cooling medium for a heating block, wherein the heat block comprises at least a base and walls to contain the liquid metal or a thermally conductive fluid
- the liquid metal or a thermally conductive fluid provides a more uniform temperature throughout the heat block than conventional heat blocks, because of faster heat transfer withm the liquid metal or a thermally conductive fluid and the ability to use convection or stirring to distribute heat
- the heat block comprises a liquid metal or thermally conductive fluid m direct contact with sample vessels In this embodiment full contact with sample vessels can be achieved, resulting in uniform heat transfer regardless of the type, size, and shape of the sample vessels Pre-formed wells are not required
- uniformity of temperature withm the heating block can be achieved, because the liquid metal or thermally conductive fluid can easily be circulated in the heating block by convection or by an external force, including but not limited to a stir bar, a pump, a vibration device, or magnetohydrodynamic (M
- the liquid metal or thermally conductive fluid is completely contained within the heat block and does not contact the sample vessels
- the container has receptacles which are designed to accept sample vessels of a desired shape and size These receptacles are designed to closely fit the sample vessels placed in the wells
- these receptacles are made out of a substantially transparent material that allows light transmitted into them to be reflected back into the sample vessel from the liquid metal
- the receptacles are made out of a reflective material that reflects substantially all of the light to enter or that is created in a sample vessel m a receptacle well back into said sample vessel.
- the receptacle may be made out of an opaque material that is neither transparent nor substantially reflective ⁇ 0038]
- the receptacle is manufactured from a flexible material, so that the wells of the receptacle conform to fit tightly with the sample vessels The tightness of the contact between the wells of the receptacle and the sample vessels increases as the heat block increases in temperature and the liquid me a or erma y con uc ive ui expan s in vo ume e recep ac es may e manu ac ure rom an opaque, transparent, semi-transparent or translucent, mate ⁇ al [0039]
- the heat block may optionally include a reservoir of liquid metal or thermally conductive fluid which may be part of the heat block, such as depression or bulge in bottom or sidewall of the heat block, or as a separate reservoir connected to the heat block by a connector, such as a tube This reservoir can optionally be connected by a pump to the heat block
- a thermal cycler body (101; 151) comprises a fan (103; 153) and a removable heat block assembly, or swap block (105; 155) ( Figure 1)
- the swap block (105; 155) is inserted into and removed from the thermal cycler body (103; 153) by optionally sliding the swap heat block on sliding rails
- the swap heat block (105; 155) comprises a liquid composition container (111; 161) and a heat sink (107;157) and optionally capped samples (109;159)
- the swap heat block ( Figure 2) comprises a receptacle with wells that seals the in the liquid composition so that the sample vessels do not contact the liquid (metal, metal alloy or metal slurry)
- the swap block (105; 155) comprises a receptacle barrier with wells (307;407) that is sealed to a liquid composition container housing (311;411), wherein the seal is liquid tight and may op iona y comprise a gas e ; igures an ur er, t e iqui composition container ousing
- the swap block (105; 155) is held together by fasteners, such as screws (301;401)
- the swap block comprises a first piece, such as a receptacle with 48 wells (307;407), that is occupied by a second piece, such as a sample vessel, including but not limited to a sample plate (305;405), a single sample vessel or a strip
- the sample vessels are placed directly into the liquid.
- the receptacles provide a ⁇ ng (e g , O ring) that functions as a squeegee to wipe clean a sample vessel bemg removed from the liquid and optionally closes to seal the liquid metal in.
- the receptacle provides a sleeve into which the sample vessel is placed (e g , sleeve composed of a pliable plastic film); in other words, the sleeve functions as a barrier between the sample vessel and the liquid metal or thermally conductive fluid
- Liquid composition such as liquid metal or thermally conductive fluids
- a pump is used to circulate the liquid metal or thermally conductive fluid in the heat block
- Any pump design which can displace the liquid metal or thermally conductive fluid is suitable, such as a positive displacement pump (including but not limited to a rotary-type pump, reciprocating-type pump, roots type pump, a syringe pump, a Wendelkolben pump or a helical twisted roots pump) a centrifugal pump, an MHD pump, or a kinetic pump
- the pump will be manufactured out of materials capable of withstanding the temperature differentials and/ or corrosive issues associated with liquid metal or thermally conductive fluids.
- a pump is utilized to circulate the liquid and/or to cause the liquid metal or thermally conductive fluid (e g , increased pressure in the reservoir chamber) to press against the receptacle walls or sample walls depending on the configuration of the thermal cycler (e g , open versus closed)
- the liquid metal or thermally conductive fluid e g , increased pressure in the reservoir chamber
- the increased pressure causes the liquid to exert pressure against the receptacle walls, thus causing such walls to press closely against the sample vessel walls, forming a junction
- Such junction enhances thermal conductivity and/or optical transmittance
- the pump may increase the level of the liquid metal or thermally conductive fluid such that it contacts a greater surface areas of the receptacle walls
- a MHD force is used to circulate the liquid metal.
- the liquid metal is exposed to a magnetic field formed in one direction and an electrical field formed in a direction perpendicular to the direction of the magnetic field.
- the liquid metal then flows in a direction perpendicular to both the direction of the magnetic field and the direction of the electrical field.
- the electrical field is formed by DC or AC current, as the frequency is modulated, the flow of the liquid metal oscillates in relation to the change in frequency, thereby achieving high uniformity of temperature throughout the heat block.
- the liquid metal or thermally conductive fluid may be circulated by a stir bar.
- the stir bar may be linked to a motor which causes it to stir, or it may be magnetically responsive and stir in response to a change in magnetic field.
- the stir bar is resistant to rapid changes in temperature or it is coated with a covering that is resistant to rapid changes in temperature.
- the stir bar is a simple horizontal bar.
- the stir bar may be fan shaped or have multiple projections (such as 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) which serve to stir the liquid metal or thermally conductive fluid.
- the thermal cycler (101) comprises a motor operatively linked to a fan (103) and a stir bar.
- the fan and stir bar are connected coaxially to the same motor and optionally turn simultaneously.
- the liquid metal or thermally conductive fluid may be circulated by a vibration device.
- the vibration device may be integrated into the thermal cycler or heat block structure, or it may be a secondary device in contact with the heat block or thermal cycler.
- the vibration device will transfer waves of vibration through the liquid metal or thermally conductive fluid, aiding in its convection and decreasing the time it takes for the metal to reach thermal uniformity.
- an acoustical device is used to vibrate the liquid metal or thermally conductive fluid, such as a piezo mixer, ultrasonic vibrator, subsonic vibrator or other sonic device.
- the vibrator may comprise speaker coils or piezos or mechanical motors. Vibratory devices also may shake the sample and PCR reagents in the sample vessels thus mixing the contents allowing the reaction to occur more efficiently
- Liquid metals or a thermally conductive fluid are flowable during the operation of a thermal cycler and have a boiling point higher than the operation temperature. Further, the liquid metal or thermally conductive fluid is preferably non-toxic under conditions of operation. Liquid metal has high heat and electrical conductivity and thus can be very responsive to heating and cooling patterns/cycles. For polymerase chain reaction (PCR), rapid heating and cooling rates are preferred, which liquid metals or thermally conductive fluids can satisfy.
- the thermal cycler comprises a sandwich liquid composition heat block ( Figure 5).
- the sandwich heat block comprises a liquid composition container (501;551;571) that is thermally coupled to Peltier heating and cooling elements (503;553;573), which is in turn thermally coupled to heat sinks (505;555;575) and optionally coupled to flanking fans (507;557).
- the liquid composition container (501;551;571) comprises openings for sample vessels ; suc as capi ary tu es n one em o iment t e samp e vesse s are in irect contact wit t e liquid composition
- the samples are physically separated from the liquid composition by thermally conductive deformable tube
- the heat sink comprises tubes or fins for increasing the radiative surface of the heat sink
- the openings for the sample vessels (559;579) comprise a rubber or plastic O- ⁇ ng which acts to form a seal around the sample vessel placed through the opemng into the liquid composition and to squeegee off the liquid composition that may adhere to the sample vessel upon its withdrawal from the heat block
- Sandwich heat block embodiments may comprise openings for at least 1 sample vessel, such as 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 192, or 384
- one or more capillary tubes (581) of a thermally conductive plastic such as that available from Cool Polymers ( Inc , 333 Strawberry Field Rd , Warwick, RI 02886 USA, http //www coolpolymers com) is inserted into a heat block comprising a liquid metal (501;551;571)
- the capillary tube (581) may penetrate through the opposing side of said heat block, providing optical access to both the top and bottom of the tube
- a sample withm the tube (581) can be excited on one end and any resulting signal can be detected on the other
- the tube (581) directly contacts the liquid metal
- the tube (581) is inserted mto a highly thermally conductive deformable tube (such as a plastic tube) withm the liquid metal comprised within the heat block.
- a sample vessel into the highly thermally conductive deformable plastic tube
- the liquid metal could then be pressurized by using a pump, wherein
- the liquid metal or thermally conductive fluid heat block may be used m a continuous PCR thermal cycler
- Continuous PCR thermal cyclers can be used when highly sensitive or high throughput PCR is desired There are many situations m which one might want to sample air, blood, water, or other medium continuously in a sensitive PCR assay This can be used to look for a variety of biological contaminants including influenza, bacterial pathogens, and any number of viral or bacterial pathogens
- Continuous PCR allows PCR to be practiced in an automated manner without the need for human interaction
- a continuously sampling PCR system can also serve as an early warning system m HVAC systems of buildings, airplanes, busses, and other vehicles, and can be used in the monitoring of blood, water, and other possibly contaminated sources
- the continuous PCR system takes an sample from a collection device, such as an air sampler, fluid sampler or other sampler, (1101) ( Figure 11)
- a collection device such as an air sampler, fluid sampler or other sampler
- condensate fluid collected on the condenser unit of an air conditioning system is used as a starting sample or a specialized gas sampling device that works through direct impaction is used to obtain a sample
- the sample is prepared (1103), which in some embodiments may include cell lysis, DNA or RNA purification, filtration, an or reverse ranscr p on.
