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EP1666150B1 - Präparieren von Nucleinsäuren - Google Patents

Präparieren von Nucleinsäuren Download PDF

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Publication number
EP1666150B1
EP1666150B1 EP20050024993 EP05024993A EP1666150B1 EP 1666150 B1 EP1666150 B1 EP 1666150B1 EP 20050024993 EP20050024993 EP 20050024993 EP 05024993 A EP05024993 A EP 05024993A EP 1666150 B1 EP1666150 B1 EP 1666150B1
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EP
European Patent Office
Prior art keywords
volume
thermocycles
amplification chamber
reaction mixture
amplification
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EP20050024993
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English (en)
French (fr)
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EP1666150A1 (de
Inventor
Martin Kopp
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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Priority claimed from EP04027624A external-priority patent/EP1658898A1/de
Application filed by F Hoffmann La Roche AG, Roche Diagnostics GmbH filed Critical F Hoffmann La Roche AG
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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
    • 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
    • B01L2300/0803—Disc shape
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • 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
    • 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
    • B01L2300/1861—Means for temperature control using radiation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break
    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes

Definitions

  • the present invention is related to a method of preparing nucleic acids from a template nucleic acid, a diagnostic device for preparing nucleic acids from a template, a computer program for controlling a method for the preparation of nucleic acids from a template nucleic acid using thermocycles, a computer program product comprising said program, an apparatus for preparing nucleic acids and a method for determining the presence or absence or amount of a template nucleic acid in a sample.
  • the sample containing the nucleic acid to be amplified is repeatedly subjected to a temperature profile reflecting the steps of primer hybridization to the target nucleic acid, elongation of said primer to prepare an extension product using the nucleic acid to be copied as a template and separating the extension product form the template nucleic acid.
  • the temperature profile is applied several times, allowing the repetition of the steps, including hybridization and elongation of a second primer capable of hybridizing to the extension product of the first primer.
  • Each repeatedly performed temperature profile is called a thermocycle.
  • the time necessary for conducting an amplification reaction to a great extent depends on the reaction volume used. For example, when conducting a PCR reaction in a 50 - 100 ⁇ l volume on a thermocycler instrument as the PCR System 9700 instrument (Applied Biosystems), a reaction time of two to four hours is needed. Most of this time is needed for changing the temperature of the reaction mixture to conduct the thermocycles. This can be speed up by several means. Firstly, the shape of the reaction vessel can be changed to get an increased surface allowing a faster heating and cooling regime. Secondly, the reaction volume can be decreased so that less volume needs to be heated and cooled. By these means, thermocyclers like the LightCycler® (Roche Diagnostics) allow to decrease the reaction time up to several minutes instead of hours.
  • reaction volumes have the disadvantage that also only small volumes of sample can be added to the reaction, which will proportionally reduce the limit of detection (LOD).
  • LOD limit of detection
  • the increased surface of such reaction chambers can inhibit the reaction.
  • WO2004/51218 there is disclosed a method for detecting different analytes wherein after a multiplex amplification of all ingredients of the reaction mixture the reaction mixture is split into aliquots and the aliquots be treated with reagents for specific amplification of specific analytes in separate reactions.
  • This method has the disadvantage that it needs additional reagents for the second amplification.
  • WO 02/20845 there is disclosed a method for avoiding primer-dimer formation by using a first amplification reaction with low primer concentration, then adding more primers and performing more amplification steps. Again, this method has the disadvantage that at a certain stage during amplification, the reaction tube must be opened to add more reagents. This is both inconvenient for the workflow in a laboratory and problematic for contamination reasons. In addition the use of a standard thermocycler does not allow very fast cycling speeds.
  • the invention is directed to a method of preparing nucleic acids from a template nucleic acid by subjecting a sample to thermocycles comprising the steps:
  • the integral heating and cooling speed preferably is at least 2 Kelvin/ second (K/s) in step a) and higher in step b), preferably at least 5 K/s.
  • the invention is directed to a diagnostic device for preparing nucleic acids from a template comprising
  • the invention is directed to a computer program for controlling a method for the preparation of nucleic acids from a template nucleic acid using thermocycles, characterized in that the computer program is set to apply a first number of thermocycles to the sample and subsequently a second number of thermocycles having a shorter cycling time on a smaller volume of a reaction mixture originating from the same sample.
  • the invention is directed to a computer program product comprising said program on a physical storage means.