- en e samp e s prepare or y a ng t e samp e to reagents (such as at least one DNA polymerase, dNTPs, buffer and a salt) and primers, (such as assay- specific primers or broadly applicable primer sets for multiple target pathogens).
- reagents such as at least one DNA polymerase, dNTPs, buffer and a salt
- primers such as assay- specific primers or broadly applicable primer sets for multiple target pathogens.
- These primers may be chosen to selectively amplify the DNA or cDNA isolated from a specific pathogen (such as a mold, virus, bacteria, parasite or amoeba), gene, other desired nucleic acid, or any combination thereof.
- the PCR sample/reagent cocktail (1107) then flows through a tube to the thermal cycling unit (1109).
- the tube is a clear or transparent.
- the tube is opaque.
- the tube is a cylinder.
- the tubes cross section comprises one or more planes forming a shape such as a triangle, square, rectangle, pentagon, hexagon, heptagon, octagon, nonagon, decagon, or other polygon.
- the volume of sample (1107) is such that it takes up a small discrete length of space in the sample vessel, the rest of which is occupied by air, gas, or non- reactive liquid, such as mineral oil.
- Compressed gas or liquid is used to push the sample into the heat block of the thermal cycler.
- the heat block is a liquid metal or thermally conductive fluid heat block, which may be heated and cooled by a variety of devices, including but not limited to thermo-coupled Peltier thermoelectric module, a conventional thermoelectric module, hot air or hot light.
- the thermal cycler uses Peltier thermoelectric modules external to the tube to heat and cool the sample as desired.
- the sample is pushed further down the tube using compressed air or liquid, exiting the thermal cycling region and passing into a detection region (1113) in which a fluorescence measurement, absorbance measurement, or other interrogation measurement can be made.
- the tube is opaque except for the detection region, which is clear or substantially transparent.
- a light source such as a coherent light source including but not limited to a laser
- fluorescent dyes such as intercalating dyes, including but not limited to ethidium bromide or Syber green and related dyes
- a photodetector such as a CCD, CMOS, or other optical detector.
- the detection electronics (1115) evaluate the signal sent from the detection region (1113) A positive PCR test will yield larger amounts of detected fluorescence than will a negative PCR test.
- the sample is pushed further down the tube, eventually to be collected as waste in the waste collector (1117).
- the tube is used for a single use only, then disposed of.
- the tube can be used to amplify and detect the presence or absence of amplification products in multiple samples.
- the samples are loaded at intervals and interspaced with a barrier of gas or liquid to prevent intermixing.
- the samples are spaced apart in the transport tube, allowing each one to be individually cycled and detected.
- the samples are spaced apart in a manner so that as one is undergoing thermal cycling another sample is in the detection region undergoing interrogation.
- multiple tubes may be used in parallel to increase sample throughput.
- the system may alert the user when amplification has occurred (a positive result), indicating that the target sequence is present.
- Gradient Heat Block the liquid metal or thermally conductive fluid heat block is designed so that it can maintain different temperatures in different zones of the heat block. This allows different sample vessels located in wells in different zones to be cycled at different temperatures simultaneously, such as during gradient PCR.
- the liquid metal or thermally conductive fluid heat block is a capa e o main aining a empera ure gra ien across or more zones, suc as , , , , , , , , , , , , , , , , , ,
- the heat block comprises a receptacle with 1 or more sample vessel wells in each temperature zone, such as 2, 3, 4, 5 ,6 ,7 ,8 ,9, 10, 11, 12, 13,14, 15,16,17,18, 19, 20, 21, 22, 23, or 24 wells
- temperature gradients in excess of 0 1°C to 20° C across the liquid metal or thermally conductive fluid heat block can be achieved
- the temperature gradient changes in a linear fashion across a single dimension
- the heat block will contain internal baffles or insulated walls which act to separate different zones of the liquid metal or thermally conductive fluid from other zones
- Each zone may further comprise an individual heat mixer (such as a pump, stir bar or MHD) or the entire heat block may be attached to a single heat mixer such as a MHD or vibration device
- each zone of the heat block may comprise individual heating and/or cooling elements such as a heat conduction element (wires, tubes), thin foil type heater, Peltier elements or cooling units
- the heat block may comprise multiple heating and cooling devices, some of which act globally across all of the liquid metal or thermally conductive fluid heat zones, and some of which act only withm a single zone
- the heat block may employ uniform heating across the entire length and width of block with additional heating and/or cooling devices adjacent to each zone m the heat block
- Such an arrangement may provide greater precision for precisely tuning the temperature of the liquid metal or thermally conductive fluid in each zone and the reaction temperature of the associated sample vessels within said zone
- Thermal conductivity [0068]
- the liquid composition heat block, containing a liquid metal or thermally conductive fluid maintains a more uniform temperature across the block in comparison to solid metal heat blocks
- Solid heating blocks show a significant variation in temperature both across the block and in sample to sample va ⁇ ation (Schoder et al , J Clin Micro Biol 2005,43 2724-2728)
- the variability of temperature at any given point in the heating apparatus is greater for the conventional solid metal thermal cycler than for the liquid metal or thermally conductive fluid heat block Variability as high as +
- the liquid metal or thermally conductive fluid heat block has a substantially uniform temperature between any two wells in the heat block receptacle with a variance from a desired temperature of no more than +/- 0.6 0 C, such as no more than +/- 0.59 0 C, +/-, O.58°C, +/- 0.57 0 C, +/- 0.56°C, +/- 0.55 0 C, +/- 0.54°C, +/- 0.53 0 C, +/- 0.52 0 C, +/- 0.51 0 C, +/- 0.5 0 C, +/- 0.49 0 C, +/-, 0.48 0 C, +/- 0.47 0 C, +/- 0.46 0 C, +/- 0.45 0 C, +/- 0.44 0 C, +/- 0.43 0 C,
- a sample vessel in the liquid metal or thermally conductive fluid heat block has a uniform temperature within the sample vessel with a variance from a desired temperature of no more than +/- 0.6 0 C, such as no more than +/- 0.59 0 C, +/-, 0.58°C, +/- 0.57°C, +/- 0.56 0 C, +/- O.55°C, +/- 0.54 0 C, +/- 0.53 0 C, +/- 0.52 0 C, +/- 0.51 0 C, +/- 0.5 0 C, +/- 0.49 0 C, +/-, 0.48°C, +/- 0.47 0 C, +/- 0.46 0 C, +/- 0.45 0 C, +/- 0.44 0 C, +/- 0.43 0 C, +/- 0.42
- the uniformity of temperature of the liquid metal or thermally conductive fluid heat block is regulated by circulating the liquid metal or thermally conductive fluid in the block. Circulation of the liquid metal or thermally conductive fluid can be created by natural convection or forced convection, such as by the intervention of a device including but not limited to a stir bar, a pump or MHD power, vibration by physical force or MHD power with DC or AC current, etc.
- the liquid metal or thermally conductive fluid heat block has a ramp rate or can change temperature at a rate substantially faster than conventional metal heat blocks, such as at a rate of at least 5-50.5 0 C per second, including but not limited to a range of at least 10-40 0 C per second, more specifically at a rate of at least 5°C, 5.5°C, 6 °C, 6.5°C,7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, 10 0 C, 10.5 0 C,
- said liquid metal or thermally conductive fluid heat block can change temperature at a rate substantially faster than conventional metal heat blocks while maintaining a uniform temperature across the heat block and/or withm a sample within said heat block
- the liquid metal heat block can increase temperature at a rate of at least 44°C per second
- the liquid metal heat block can decrease temperature at a rate of at
- a series of PCR cycles may be performed faster than conventional solid block thermal cyclers
- a single simple PCR cycle normally includes a denaturation step, a hybridization step and an extension step, each performed at a specific temperature
- a series of 30 PCR cycles may be completed in 1 to 20 minutes, such as 1 mm, 2 mm, 3 mm, 4 mm, 5 min, 6 min, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 min, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm
- the length of time of a PCR run is dependent not only on the speed and temperature uniformity of the thermal cycler It will be clear to a practitioner in the art that the length of time of the PCR run can be varied depending on the characteristic of the desired PCR product
- the composition preferably is liquid at least in the range between primer annealing and duplex dissociation
- Primer annealing occurs typically at around 55° C but can be as high as about 70° C or as low as about 45°C
- Duplex dissociation occurs typically around 94° C but can be lower depending on factors such as the length and percentage of guanine-cytosme base pairings (GC content) in the amplicon
- the liquid composition has a melting temperature (i e , transition from solid to liquid at) no greater than 70° C
- the liquid composition has a melting temperature no greater than 60° C
- the liquid composition has a melting temperature no greater than 50° C
- the liquid composition has a melting temperature no greater 40° C
- a variety of liquid metal compositions may be used in
- a toxic liquid metal may be used in the present invention, such as mercury, mercury alloys or Woods Metal.
- Woods metal which comprises about 50% Bi, 25% Pb, 12.5% Sn, 12 5% Cd has a high working temperature range (70-350 0 C)
- a heat block comprising a liquid metal such as woods metal can be used to boil biological samples or in any other laboratory technique requiring a stable high temperature heat block
- the liquid composition is a liquid metal, liquid metal alloy or metal containing slurry
- the expansion coefficient of the liquid metal will vary according to the precise components of a liquid metal, metal alloy or slurry that are contemplated for use in thermal cyclers of the invention
- a liquid metal, liquid metal alloy or metal slurry it expands in volume during PCR by about 0 1, 0 2, 0 3, 0 4, 0 5, 0 6, 0 7, 0 8, 0 9, 1 0, 1 1, 1 2, 1 3, 1 4, 1 5, 1 6, 1 7, 1 8, 1.9 ,2 0, 2 1, 2 2,
- a liquid metal, liquid metal alloy or metal containing slurry is designed to expand a sufficient amount to compress the junction between a container receptacle and a sample vessel, forming a tight contact
- This contact provides enhanced thermal conductivity, as well as optical transmittance (e g , reflectance of light back into the sample vessel)
- This tight contact increases the thermal conductivity between the liquid metal and the sample vessel by decreasing the air space separating the receptacle from the sample vessel
- gallium exhibits a fairly uniform expansion
- the heat block comprise a liquid composition which is a thermally conductive fluid
- thermally conductive fluids includes waxes and oils which have a working range of temperatures suitable for use in a thermal cycler
- the waxes and oils should have a relatively low melting temperature, in the range of 30-55 0 C and maintain their stability at temperatures in at least in the range of 30-110 0 C
- waxes and oils are suitable for use as thermally conductive fluids, including compounds such as, but not limited to hydrocarbon and silicon compounds and mixtures therof Examples include silicone oil, carboxy-modified silicone oil, mineral oil, dibutyl phthalate, polydiethylsiloxanes, polydimethylsiloxanes, .
- the thermally conductive fluid may comprise additives which act to modify its stability, viscosity, expansion coefficient, opacity, and/or reflectivity.
- additives include but are not limited to plastics, minerals aqueous fluids, antifreezes or metals.
- a liquid metal or thermally conductive fluid is used that reflects substantially all of the light it receives.
- the liquid metal or thermally conductive fluid is a component in a heat block of a thermal cycler connected to an optical assembly that is capable of exciting florescent molecules in a sample vessel and detecting the signal.
- the sample vessels may be directly immersed in the liquid metal or thermally conductive fluid of the heat block, or be separated from the liquid metal or thermally conductive fluid by a receptacle that is thermally conductive.
- the receptacle may be manufactured with substantially transparent or reflective material.
- the liquid metal or thermally conductive fluid or reflective sample vessel surface reflects back substantially all of the light it receives which can then be detected by a light detection device, including but not limited to CCD devices, CMOS devices, LED devices, PN photodiodes, PIN photodiodes, or photovoltaic cells.
- a transparent receptacle a transparent sample vessel may be used that consists of the same plastic as the receptacle, or is manufactured from a transparent material with a similar index of refraction as the receptacle.
- the level of liquid metal or thermally conductive fluid is higher than the level of the sample in the sample vessel. In other words when viewed from the side, the volume of sample and PCR reaction cocktail in the sample vessel is below the level of the liquid metal or thermally conductive fluid.
- the liquid metal or thermally conductive fluid will then substantially reflect any light which enters or is created in the sample vessel.
- the liquid metal or thermally conductive fluid heat block is a component in a real time thermal cycler comprising an optical assembly comprising a multi-channel detection apparatus, such as multiple PIN diodes.
- the apparatus may comprise multiple single channels dedicated detecting light from individual wells in the receptacle.
- the liquid metal or thermally conductive fluid acts as an optical buffer which substantially prevents the light transmitted into or out of a first sample vessel from being detected by a detection channel associated with a second sample vessel.
- sample vessels includes reaction vessels of a variety of shapes and configurations.
- sample vessels can be used to contain reaction mixtures, such as PCR reaction mixtures, reverse transcription reaction mixtures, real-time PCR reaction mixtures, or any other reaction mixture which requires heating, cooling or a stable uniform temperature.
- the sample vessels are round or tubular shaped vessels.
- the sample vessels are oval vessels.
- the sample vessels are rectangular or square shaped vessels. Any of the preceding embodiments may further employ a tapered, rounded or flat bottom.
- the sample vessels are capillary tubes, such as clear glass capillary tubes or coated capillary tubes, wherein the coating
- sample vessels are slides, such as glass slides.
- sample vessels are sealed at the bottom.
- the samp e vesse s are coa e , a eas ex erna y, wi an i-a esion coa ings suc as e on or si ane so as o reduce the adherence of a liquid composition, such as a liquid metal or thermally conductive fluid.
- sample vessels are coated, at least internally, with a material for preventing an amplicon from sticking to the sample vessel walls, such as a fluo ⁇ nated polymer or BSA [0086]
- sample vessels are manufactured and used as individual vessels
- sample vessels are linked together in a horizontal se ⁇ es comp ⁇ sing a multiple of individual vessels, such as 2, 4, 6, 10, 12, 14 or 16 tubes
- sample vessels are linked together to form a sheet, plate or tray of vessels designed to fit into the top of the heating block of a thermal cycler so as to occupy some or all available reaction wells.
- the trays or sheets may comprise at least 6, wells, 12 wells, 24 wells, 36 wells, 48 wells, 54 wells, 60 wells, 66 wells, 72 wells, 78 wells, 84 wells, 90 wells or 96 wells, 144 wells, 192 wells, 384 wells, 768, or 1536 wells.
- the sample vessels have caps attached to their open end by a linking element, such as a plastic strip which is optionally hmged.
- the sample vessels lack an attached cap.
- the sample vessels are designed to hold a maximum sample volume, such as 10 ul, 20ul, 30ul, 40ul, 50 ul, 60ul, 70ul, 80ul, 90ul, 10OuI, 200ul, 250ul, 500ul, 750ul, 100OuI, 1500ul, 2000ul, 5mL, or
- PCR real-time polymerase chain reactions
- sample vessels manufactured from materials chosen for then * optical clarity and for their known non-interaction with the reactants, such as glass or plastic
- the sample vessels are designed so that light can enter and leave through the top portion of the sample vessel, which may be covered a cap capable of transmitting light.
- the sample vessels are designed so that light can enter and leave the sample vessel through the bottom of the sample vessel
- the sample vessels are manufactured from a transparent or translucent material capable of transmitting light
- the sample vessels are designed so that light is directed to exit through a single surface, such as the top or bottom.
- sample vessels are manufactured from materials that are substantially internally reflective, such as reflective plastic, coated plastic (such as with metal or other reflective substances), coated glass (such as with metal or other reflective substances), doped glass (manufactured with the addition of molecules that increase the reflectivity of the glass), or metal, including but not limited to stainless steel, chromium, or other substantially non-reactive metals.
- substantially internally reflective such as reflective plastic, coated plastic (such as with metal or other reflective substances), coated glass (such as with metal or other reflective substances), doped glass (manufactured with the addition of molecules that increase the reflectivity of the glass), or metal, including but not limited to stainless steel, chromium, or other substantially non-reactive metals.
- Metal has a high strength that allows for much thinner sidewalls than glass or plastic This decreases the thermal barrier between the reactants and external hot/cold sources, allowing for better thermal control, greater spatial temperature uniformity, and more rapid temperature changes, all of which can produce faster, more efficient polymerase chain reactions
- the high reflectivity of the internal metallic surface aids in the collection of fluorescent light
- the interior of the metal sample vessels is polished or electro-polished.
- the metal sample vessels are manufactured from a larger cylindrical or rectangular stock. First the stock is pulled to the desired diameter Alternatively the standard methods of syringe production may be used A continuous tube (such as a rectangular or cylindrical tube) is generated, which must be both cut and sealed to produce pieces of a desired length , such as about IcM, 2cM, 3cM, 4cM,
- the diameter of the sealed end does not exceed the diameter of the tube itself
- the tube is next cut and sealed
- the tu e may e e t roug a groove in a meta ic oc igure n one embodiment the groove width is very close to the outer diameter size of the metal tube
- a cutting blade (1307) is lowered with its edge flush with the metal block, perpendicular to the groove
- the blade (1307) is shaped with a tapered edge (1309)
- the blade is shaped with an edge such that both cuts and crimps shut the end of the metal tube, and the groove walls prevent the seal from flaring, forcing its diameter to remain close to the diameter of the bulk tube After crimping, the vessel may require further sealing In one embodiment this can be accomplished with a small weld or braze or solder In
- the metal sample vessel may be pulled to the desired diameter and cut into desired lengths One end of each piece is then forced into a hemispherical cup having a similar diameter to the tube itself This motion may round off and c ⁇ mp closed the metal tube
- a pin is inserted through the open top of the metal tube to aid in crushing the seal end of the tube into the receiving cup The tube can be c ⁇ mp sealed in order to create a vessel with one closed end and one open end suitable for PCR
- top of the sample vessel it is generally desirable to cover the top of the sample vessel to prevent evaporation during PCR thermal cycling This can be accomplished by layering the top of the sample vessel with a layer of a non-reactive liquid, such as mineral oil, sealing the sample vessel (such as by heat sealing a capillary tube) or by closing the sample vessel with a cap
- a cap is used The cap may be manufactured using any suitable material (such as glass or plastic) that forms a seal with the sample vessel and acts as a vapor barrier
- the cap is a plastic cap This cap may be opaque, translucent or substantially transparent
- the cap is optically transparent and is suitable for use with a thermal cycler comprising a liquid metal or thermally conductive fluid heat block, and an optical assembly
- the cap is partially or completely coated with an opaque coating
- the cap is partially or completely coated with a reflective coating, such as a mirror coating
- the extraction of light from a sample vessel or vessel containing a material which fluoresces when pumped with an excitation wavelength can be difficult to accomplish especially when the heat block occludes the vessel
- a sample vessel cap (303;403) which is made of a clear material, such as plastic or glass, and which optionally comprises a lightguide that can be optically coupled (with lenses, filters, beam splitters, e g FIG.