  • the invention is directed to an apparatus for preparing nucleic acids comprising
  • a method for determining the presence or absence or amount of a template nucleic acid in a sample comprising the above described nucleic acids preparation method and detecting the formation of nucleic acids as a measure of the presence or absence or amount of nucleic acids to be determined, is also disclosed.
  • One aspect of the present invention is directed to a method of preparing nucleic acids from a template nucleic acid.
  • a first amount of a sample is subjected to a first number of thermocycles to prepare a first amount of a first reaction mixture.
  • An aliquot/ partial amount of that first reaction mixture is then subjected to a second number of thermocycles to prepare a second amount of a second reaction mixture.
  • the time per thermocycle can be decreased compared to the time necessary for thermocycling the first reaction mixture because of the reduced thermal diffusion distance.
  • the first few thermal cycles are the most critical for the specificity of the amplification and need therefore very precise themperature levels without major over- respectively undershooting.
  • a reaction volume of around 5 to 200 ⁇ l in the first reaction step provides sufficient volume to add enough of a nucleic acid preparation derived from a sample material to be analysed so that also very sensitive amplification methods are possible.
  • the second part of additional 40-50 cycles is mainly needed to create a detectable signal level. According to these needs the cycler, the amplification chamber and the feedback control can be adjusted either to very accurate temperature levels or speed.
  • the well confined, compact second amplification volume leads to a highly sensitive optical setup. This principle is being illustrated in Figure 1 .
  • This method preferably is being based on the PCR-method, but also other methods can be used, such as linear or exponential nucleic acid amplification methods. Exponential amplification methods are well known in the art. Especially suitable are methods like PCR ( US 4,683,202 ) and LCR ( US 5,185,243 , US 5,679,524 and US 5,573,907 ), in which the reaction mixture is repeatedly subjected to different temperatures (thermocycles).
  • the amount of sample, first and second number and length of thermocycles depend on the concrete purpose and amplification method used.
  • the first amount of sample typically has a volume of 5 ⁇ l to 200 ⁇ l, preferably 5 ⁇ l to 50 ⁇ l.
  • the further reagent necessary for conducting an amplification reaction can be added to the sample in dry form, for example as a deposit in the first amplification chamber, which deposit is solubilized by addition of the sample.
  • These reagents can also be added in solution, typically in a volume of 2.5 to 100 ⁇ l, more preferably in a volume of 2.5 to 25 ⁇ l.
  • the sample is then subjected in a first amplification chamber to a first number of thermocycles, which are typically 3 to 15 thermocycles, more preferably 5 to 8.
  • thermocycle greatly varies between the different amplification methods.
  • PCR it typically varies between 20 seconds to 5 minutes, more preferably 20 to 120 seconds.
  • an amplification chamber is used having an integral heating and cooling speed of at least 2 Kelvin/ second, more preferably between 4 to 7 K/s.
  • the integral heating respectively cooling speed can be described as the temperature step divided by the time needed to switch from one temperature level to the next temperature level. This is the relevant parameter in thermocycler instruments that can lead to faster PCR protocols. Typically these steps are from 95°C to 60°C, 60°C to 72°C and 72°C to 95°C. Therefore, in the context of the present invention integral heating and cooling speed is understood as the speed of a given amplification chamber and a given reaction volume in the temperature range of around 60°C and 95°C. This integral heating and cooling speed is affected by the means used in the thermocycler for heating and cooling as well as by the size of the amplification chamber which determines the volume of the reaction mixture to be amplified.
  • thermocycler with an amplification chamber having a small volume allows short cycling times.
  • Conventional thermocyclers based on Peltier technology (Applied Biosystem 9700) with a mounted aluminum block have typically integral ramping speeds smaller than 2-3 K/s and alone do therefore not allow taking full benefit of the herein proposed concept.
  • thermocycler that yields a heating and cooling speed of 5-6 K/s like the LightCycler or instruments equipped with high performance Peltier elements first benefits could be seen. Even more benefit is achievable using thermocyclers that allow ramping speeds above 10 K/s in particular for the cooling rate.
  • a partial amount of said first amount of reaction mixture is then subjected in a second amplification chamber to a second number of thermocycles to prepare a second amount of a second reaction mixture.
  • the volume of said partial amount of said first amount of reaction mixture typically has a volume of 00.5 to 5 ⁇ L, more preferably 0.1-2 ⁇ L.