- the light emitter may be a coherent source such as a laser or a non-coherent source such as an LED
- an advantage of one of the exemplary embodiments of the invention is that light emitted from a label/dye in the sample vessel is reflected back into the sample vessel based on the reflective property of the liquid metal or thermally conductive fluid, liquid metal alloy or metal slurry
- the amount of reflection is further enhanced by forming a better contact between the sample vessel and receptacle well (e g , using the volume expansion to form a tight junction, or by using a pump to increase the pressure inside the liquid containing chamber, so that the liquid exerts additional pressure on the walls of the receptacle wells to form a tight junction with the sample vessels)
- a simi ar e ect or ig t re ection is e ecte y uti izing t e specia ize samp e vessels of the invention, where a sample vessel is comprised of an opaque composition (e ecte y uti izing t e specia ize samp
- cap implements of the invention are comprised of a material that is optically clear, characterized by having low or no autofluorescence and forms a good seal with the sample vessel Furthermore, m some embodiments, the cap provides of an extrusion (e g , hghtguide) that protrudes a certain length into the sample vessel Examples of material that can be utilized in composing the cap include but are not limited to acrylics, polycarbonates, polystyrenes, styrene block copolymers
- the cap and/or extrusion can be of a geometric shape which includes but is not limited to a polygon, elliptical, circle, square, rectangle, triangle, or any shape that can be obtained through injection molding (as known in the art)
- the sample vessels, as well as the receptacle wells can also be of any of the shapes desired
- the cap, sample vessel, and receptacles are of the same geometric shape
- the cap extrusion protrudes to a level in the sample vessel that is above, at or below the level (e g , line) at which the liquid metal or thermally conductive fluid, liquid metal alloy or liquid metal slurry (collectively "liquid”) is positioned.
- the protrusion is below the level of the liquid is positioned
- the light will travel the extrusion into the sample vessel and the resulting signal emitted from a label/dye in the sample travels back up the extrusion, this light is emitted from a substantially discrete portion of the cap where it is detected by a photodetector, such as a photodiode (e g , FIG6 )
- a photodetector such as a photodiode (e g , FIG6 )
- the tip of the cap is below the surface level of the liquid metal or thermally conductive fluid If viewed from the side it would be apparent that the tip of the cap was located below the level of the liquid metal or thermally conductive fluid m which the sample vessel was located so that it collects substantially all of the light This is the case for embodiments where the sample vessel is directly immersed in the liquid metal or thermally conductive fluid as well as embodiments wherein the sample vessel is placed mside a receptacle well
- the cap shape and surface quality can be designed to maximize optical coupling from the cap into the liquid
- the cap may be illuminated with a fiber bundle, or by a free space solution ie ree space sys em may inc u es coup ing enses, an /or a eamsp i er e op ica sys em may simultaneously excite the fluorescing liquid, and detect fluorescence
- the plastic cap may be fabricated either through cast plastic, or injection molding [00106]
- the cap readily absorbs the heat emitted from the thermal cycler/liquid so that the cap is heated to a temperature that reduces or eliminates condensation
- the upper area/cap of a sample vessel would be at a cooler temperature than the lower portions of the sample vessel, thus reaction liquid would condense onto the colder surface thereby causing changes in the chemistry of the reaction (e g , changes in concentrations leading to inefficient reactions or faulty results)
- the sample vessel is designed so that the sample vessel extends far enough below the surface level of the liquid so that the top entrance of the sample vessel is far enough from the surface of the sample/PCR cocktail that condensation is prevented
- a lid or downward pressure on the cap forces the sample vessel into a tighter contact with the receptacle, which may retard evaporation
- Means for heating and cooling the composition [00108] The liquid metal or thermally
- the liquid metal or thermally conductive fluid heat block is thermally coupled to a heating component, such as a multi-zone heater and a bias coolmg system
- a heating component such as a multi-zone heater and a bias coolmg system
- the bias cooling system provides a small constant flow of chilled coolant through bias cooling channels in the attached to the base or sides of the heat block This causes a constant, small heat loss from the heat block, which is compensated by a multi-zone heater
- the heater is thermally coupled to the sample block for incubation segments where the temperature of the sample block maintained at a steady value
- the constant small heat loss caused by the bias cooling flow allows the control system to implement proportional control both upward and downward in temperature in small increments This means both heating and cooling at controlled, predictable, small rates is available to the temperature control system to correct for block temperature errors
- the multi-zone heater may be controlled by a CPU
- the heating element is comprised of one or more wire components, such as heat coils (FIG. 10) Such wire components are arranged to provide optimum heating and to reduce or eliminate edge effects
- the wire elements are arranged at the perimeter of the heating block
- wire elements are spaced equidistantly, or in a increasing/decreasing distance gradient from the inside out to the outer perimeter to provide optimum ea ing an re uce or e imina e e ge e ec s n one em o imen e wires are arrange a e o om o e block in a checkerboard pattern
- the wires are arranged in concentric circles or ovals along the bottom of e heat block
- the wires are arranged along the base and sides of the heat block
- the wires are arranged in pattern which allows the liquid metal to flow around all sides of the wires, such as a suspended, stacked or 3 dimensional arrangement comprising more than one checkerboard layer
- the wires are located more closely together near the edges of the heat block than m the interior of the heat block (such as variable spacing) so as to counteract the tendency of the outer edges of
- a coolant control system continuously circulates a chilled liquid coolant such as a mixture of automobile antifreeze and water through bias cooling channels attached to the heat block via input and output tubes
- the coolant control system also controls fluid flow through higher volume ramp cooling fluid flow paths in the heat block
- the ramp cooling channels are used to rapidly change the temperature of the heat block by pumping large volumes of chilled liquid coolant through the block at a relatively high flow rate
- the liquid coolant used to chill the heat block consists mainly of a mixture of water and ethylene glycol
- the liquid coolant is chilled by a heat exchanger which receives liquid coolant which has extracted heat from the sample block via an input tube
- the heat exchanger receives compressed liquid refrigerant (such as freon or ethanol) via an input tube from a refrigeration unit
- This refrigeration unit generally includes a compressor, a fan and a fin tube heat radiator
- the refrigeration unit compresses ref ⁇ gerant received from the heat exchanger
- the heated refrigerant is cooled and condensed to a liquid in the fin tube condenser
- the pressure of the refrigerant is maintained above its vapor pressure in the fin tube condenser by a flow restrictor capillary tube
- the output of this capillary tube is coupled to the input of the heat exchanger.
- the pressure of the ref ⁇ gerant is allowed to drop below its vapor pressure, allowing it to expand In this process of expansion, heat is absorbed from the warmed liquid coolant circulating in the heat exchanger and this heat is transferred to the ref ⁇ gerant thereby causing the refrigerant to boil
- the warmed ref ⁇ gerant is then extracted from the heat exchanger and is compressed and again circulated through the fin tube condenser
- the fan blows air through the fin tube condenser to cause heat in the refrigerant from tube to be exchanged with the ambient air
- the refrigeration is capable of extracting at least 400 watts of heat at 30° C and 100 watts of heat at 10° C from the liquid coolant to support the rapid temperature cycling
- bias cooling may be eliminated or may be supplied by other means such as by the use of a cooling fan and cooling fins formed m the metal of the sample block, Peltier junctions or constantly circulating water (such as distilled or tap water)
- Peltier devices or elements also known as thermoelectric (TE) modules, are small solid-state devices that function as heat pumps
- a typical Peltier unit is a few millimeters thick by a few millimeters to a few centimeters square It is a sandwich formed by two ceramic plates with an array of small Bismuth Telluride (Bi 2 Te 3 ) cubes ("couples") in between When a DC current is applied heat is moved from one side of the device to the other where it can be removed by a heat sink The "cold" side may be used to cool an electronic device such as a microprocessor or a photodetector If the current is reversed the device changes e irec ion in w ic e ea is move e ier evices ac moving par s, o no require re rigeran , o not produce noise or vibration, are small in size, have a long life, and are capable of precision temperature control Temperature control may be provided by using a temperature sensor feedback
- a thermal cycler comprises a liquid metal or thermally conductive fluid heat block thermally coupled to a heating component which is a Peltier element, in order to obtain a desired temperature profile (a temperature curve during a defined time interval) in the liquid metal or thermally conductive fluid heat block ( Figures 1-4)
- a desired temperature profile a temperature curve during a defined time interval
- the Peltier element depending on the temperature to be obtained, is used as a cooling or a heating element within a temperature profile
- the thermal cycler may further comprise an electric resistance heater and a Peltier element used in combination to obtain the required speed of the temperature changes in the liquid metal or thermally conductive fluid heat block and the required precision and homogeneity of the temperature distribution
- the thermal cycler contains at least one Peltier element that forms part of the thermal cycler for cyclic alteration of the temperature of a heat block comprising a liquid composition
- At least one heat transfer surface of the Peltier element is in thermal contact over a large area with the bottom surface of the liquid metal, heat block plate, or heat spreader and the other heat transfer surface is in contact over a large area with a cooling member for heat dissipation
- the cooling member may be a metal such as aluminum or copper
- the thermal cycler may further comprise a fan for heat dissipation which may be optionally switchable
- the liquid metal or thermally conductive fluid heat block and a Peltier element are joined to form a discrete unit ( a swap block) which can be removed from the body of the thermal cycler
- a heat block comprising a liquid composition is thermally coupled to a plurality of Peltier devices situated adjacent one another on a first side of said heat block
- a heat block comprising a liquid composition (501;551;571) is thermally coupled to a plurality of Peltier devices (503;553;573), wherein at least one Peltier device is situated on a first side of said reservoir and at least a second Peltier device is situated on a second side of said reservoir
- at least one Peltier device from a commercially available source, such as Marlow Industrries or Nextreme Thermal Solutions is coupled to a heat block comprising a liquid composition (Nextreme Thermal Solutions, 3040 Cornwallis Road, P O Box 13981, Research Triangle Park, NC 27709-39810)
- the Peltier element is protected from thermodynamic mechanical tension peaks by a central spring-biased securing means which presses the Peltier element and holds it against the heat block
- the Peltier element may be resihently clamped between the heat transfer surfaces of the heat block and the cooling member
- the contact surface of the cooling member can be pressed, for example, by a pressure spring, or similar device, against the Peltier element
- the sp ⁇ ng tension can be adjusted via a screw, a spring washer and a ball and socket joint, which further increases the degrees of freedom of the cooling member n an a erna ive em o imen e e ier e emen is use exc usive y as a co -pro ucing nea remova element. That is, it is only used for cooling a heater unit. This will prolong the useful life of the Peltier element.
- the thermal cycler may incorporate an electric resistance heater disposed around the heat block and along the periphery of the outer wall of the heat block.
- the Peltier element may be used only for cooling. This relieves the Peltier element from mechanical thermal stress and thus contributes to prolonging the service life of the Peltier element in the thermal cycler.
- Lids [00126]
- the liquid metal or thermally conductive fluid heat block is part of a thermal cycler that optionally includes a lid, such as a hinged lid.
- a lid can be fastened to the block by various means (e.g., clip, spring, screws, etc.).
- the lid contains a closing and pressing device for securing the sealed sample vessels positioned in the receptacle of the liquid metal or thermally conductive fluid heat block.
- the lid may seal the sample vessels as it closes.
- a lid may have a spring held pressure plate, which presses each sample vessel with a defined force into the wells of the receptacle of the liquid metal or thermally conductive fluid heat block.
- the lid may further comprise recesses for holding the cap-shaped lids sample vessels and/or openings for piercing by pipetting needles in the pressure plate coaxially with the sample vessels.
- the spring element may comprise a corrugated washer.
- a safety ring prevents the pressure plate from falling out when the hinged lid is opened.
- the lid comprises a heating element.
- the lid of the thermal cycler incorporates a detection mechanism capable of detecting light.
- This lid may further comprise a heating element.
- PCR PCR
- the heated lid comprises a cover that is sized to cover the entire top area of the receptacle
- the lid may have at least two thin plates made of rigid, heat tolerant material, such as ceramics, glass, or silicon rubber. Sandwiched between the plates is an electrical resistive heating element, which in one embodiment may be in the form of a small diameter Nichrome wire or formed by depositing resistive materials such as Nichrome or stannous oxide on one of the plates. For example, a 36 gauge Nichrome wire with a resistivity of 12 Ohms per foot may be used to provide sufficient heating to the cover.
- the heating element may be configured in a serpentine fashion across the area of the plate so as to provide uniform heating across the cover.
- a filler material such as epoxy may be used to secure the plates and to fill the voids between the plates.
- the heating element is connected to a variable power supply, which can be controlled to provide current for heating the cover to a desired temperature.
- the leads between the power supply and the heating element may be flexible and configured to avoid stress in the leads so that the cover can be moved without restriction, e.g. by a robotic means in an automated laboratory workstation.
- a temperature sensor may be provided on the cover to measure its temperature and provide feedback for controlling the power supply for obtaining a desired temperature. .
- n an a erna ive em o imen i is con emp a e a or si ua ions in w ic e empera ure o e samp e vessels is below ambient temperature, it may be desirable to cool the cover to a temperature below ambient but above the temperature of the substance vapor. This is to maintain minimum temperature differential between the cover and the substance so that the temperature of the cover would not affect the controlled temperature of the substance in the receptacle
- the devices of the invention are configured to provide a means of measuring detectable labels m sample vessels comprising a reaction (e g , real time PCR)
- a reaction e g , real time PCR
- Various embodiments of the devices of the invention are fully compatible with detection optics, so that rapid nucleic acid amplification/detection can be carried out
- the thermal cyclers of the present invention by providing uniform temperature, rapid temperatures and increased signal reflectivity, during PCR or related processes, enable more accurate amplification, detection and measurement.
- one advantage is that less expensive optical assemblies can be utilized with a thermal cycler of the invention, and yet obtain as accurate or more accurate real-time measurements Therefore, a thermal cycler of the invention enables rapid, accurate, and reliable DNA amplification and detection, with less expensive optics, as well as conventional optic assemblies
- the measuring optic includes a light source, which for example is formed by a light emitting diode
- the light source is directed toward the measuring field (e g , sample vessel/tube)
- the light source is controlled by a evaluation and control circuit which can be operably linked to a computer, containing computer executable logic for controlling optic measurements
- the measuring optic further includes a detector which can receive light from the entire measuring field The detector is connected with the evaluation circuit/computer
- Optical assemblies useful in various embodiments of the invention comprise those having a CCD imager, a CMOS imager, a line scanner, at least one photodiode, at least one phototransistor, at least one photomultiplier tube, at least one avalanche photodiode, a microlaser, or a q-switched laser.
- the reader can be a reflectance, transmission, epifluorescence/fluorescence, chemo- bioluminescence, magnetic or amperometry reader (or two or more combinations), PIN diode, or other readers known in the art depending on the signal that is to be detected from a sample tube [00135]
- the light sources that can be coupled into an optical assembly of the invention include LEDs, laser diodes, VCSELs, VECSELs, DPSS lasers or fiber optic connections that can be subsequently coupled to light sources such as large laser systems, laser diodes or lamps
- the diode is a laser diode Laser diodes can be used for illumination, photodiode detectors have excellent sensitivity, and most materials have minimal autofluorescence in the pertinent spectral region Any conventional, LED or photodiode may be utilized, such as the PIN photodiodes from Pacific Silicon Sensors (5700 Corsa Avenue, Westlake Village CA, 91362)
- optical assemblies are configured to provide excitation(hght source)/emission (detector) at wavelengths such as- 365/460, 470/510, 530/555, 585/610, 625/660, 680/712 nm
- various filter windows are utilized at +-5-20nm
- Various fluorescence filter sets are commercially available (e.g., Omega Filters, Omega Optical, Inc , or INTORR), including dye-specific filters for single or multi-label fluorescence
- an optical assembly is configured with light sources having the specifications provided in Table 1
- FIG. 3 provides one example of an optical assembly, where a PIN photodiode is the optical detector.
- the optical assembly for detecting fluorescence comprises a light source 603 that provides lights passing through an excitation filter 605 which is directed into a sample vessel by a dichroic reflector/mirror 611 through an objective lens 609 which focuses the light beam into the sample vessel.
- the same lens collects fluorescence generated by the constituents of the sample (e.g., SYBR Green or Cy5; intercalating dyes disclosed herein), which emission passes the dichroic filter 611 and directed back up through a barrier filter 613, focus lens 615 and into the detector module PIN photodiode 617.
- the output is digitized and displayed as a graph electronically or displayed on paper or recorded 619. Furthermore, the use of epifluorescence provides an additional advantage in that there are no size constraints, thus a numeric aperture aspheric or ball-type lenses can be used as a collection optic.
- additional excitation filters can be positioned for increased spectral conditioning.
- the light sources is a Cree XLamp and the optical detector is a T5 and/or Tl 8 PIN diode.
- an optical assembly emits and detects light through the same portion of a sample vessel (e.g., cap).
- an optical assembly light source emits light from one portion of a sample vessel (e.g., into a PCR reaction), while emissions are detected from another portion of the sample vessel (e.g., where light source is at bottom and detector is at top, or vice versa).
- the reader is a LED reader which detects a fluorescence signal.
- the fluorescence signal is excited by a light emitting diode that emits in the region of the optical spectrum and within the absorbance peak of the signal (e.g., fluorescent label).
- the emitted fluorescence signal is detected by a photodiode.
- the wavelength of the signal detected may be limited using a long pass filter which blocks stray emitted light and transmits light with wavelengths at and above the peak emission wavelength of the fluorescence emitting label.
- the long pass filter may be replaced by a band pass filter.
- the excitation light may be limited by a band pass filter.
- the excitation source and the detector are mounted in a single machine (such as the body of a thermal cycler), molded block (e.g., FIGs. 1-2), for simplified reading of the fluorescent signals generated in the sample tubes.
- Cy5 is a popular red-emitting fluorophore with a very high extinction coefficient.
- Common forms of such a fluorophore include the N-hydroxysuccinimide ester of or the related dye, Cy5.5. These yes are in o car ocyanine yes t at are use common y m ow cytometry and automated fluorescence sequencers and are available from Amersham (Pittsburg, Pa ) Cy5 is commercially available as amidites for direct, automated incorporation into oligonucleotides.
- samples labeled with Cy5 are processed utilizing the devices of the invention For example, working in the red/infrared region of the spectrum is advantageous when choosing optical components for instrumentation.
- real-time measurements of PCR amplification can be made utilizing SYBR Green.
- a system comprising a cycler of the invention further comprises a collection of photodetectors, in which each photodetector provides an output signal.
- the system also comprises at least one light source. The light source is positioned such that light emitted passes through a corresponding well retained m or otherwise provided by a multi-well plate (305;405) or strip of sample vessels and to a corresponding photodetector or a collection of photodetectors.
- the system also includes a processor or other means for analyzing the output signals from the plurality of photodetectors.
- the devices of the invention obviate the need to utilize expensive fluorochromes or dye markers, which require large integrated light sources and detection optics on the system.
- an integrated real-tune PCR system can cost $90,000 (Applied Biosystems ABI PRISM.RTM. 7700 Sequence Detection System) as compared to $7,500 for a non-real time PCR system (GeneAmp 9700) Both systems perform PCR amplification for 96 wellplates but the GeneAmp 9700 requires a separate spectrophotometer or a fluorescent wellplate reader for DNA concentration measurement
- LEDs or lasers of different wavelength could be integrated into a single package or several packaged LEDs/laser can be very closely spaced to excite one well/sample tube.
- the LED actually represents several LEDs with different wavelengths, but very closely spaced. Therefore, in further embodiments, the detectors are similarly configured.
- light sources for optics configurations of the invention can be an LED or a laser diode.
- an LED is activated to emit light of a first wavelength or first set of wavelengths, which excites a dye or label in a sample that which emits a signal.