  • the partial amount of said reaction mixture is subjected to less than 50 thermocycles , more preferably between 20-40 thermocycles.
  • the smaller volume of said partial amount of said first reaction mixture allows a higher integral heating and cooling speed of said second amplification chamber (at least 5 K/s, preferably between 8 to 12 K/s) and the length of a thermocycle can be less than the length of thermocycle in the first round of amplification and usually varies between 5 - 30 seconds.
  • sample can be derived from human, animal and elsewhere in nature.
  • samples especially in diagnostic approaches, are blood, serum, plasma, bone marrow, tissue, sputum, pleural and peritoneal effusions and suspensions, urine, sperm and stool.
  • the nucleic acids were purified from the samples prior to amplification, so that a more or less pure nucleic acid sample can be added to the amplification reaction.
  • Methods for purifying nucleic acids are well known in the art. Beside laborious methods as described in Sambrook et al (Molecular Cloning - A Laboratory Manual, Coldspring Harbour Laboratory Press (1989 )) also commercial kits are available for this purpose (for example, MagNAPure®, Roche Diagnostics).
  • the sample according to the present invention can be a sample directly derived from a donor, especially for cases where a further purification of the nucleic acids present in a sample is not needed as well as purified samples containing nucleic acids preparations from a donor sample.
  • Another aspect of the present invention is directed to a method for preparing and/ or detecting nucleic acids from a sample as described above in which the purification of the nucleic acids present in a sample is integrated preferably in the first amplification chamber of the device.
  • Devices and methods in which the nucleic acids present in a sample are purified in the same reaction chamber as used for conducting a nucleic acid amplification reaction are known in the art. For example in WO 03/106031 integrated devices are described in which binding matrices like glass fleeces are used for capturing of nucleic acids present in a sample. Following the sample preparation the amplification reaction can be conducted in the same reaction chamber used for nucleic acid sample preparation. Such an approach can be combined with the methods and devices of the present invention.
  • the nucleic acids of a sample can be purified and can be subjected to a first number of thermocycles to prepare a first amount of a first reaction mixture in a first amplification chamber of a device. An aliquot of said first reaction mixture can then be transferred to the second amplification chamber for the second number of thermocycles to prepare a second amount of a second reaction mixture.
  • Such methods and devices have several unexpected advantages. First, as already described the second amplification step allows much faster thermocycling due to the smaller reaction volume. Secondly, also in case the nucleic acids of the sample are still partially bound to the binding matrix used for sample preparation and are not completely eluted from said matrix these nucleic acids can still be amplified because the binding matrix is present during the first number of thermocycles.
  • thermocycle is defined as a sequence of at least two temperatures, which the reaction mixture is subjected to for defined periods of time. This thermocycle can be repeated. In PCR methods usually three different temperatures are used. At around 45 - 70 °C the primers are annealed to the target nucleic acids. At a temperature at around 72 °C the primers bound to the target are elongated by a thermostable polymerase and subsequently at around 90 - 100°C, the double-stranded nucleic acids are being separated. In the PCR method, this thermocycle is usually repeated around 30 to 50 times. The time necessary for changing the temperature within the reaction mixture mainly depends on the volume and the shape of the reaction vessel and usually varies from several minutes down to a fraction of a second.
  • first and second amplification chamber Prior to subjecting a partial amount of the first reaction mixture to a second number of thermocycles, it is preferred to transfer this partial amount of the reaction mixture to a second amplification chamber. This can be done manually by using a pipette. However, in view of the contamination risk, it is preferred if this is being automated in the device for example by pumps and valves.
  • the first and second amplification chamber can be separated from each other by channels, valves, hydrophobic barriers and other means.
  • Technical means for such integrated devices are known to an expert (see for example Lee et al., J. Micromech. Microeng. 13 (2003) 89-97 ; Handique et al., Anal. Chem. 72 4100-9 ; Hosokawa et al., Anal. Chem.
  • first and second amplification reaction chambers are two compartments in one unseparated reaction chamber without physical separation of both reaction mixtures.
  • the reaction mixture contains all ingredients necessary for conducting the amplification method of choice. Usually, these are primers allowing specific binding of the target nucleic acid to be amplified, enzymes like polymerases, reverse transcriptases and so on, nucleotide triphosphates, buffers, mono and divalent cations like magnesium.
  • the ingredients depend on the amplification method and are well known to the expert.