- the signal is detected by the appropriate photodetector and then, or concurrently, a second LED is activated, which emits light having a different wavelength or set of wavelengths from the first LED.
- This light excites a different dye or label in a sample, which emits a signal that is detected by the appropriate photodetector.
- the second LED is de-activated and then, or concurrently, a third LED is activated.
- the third LED emits light having a different wavelength or set of wavelengths from the first
- LED and second LED This light excites a different dye or label in a sample, which emits a signal that is detected by the approp ⁇ ate photodetector. These measurements are performed very rapidly and processed by a computer generated display.
- the collection of closely spaced light sources can be configured to sequentially emit light as described [00149]
- different light sources are configured in the optical assembly and separately detected using optical detectors, wherein detection is at the same time. n one em o imen , e evices o e inven ion are con igure wi an array ot light sources and lig detectors.
- the light source array includes a plurality of light sources disposed on a suitable substrate or mounting component and arranged in a particular configuration, which typically is a grid pattern.
- the well array comprises a plurality of sample wells (FIG. 1,2), also supported and positioned on or within a suitable retaining substrate.
- the photodetector array includes a plurality of photodetectors similarly mounted and arranged to receive light emitted from an array of sample vessels (305;405) such as through sample vessel caps (303;403) (FIG. 3).
- a rail system comprising multiple separate light sources (of the same or multiple different wavelengths) can be configured over a row of samples in a multi-row arrangement, along with the corresponding number of detectors.
- the number of light sources and corresponding detectors can be 8 separate light sources of perhaps multiple wavelengths, detected by 8 different detector setup each which can detect multiple wavelengths emitted from an excited sample. Therefore, the rail system can be operated to move back and forth interrogating successive rows of sample vessels.
- Various commercially available filters can be utilized as described herein above.
- the detectors comprise optics made cheaply through injection molding.
- higher power LED's can be used.
- Avalanche photodiodes e.g. SiC, GaN, GaAs or Si or blue enhance Si photodiodes can also be used.
- the sensitivity of avalanche photodiodes is much greater than simple photodiodes because the signal is amplified by the avalanche process.
- Avalanche photodiodes have a typical gain of between 10 and 10000, which means that the signal is amplified by a factor of 10 to 10000.
- Using improved measurement techniques like lock-in amplifiers can also extend the dynamic range of the measurements.
- an LED light source contemplated is a GaN-based ultra-violet LED.
- the light source is cree xlamp 750 mwatt ultra bright LEDs.
- a single wavelength emission is utilized. No additional grating or filter or complex optics are required to select the desired wavelength and focus the light. That significantly simplifies the experimental setup and reduces the cost of assembly.
- Another advantage is the low cost of LED's. Semiconductor LED's can be mass-produced and made extremely inexpensive. Current GaN-based violet, blue and green LED's cost on the order of 10 to 50 cents per packaged device and a similar price range is to be expected for future mass-produced GaN-based LED's. Multiple wavelength LED's can be either integrated as hybrid LED chips or special LED's could be developed, whose emission can be switched between two or more wavelengths. [00154] Yet another advantage relates to the high output power of LED's.
- LED's output power levels in the range of 10 mW are within reach in the near future.
- the typical footprint of an LED is 200 um x 200 um, which means that the light intensity can be concentrated to a smaller area and reduces the amount of additional optics (e.g. lenses) needed to concentrate the light. That is a considerable advantage for high throughput PCR systems with a large number of wells per plate (48, 96, 384, or 1536 wells) and consequently smaller wells. Even with 48, 96 or 384 wells, well dimensions are still large enough to fit several LEDs in the area of a single well in a wellplate.
- LED arrays can also be arranged in one- or two-dimensional LED arrays. This enables the measurement of the fluorescence in multiple wells at the same time assive para e processing signi cant y re uces t e measurement time and accelerates t e throughput This can be a significant cost/time factor in the operation of a PCR system, particularly with the trend going to higher-density wellplates
- LED's can be pulsed In order to avoid bleaching or heating of the DNA molecules, short, but intense pulses can be produced by LED's LED's can be turned on and off in a very short time scale (about 1 ns to 100 ns), depending on the design, the size and the packaging of the LED The LED pulses can also be synchronized with a photodetector readout using common lock-in techniques to achieve better signal to noise ratios and higher sensitivity ranges. In addition, LED's do not require any warm-up time before stable light output is achieved This is in contrast to deuterium and Xenon lamps, which require at least several minutes of warm-up time, before stable operation is achieved
- a method of performing a polymerase chain reaction assay with fluorescence detection comprises providing a system that includes a multi-well plate, a thermal cycler, a photodetector that provides an output signal, at least one light source positioned to pass light through the multi-well plate and to the photodetector, and a means for analyzing the output signal of the photodetector
- the method also comprises obtaining samples upon which the polymerase chain reaction assay is to be performed Additionally, the method comprises depositing samples in the multi-well plate And, the method comprises performing a polymerase cham reaction in the samples
- the method includes emitting light from the light source(s) such that light passes through the samples to the photodetector And, the method comprises analyzing the output of the photo
- a method of performing a polymerase cham reaction assay is provided The method is based upon using light as follows The method mc u es provi ing a sys em inc u ing i a mu i-we p a e a ap e o re ain a piuranry or samples, ⁇ a thermal cycler, (iii) a photodetector that provides an output signal, (iv) a plurality of light sources positioned such that light emitted passes through the multi-well plate to the photodetector, and (v) a means for analyzing the output signal of the photodetector upon detecting ultra-violet light.
- the method also comprises obtaining samples upon which the polymerase chain reaction assay is to be performed.
- the method further comprises depositing the samples in the multi-well plate.
- the method also comprises performing a polymerase chain reaction in the samples.
- the method further comprises emitting light from the plurality of light sources such that the light passes through the samples to the photodetector.
- the method also includes analyzing the output signal of the photodetector.
- turn on/off times for LEDs are typically on the order of a few nanoseconds or tens of nanoseconds. In other embodiments, laser diodes are even faster with sub nanosecond switching times.
- a PCR system is provided and related assays and techniques that do not require the use of fluorophores and associated detection light sources and optics otherwise required.
- LED could contain a 260 ran and a 280 ran LED. To perform an absorbance measurement, first the 260 run LED would be turned on and the absorbed light at 260 ran detected by the photodetector. The
- the optical configuration can reduce the cost of fluorescent primers and Taqman probes utilized with many real-time PCR systems.
- an array of inexpensive ultra-violet LED's, emitting 260 nm and 280 nm wavelength light are incorporated in a PCR system with the format of a wellplate in thermal contact (directly/indirectly) with a liquid medium (e.g., liquid metal or a thermal fluid).
- the arrays of emitter and detectors have a configuration, that may correspond to the arrangement of sample vessels in the liquid composition heat block of the thermal cycler, for example, like the sample vessels (305;405) in FIG. 3.
- a plurality of ultra-violet light emitting units, such as LED's are positioned with respect to a plurality of wells FIG.
- LEDs sometimes exhibit luminescence at longer wavelengths from recombination through defects in the LEDs active regions or recombination of carriers outside the active region of the LED. This unwanted luminescence is typically 3 to 4 orders smaller than the main luminescence peak. Nevertheless it may be desirable to suppress this luminescence even further by using optical band-pass filters.
- optical assembly systems known in the art can be configured for use with a thermal cycler device of the invention. Examples of such optical assemblies as well as reagents useful in detecting reaction products are disclosed in US Patent Application Nos.
- a control assembly is operatively linked to a thermal cycler of the invention
- a control assembly for example, comprises a programmable computer comprising computer executable logic that functions to operate any aspect of the devices, methods and/or systems of the invention
- the control assembly can turn on/off motors, fans, heating components, stir bars, contmuous flow devices and optical assemblies
- the control assembly can be programmed to automatically process samples, run multiple PCR cycles, obtain measurements, digitize measurements into data, convert data into charts/graphs and report
- Computers for controlling instrumentation, recording signals, processing and analyzing signals or data can be any of a personal computer (PC), digital computers, a microprocessor based computer, a portable computer, or other type of processing device
- a computer comprises a central processing unit, a storage or memory unit that can record and read information and programs using machine -readable storage media, a communication terminal such as a wired communication device or a wireless communication device, an output device such as a display terminal, and an input device
- control assembly generates the sampling strobes of the optical assembly, the rate of which is programmed to run automatically
- timing is adjustable for shining a light sources and operating a detector to detect and measure signals (e g , fluorescence)
- an apparatus comprising a control assembly further comprises a means for moving sample vessels into apertures, such as wells in the receptacle of a heat block comprising a liquid composition
- said means could be a robotic system comprising motors, pulleys, clamps and other structures necessary for moving sample vessels
- Sample preparation station In some aspects of the invention, the devices/systems of the invention are operatively linked to a robotics sample preparation and/or sample processing unit
- a control assembly can provide a program to operate automated collection of samples, adding of reagents to collection tubes, processing/extracting nucleic acids from said tubes, optionally transferring samples to new tubes, adding necessary reagents for a
- a thermal cycler comprising a liquid metal or a thermally conductive fluid heat block can be used for disease diagnosis, drug screening, genotyping individuals, phylogenetic classification, environmental surveillance, parental and forensic identification amongst other uses Further, nucleic acids can be obtained from any source for experimentation using a liquid metal or a thermally conductive fluid heat block.