  • the nucleic acid products prepared in the first and second reaction mixture can be detected by procedures known in the art, for example by detecting the length of the products in an agarose gel.
  • sequence specific oligonucleotide probes By using sequence specific oligonucleotide probes, a further level of specificity can be achieved, for example by conducting a Southern or dot blot techniques.
  • the detection probe or other detection means are already present in the reaction mixture during generation of the amplified nucleic acids.
  • the probe is being degraded by the processing polymerase when elongating the primes.
  • labels can be used for detection. Examples are fluorescence labels like fluorescein, rhodamine and so on.
  • Another aspect is directed to a method for determining the presence or absence or amount of a template nucleic acid in a sample comprising
  • nucleic acids can either be determined after completion of steps a) and b), or during the amplification steps a) and/ or b).
  • multiple second reaction mixtures can be derived from the first reaction mixture. This allows subjecting more than one partial amount of the first reaction mixture to a second number of thermocycles and therefore allowing a multiplex reaction protocol. This can be in the simplest case a parallel reaction of the same mixture satisfying the results obtained in this method. In case different primers and/or probes are added to the partial amount of the first reaction mixture, a real multiplex detection method, for example for detecting different alleles of a target is possible.
  • Example 3 A possible device for the methods of the present invention is described in Example 3.
  • the method of the present invention is not restricted to certain devices. It can be conducted by hand using commercially available thermocyclers like Applied Biosystems 9700'er system and the LightCycler (Roche Diagnostics). However, this method is especially suited for functionally integrated devices be based on technologies as for example described in Micro Total Analysis Systems, Proceedings uTAS'94, A van den Berg, P Berveld, 1994 ; Integrated Microfabricated Biodevices, M J Heller, A Guttman, 2002 ; microsystem Engineering of Lab-on-a-Chip devices, O Geschke, H Klank, P Tellemann, 2004; US 2003/0152492 and US 5,639,423 .
  • Such devices usually have an automated liquid transport, which allows transporting of a sample between reaction chambers, means for thermocycling, reagents which are either preloaded in the device or which can be added automatically, and means for detecting the reaction product.
  • the reaction is being controlled by computer means and a computer program for controlling.
  • Another aspect of the present invention is a diagnostic device for preparing nucleic acids from a template comprising
  • the smaller size of the volume of the second amplification chamber allows decreasing the time necessary for each thermocycle.
  • standard thermocyclers like the PCR System 9700 (Applied Biosystems) the size of the volume of the amplification chambers is not changed and, in addition most often metal blocks are used for thermocycling which does not allow to decrease the time necessary for a thermocycle to less than a few minutes. Therefore, taking an aliquot of an amplification reaction and using a faster thermocycler like the LightCycler for a second amplification reaction allows decreasing the overall reaction time without decreasing the sensitivity of the assay.
  • Amplification chambers suitable for a diagnostic device of the present invention basically are known in the prior art. These chambers do provide space for containing the reaction mixture.
  • This chamber can be for example a thin-wall plastic tube which is fitted into a bore hole in the metal block of a thermocycler such as the Perkin Elmer 9700er instrument or the inner volume of the glass capillary which can be placed into the LightCycler instrument.
  • the volume of the amplification chamber is defined by the maximal volume of a reaction mixture which can be used in the reaction.
  • the reaction mixtures can be heated by using for example heating elements like Peltier- or resistance-heating elements.
  • heating elements like Peltier- or resistance-heating elements.
  • active cooling elements or passive cooling elements like heat sinks can be used.
  • heat sinks For conducting the heat and cool to the reaction mixture contained in the amplification chamber several means are known. In many conventional thermocyclers metal blocks containing the amplification chambers are used for providing the heat and cool to the reaction mixture. In the LightCycler format a hot air stream floating around the glass capillary provides this function.
  • the diagnostic device of the present invention has at least two amplification chambers as described above. These chambers can either be situated in one instrument or separated on two different instruments, whereby the transfer of an aliquot of the first reaction mixture to the second amplification chamber can be done by manual pipetting or, preferably, is being automated.
  • the apparatus according to the invention has a receptacle to contain the device. It also comprises means heating and cooling the chambers and also for controlling the temperature of the amplification cycles during the thermocycles, preferably a unit for controlling loaded with a computer program as described below.
  • a further aspect of the present invention is a computer program for controlling a method for the preparation of nucleic acids from a template nucleic acid using thermocycles, characterized in that the computer program is set to apply a first number of thermocycles to the sample and subsequently a second number of thermocycles having a shorter cycling time on a different volume of a reaction mixture originating from the same sample.