- a test sample can be biological and/or environmental samples
- Biological samples may be derived from human, other animals, or plants, body fluid, solid tissue samples, tissue cultures or cells derived therefrom and the progeny thereof, sections or smears prepared from any of these sources, or any other samples suspected to contain the target nucleic acids
- Exemplary biological samples are body fluids including but not limited to blood, urine, spmal fluid, cerebrospinal fluid, smovial fluid, ammoniac fluid, semen, and saliva
- Other types of biological sample may include food products and ingredients such as vegetables, dairy items, meat, meat by-products, and waste
- Environmental samples are derived from environmental mate ⁇ al including but not limited to soil, water, sewage, cosmetic, agricultural, industrial samples, air filter samples, and air conditioning samples
- a thermal cycler comprising a liquid metal or a thermally conductive fluid heat block can be used in any protocol or experiment that requires either thermal cycling or a heat block that can accurately maintain a uniform temperature
- said thermal cycler can
- the apparatus of this invention allows one to perform PCR with increased speed and specificity, particularly in the context of real time PCR
- a composition with high thermal conductivity such as a liquid metal
- measuring signal from a discrete portion of the test receptacle, such as the top relieves one of the need to remove sample vessels from the heating composition for measurement This also preserves temperature control and allows measurements to be made in real time with the heating cycles
- a reflecting mate ⁇ al that prevents escape of signal except from the discrete location allows less sensitive detectors to be used as more light can be collected for measurement
- PCR reaction conditions typically comprise either two or three step cycles Two step cycles have a denaturation step followed by a hybridization/elongation step Three step cycles comprise a denaturation step followed by a hybridization step during which the primer hybridizes to the strands of DNA, followed by a separate elongation step
- the polymerase reactions are incubated under conditions m which the primers hybridize to the target sequences and are extended by a polymerase
- the amplification reaction cycle conditions are selected so that the primers hybridize specifically to the target sequence and are extended.
- Primers are typically designed so that all of the primers participating in a particular reaction have melting temperatures that are within at least 5°C, and more typically within 2°C of each other. Primers are further designed to avoid priming on themselves or each other. Primer concentration should be sufficient to bind to the amount of target sequences that are amplified so as to provide an accurate assessment of the quantity of amplified sequence. Those of skill in the art will recognize that the amount of concentration of primer will vary according to the binding affinity of the primers as well as the quantity of sequence to be bound.
- the liquid metal or thermally conductive fluid heating block may be used for PCR, either as part of a thermal cycler or as a heat block used to maintain a single temperature.
- a sample comprising a DNA polynucleotide and a PCR reaction cocktail is denatured by treatment in a liquid metal or thermally conductive fluid heat block at about 90-98 0 C for 10-90 seconds.
- the denatured polynucleotide is then hybridized to oligonucleotide primers by treatment in a liquid metal or thermally conductive fluid heat block at a temperature of about 30-65 0 C for 1-2 minutes.
- Chain extension then occurs by the action of a DNA polymerase on the polynucleotide annealed to the oligonucleotide primer. This reaction occurs at a temperature of about 70-75°C. for 30 seconds to 5 minutes in the liquid metal or thermally conductive fluid heat block. Any desired number of PCR cycles may be carried out depending on variables including but not limited to the amount of the initial DNA polynucleotide, the length of the desired product and primer stringency. [00182] In another embodiment, the PCR cycle comprises denaturation of the DNA polynucleotide at a temperature of 94°degree C for about 1 minute.
- the hybridization of the oligonucleotide to the denatured polynucleotide occurs at a temperature of about 37°-65° C for about one minute.
- the polymerase reaction is carried out for about one minute at about 72. degree. C. All reactions are carried out in a multiwell plate which is inserted into the wells of a receptacle in a liquid metal or thermally conductive fluid heat block. About 30 PCR cycles are performed.
- the above temperature ranges and the other numbers are not intended to limit the scope of the invention. These ranges are dependant on other factors such as the type of enzyme, the type of container or plate, the type of biological sample, the size of samples, etc.
- One of ordinary skill in the art will recognize that the temperatures, time durations and cycle number can readily be modified as necessary.
- Revere transcription refers to the process by which mRNA is copied to cDNA by a reverse transcriptase (such as Moloney murine leukemia virus (MMLV) transcriptase Avian myeloblastosis virus (AMV) transcriptase or a variant therof) composed using an oligo dT primer or a random oligomers (such as a random hexamer or octamer).
- MMLV Moloney murine leukemia virus
- AMV myeloblastosis virus
- a reverse transcriptase that has an endo H activity is typically used. This removes the mRNA allowing the second strand of DNA to be formed.
- Reverse transcription typically occurs as a single step before PCR.
- the RT reaction is performed in a liquid metal or thermally conductive fluid heat block by incubating an RNA sample a transcriptase the necessary u or a u an our a a ou , we y mcuoation ior aoou minutes at about 45°C followed by incubation at about 95°C.
- the cDNA product is then removed and used as a template for PCR
- the RT step is followed sequentially by the PCR step, for example in a one-step PCR protocol
- all of the reaction components are present in the sample vessel for the RT step and the PCR step
- the DNA polymerase is blocked from activity until it is activated by an extended incubation at 95°C for 5-10 minutes.
- the DNA polymerase is blocked from activity by the presence of a blocking antibody that is permanently inactivated during the 95 0 C incubation step
- a blocking antibody that is permanently inactivated during the 95 0 C incubation step
- Real Time PCR In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is used to simultaneously quantify and amplify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample, and if it is present, the number of copies in the sample.
- QRT-PCR quantitative real time polymerase chain reaction
- kinetic polymerase chain reaction kinetic polymerase chain reaction
- real-time polymerase chain reaction is combined with reverse transcription polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type
- mRNA messenger RNA
- the amplified products are directly visualized with detectable label such as a fluorescent DNA-bindmg dye
- the amplified products are quantified using an intercalating dye, including but not limited to SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste,
- SYBR gold ethidium bromide, ac ⁇ dmes, proflavine, ac ⁇ dine orange, ac ⁇ flavine, fluorcoumanm, elhpticine, daunomycm, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypy ⁇ dyls, anthramycm
- a DNA binding dye such as SYBR Green binds all double stranded (ds)DNA and an increase in fluorescence intensity is measured, thus allowing initial concentrations to be determined
- a standard PCR reaction cocktail is prepared as usual, with the addition of fluorescent dsDNA dye and added to a sample.
- the reaction is then run in a liquid heatblock thermal cycler, and after each cycle, the levels of fluorescence are measured with a camera
- the dye fluoresces much more strongly when bound to the dsDNA (i e PCR product)
- the amount of the dye intercalated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products
- one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using the optical systems of the present invention or other suitable instrument in the art
- the dsDNA concentration in the PCR can be determined
- the results obtained for a sequence of interest may be normalized against a stably expressed gene ("housekeeping gene") such as actm, GAPDH, or 18s rRNA [00190J
- labels/dyes detected by systems or devices of the invention The term "Label” or "dye” refers to any substance which is capable of producing a signal that is detectable by visual or instrumental
- detectable molecules include but are not limited to fluorophores as well as others known in the art as described, for example, in Principles of Fluorescence Spectroscopy, Joseph R Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the 6 th Edition of the Molecular Probes Handbook by Richard P. Hoagland
- Intercalating dyes are detected using the devices of the invention include but are note limited to phenanthridines and acridines (e g , ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer- 1 and -2, ethidium monoazide, and ACMA), some minor grove binders such as indoles and imidazoles (e g , Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI), and miscellaneous nucleic acid stains such as ac ⁇ dme orange (also capable of intercalating), 7-AAD, actinomycin D, LDS751, and hydroxy stilbamidine All of the aforementioned nucleic acid stains are commercially available from suppliers such as Molecular Probes, Inc.
- nucleic acid stains include the following dyes from Molecular Probes cyanme dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-I, POPO-3, YOYO-I, YOYO-3, TOTO-I, TOTO-3, JOJO-I, LOLO-I, BOBO-I, BOBO-3, PO-PRO-I, PO-PRO-3, BO-PRO-I, BO-PRO-3, TO-
- Other detectable markers include chemiluminescent and chromogenic molecules, optical or electron density markers, etc.
- labels comprise semiconductor nanocrystals such as quantum dots (i e , Qdots), described inU S Pat No 6,207,392 Qdots are commercially available from Quantum Dot . o ⁇ ora ion.
- e semicon uc or nanocrys a s use u in e prac ice o e invention include nanocrys a s of Group H-VI semiconductors such as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixed compositions thereof; as well as nanocrystals of Group IH-V semiconductors such as GaAs, InGaAs, InP, and InAs and mixed compositions thereof.
- Group H-VI semiconductors such as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS
- Group IV semiconductors such as germanium or silicon, or the use of organic semiconductors, may also be feasible under certain conditions.
- the semiconductor nanocrystals may also include alloys comprising two or more semiconductors selected from the group consisting of the above Group HI-V compounds, Group II- VI compounds, Group IV elements, and combinations of same.
- other luminescent labels such as sequence specific probes can be employed in the amplification reaction to facilitate the detection and quantification of the amplified product. Probe based quantitative amplification relies on the sequence-specific detection of a desired amplified product.
- fluorescent oligonucleotide probes are used to quantify the DNA.
- Fluorescent oligonucleotides (primers or probes) containing base-linked or terminally-linked fluors and quenchers are well-known in the art. They can be obtained, for example, from Life Technologies (Gaithersburg, Md.), Sigma-Genosys (The Woodlands, Tex.), Genset Corp. (La Jolla, Calif), or Synthetic Genetics (San Diego, Calif.).
- Base-linked fluors are incorporated into the oligonucleotides by post-synthesis modification of oligonucleotides that are synthesized with reactive groups linked to bases.
- fluorescein fluorescein isothiocyanate (FITC), carboxy tetrachloro fluorescein (TET), NHS-fluorescein, 5 and/or 6-carboxy fluorescein (FAM), 5- (or 6-) iodoacetamidofluorescein, 5- ⁇ [2(and 3)-5-(Acetylmercapto)-succinyl] amino ⁇ fluorescein (SAMSA- fluorescein), and other fluorescein derivatives, rhodamine, Lissamine rhodamine B sulfonyl chloride, Texas red sulfonyl chloride, 5 and/or 6 carboxy rhodamine (ROX) and other r
- fluorophores Cascade Blue.TM. fluorophores such as 8-methoxypyrene-l,3,6- trisulfonic acid trisodium salt, Lucifer yellow fluorophores such as 3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins derivatives, Alexa fluor dyes (available from Molecular Probes, Eugene, Oreg.) and other fluorophores known to those of skill in the art.
- Alexa fluor dyes available from Molecular Probes, Eugene, Oreg.
- fluorophores for a general listing of useful fluorophores, see also Hermanson, G. T., BIOCONJUGATE TECHNIQUES (Academic Press, San Diego, 1996). [00196] Embodiments using fluorescent reporter probes produce accurate and reliable results. Sequence specific
- RNA or DNA based probes are used to specifically quantify the probe sequence and not all double stranded DNA. This also allows for multiplexing - assaying for several genes in the same reaction by using specific probes with different-colored labels.
- PCR is carried out in a thermal cycler comprising a the liquid metal or thermally conductive fluid heat block comprising a liquid composition.
- the thermal cycler further comprises an optical assembly.
- the liquid metal or thermally conductive fluid heat block rapidly and uniformly modulates the temperature of samples contained within sample vessels to a ow e ec ion o amp i ica ion pro uc s in rea ime.
- ano er em o iment trie detection is via a nonspecific nucleic acid label such as an intercalating dye, wherein the signal index, or the positive fluorescence intensity signal generated by a specific amplification product is at least 3 times the fluorescence intensity generated by a PCR control sample, such as about 3.5, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5,10, 10.5, or 11.
- the thermal cycler may modulate the sample temperature by more than 10 0 C per second, such as 10.5 0 C per second.
- an RNA based probe with a fluorescent reporter and a quencher held in adjacent positions is used.
- the close proximity of the reporter to the quencher prevents its fluorescence, it is only after the breakdown of the probe that the fluorescence is detected. This process depends on the 5' to 3' exonuclease activity of the polymerase used in the PCR reaction cocktail.
- the reaction is prepared as usual, with the addition of the sequence specific labeled probe the reaction commences. After denaturation of the DNA the labeled probe is able to bind to its complementary sequence in the region of interest of the template DNA.
- the polymerase is activated and DNA extension proceeds. As the polymerization continues it reaches the labeled probe bound to the complementary sequence of DNA.
- the polymerase breaks the RNA probe into separate nucleotides, and separates the fluorescent reporter from the quencher. This results in an increase in fluorescence as detected by the optical assembly. As PCR progresses more and more of the fluorescent reporter is liberated from its quencher, resulting in a well defined geometric increase in fluorescence. This allows accurate determination of the final, and initial, quantities of DNA.
- devices of the invention can be utilized for in vitro diagnostic uses, such as detecting infectious or pathogenic agents.
- PCR is conducted using a device of the invention to detect various such agents, which can be any pathogen including without any limitation bacteria, yeast, fungi, virus, eukaryotic parasites, etc; infectious agent including influenza virus, parainfluenza virus, adenovirus, rhinovirus, coronavirus, hepatitis viruses A, B, C, D, E, etc, HIV, enterovirus, papillomavirus, coxsackievirus, herpes simplex virus, or Epstein-Barr virus; bacteria including Mycobacterium, Streptococcus, Salmonella, Shigella, Staphylococcus, Neisseria, Pseudomonads, Clostridium, or E. coli. It will be apparent to one of skill in the art that the PCR, sequencing reactions and related processes are readily adapted to the devices of the
- One advantage of the devices of the invention is the capability to perform ultra fast PCR, which provides relatively faster times for diagnostic purposes. For some applications (e.g., detection of biothreat agents, intra-operative diagnostic testing), rapid PCR is a benefit. One important requirement for rapid real-time
- PCR are a thermal cycler that allows rapid heating, cooling, and thermal transfer, and a signal generation system that is compatible with the short cycle times associated with fast PCR.
- the thermal cyclers of the invention can provide rapid cooling (e.g., 5-17°C/second) and rapid heating (e.g., 10- 44°C/second).
- the devices of the invention can provided cycle times as low as about 2 seconds if desired.
- ultra fast PCR processes can be conducted by coupling the devices of the invention with reagents known in the art to facilitate faster results, in both amplification and time required to produce a detectable signal.
- Such reagents are known in the art, such as disclosed in U.S. Patent Application No. 2005/0164219. one of these that we have used is a faster polymerase such as KOD. or examp e, specia ize a e e primers can provi e signa genera ion a is instantaneous
- Stainless steel PCR reaction sample vessels have been demonstrated as a suitable vessels in which to conduct polymerase chain reactions
- the vessels used for initial tests were manufactured from stainless steel tubes with a final outer diameter of 061 niches A two-inch length of sample vessel was closed at one end with a press-fit stainless steel plug
- Example 2 [00211] Realtime PCR
- liquid metal or thermally conductive fluid heat block is well suited for real time PCR reactions because of the fast temperature ramping, thermal uniformity and reflectivity of the liquid metal or thermally conductive fluid
- Sample vessels are prepared by placing PCR reaction components into the sample vessels and sealing the vessels to prevent spillage or cross-contamination
- the reaction components include buffer, target nucleic acid, appropriate primers and probes, nucleotides, polymerases, as well as optional additional components
- four fluorescent probes are included, each adapted to detect a different target sequence, and a particular reaction vessel may include any one or more of the fluorescent probes Each probe advantageously responds to light of a different incident wavelength and emits light of a different wavelength
- a detection module is mounted above the heat block
- the detection module includes four detection channels Each channel is optimized for a different one of the fluorescent probes included in sample vessels
- the sample vessels are placed into the heat block receptacle wells
- the lid assembly is closed and positioned over the heat block
- Each channel of detection module is calibrated
- Calibration is performed by operating stepper motors to position the detection module such that at least one of its channels is in optical communication with a calibration location
- Each calibration location provides a known fluorescent response Accordingly, ca i ration measuremen s can e use to correct su sequen samp e measurements tor variations or fluctuations m detector response
- Numerous calibration techniques are known in the art When a detection module with multiple channels is used, each channel may be independently calibrated
- a PCR cycle is performed.
- the thermal cycler controls the liquid metal or thermally conductive fluid heat block to regulate the temperature of the sample vessels thereby holding the sample vessels at desired temperatures for desired lengths of time to complete a two-step or three-step PCR cycle
- the optical assembly scans and interrogates the sample vessels
- the LED or other light source such as a laser
- the LEDs of different channels are operated in parallel, in an alternative embodiment, they are operated sequentially so as to avoid reflected LED light from one channel causing false signals in the photo detector of another channel
- the operation of optical assembly may be controlled by an external computer or by a controller built into the thermal cycler
- the measurements are synchronized with the operation of the thermal cycler, so that measurements are identifiable as corresponding to particular times in the PCR process
- a controller component of the thermal cylcer may automatically turn on and off one or more fans that are optionally attached to the thermal cycler or coupled to one or more heat sink
- the resulting fluorescence is detected by the corresponding photodiode or other detector of the channel, which is read out to the external computer
- the detectors may be read out in various ways For instance, a peak signal may be detected, the signal may be integrated over a time interval, or the decay of the fluorescent signal after the LED has been deactivated may be measured [00218] These steps are repeated with the position of the detection module being changed each time so that each channel of detection module eventually interrogates each of the sample vessels In one embodiment, scanning and interrogating four channels for each of 96 sample wells takes about 15 seconds.
- the external computer may execute a program that enables a user to view measurement data as they are collected, in graphical and/or tabular form
- the real-time fluorescence measurements from process are used to detect and quantify the presence of each target sequence
- Such measurements may also be used for purposes such as determining reaction rates and adjusting reaction parameters for improved efficiency, as well as determining when additional reaction cycles are no longer needed in a particular experiment (e g , when a sufficient quantity of a target sequence has been produced)
- process is illustrative and that variations and modifications are possible Steps described as sequential may be executed in parallel, order of steps may be varied, and steps may be modified or combined
- fluorescence measurements may be performed at any point during a PCR cycle, performed multiple times during each PCR cycle (including substantially continuous scanning of the sample wells), or not performed until after some number of PCR cycles Any number of distinguishable fluorescent probes may be used in a single reaction vessel, and the detection module may be adapted to mclude at least as many channels as
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Abstract
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Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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EP12178744.4A EP2535427A3 (en) | 2006-05-17 | 2007-05-17 | Thermal cycling system |
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US80117806P | 2006-05-17 | 2006-05-17 | |
US83249206P | 2006-07-21 | 2006-07-21 | |
US87308406P | 2006-12-06 | 2006-12-06 | |
US87317206P | 2006-12-06 | 2006-12-06 | |
PCT/US2007/069197 WO2008070198A2 (en) | 2006-05-17 | 2007-05-17 | Thermal cycling system |
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EP2027251A2 true EP2027251A2 (en) | 2009-02-25 |
EP2027251A4 EP2027251A4 (en) | 2010-05-05 |
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EP07783905A Ceased EP2027251A4 (en) | 2006-05-17 | 2007-05-17 | Thermal cycling system |
EP12178744.4A Withdrawn EP2535427A3 (en) | 2006-05-17 | 2007-05-17 | Thermal cycling system |
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EP12178744.4A Withdrawn EP2535427A3 (en) | 2006-05-17 | 2007-05-17 | Thermal cycling system |
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Also Published As
Publication number | Publication date |
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WO2008070198A3 (en) | 2009-04-16 |
WO2008070198A2 (en) | 2008-06-12 |
EP2027251A4 (en) | 2010-05-05 |
EP2535427A2 (en) | 2012-12-19 |
EP2535427A3 (en) | 2013-04-24 |
JP2009537152A (en) | 2009-10-29 |
WO2008070198A8 (en) | 2008-12-24 |
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