  • a more preferred aspect of the present invention is directed to a computer program for controlling the methods for preparation of nucleic acids as described above.
  • Such computer programs can be stored on physical storage mean, such as a diskette or a CD.
  • a further aspect of the present invention is an apparatus for preparing nucleic acids comprising
  • thermocycle The exact time needed for the shortened thermocycle is defined on one side by the temperature profile and on the other side by the thermal diffusion distance.
  • the heated volume has a cubical shape and is in contact with the heat source/sink via a single wall. Most of the time during PCR will be consumed due to heat diffusion from this single wall through the water to reach a homogeneous temperature distribution.
  • the thermal diffusion time scales with the second power of the side length of the cubical volume, therefore reducing the volume by a factor of two reduces the diffusion time by the factor of 2 2/3 .
  • a typical run of 50 thermocycles has been assumed.
  • Figure 3 shows a scheme of a device having two amplification chambers and thermocycler elements, which is suitable for conducting the methods of the present invention.
  • the two chambers are in physical contact via a narrow section which could be implemented as a hydrophobic valve.
  • the second amplification chamber is not filled spontaneously when the first amplification chamber is being is filled.
  • an aliquot of the first reaction mixture is being transferred to the second amplification chamber, for example by spinning the device or by applying hydrostatic pressure.
  • Figure 4 depicts a modification of a Light-Cycler® tube, characterized by narrow tube widening to the top of the tube. The wide section and the narrow section are separated from each other by a hydrophobic section (valve). After running the first few cycles in the upper half of the tube, an aliquot is spun down into the lower section of the tube allowing now much faster cycling profile. This of course requires some modification of the instrument to allow a centrifugation step within the cycling program. However this centrifugation step can also be conducted using available centrifuges without requiring modifications of the present Light-Cycler® device.
  • Figure 5 shows a scheme of a disk-shaped device having one reaction chamber for conducting the first number of thermocycles with a higher reaction volume and subjecting more than one partial amounts of that first reaction mixture to a second number of thermocycles, and, therefore allowing a multiplex reaction method.
  • the reaction liquid can be transported by spinning the disk device and applying centrifugal force, but also other methods like pneumatic force, vacuum and so on can be used.
  • it is advisable to reversibly block liquid connection between the first and second reaction chamber for example by valves, hydrophobic vents and so on.

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Claims (17)

  1. Verfahren zur Herstellung von Nucleinsäuren aus einer Matrizennucleinsäure, indem eine Probe Thermozyklen unterzogen wird, umfassend die folgenden Schritte:
    a) Unterziehen einer ersten Menge der Probe einer ersten Anzahl von Thermozyklen in einer ersten Amplifikationskammer, um eine erste Menge eines ersten Reaktionsgemisches herzustellen,
    a) Unterziehen einer Teilmenge des ersten Reaktionsgemisches einer zweiten Anzahl von Thermozyklen in einer zweiten Amplifikationskammer, um eine zweite Menge eines zweiten Reaktionsgemisches herzustellen,
    wobei das Volumen der zweiten Amplifikationskammer kleiner als das Volumen der ersten Amplifikationskammer ist.
  2. Verfahren nach Anspruch 1, wobei in Schritt a) die erste Menge von Proben der ersten Anzahl von Thermozyklen bei
    einer integralen Heiz- und Kühlgeschwindigkeit von mindestens 2 Kelvin/Sekunde (K/s) unterzogen wird, und
    wobei in Schritt b) die Teilmenge des ersten Reaktionsgemisches der zweiten Anzahl von Thermozyklen
    bei einer integralen Heiz- und Kühlgeschwindigkeit unterzogen wird, die höher als in Schritt a) ist und die ungefähr 5 K/s beträgt.
  3. Verfahren nach einem der Ansprüche 1 bis 2, wobei die integrale Heiz- und Kühlgeschwindigkeit in Schritt a) 4 bis 7 K/s und in Schritt b) 8 bis 12 K/s beträgt.
  4. Verfahren nach einem der Ansprüche 1 bis 3, wobei das Volumen der ersten Menge der Probe in der ersten Amplifikationskammer ein Volumen von 5 bis 200 µl aufweist.
  5. Verfahren nach einem der Ansprüche 1 bis 4, wobei das Volumen der Teilmenge des ersten Reaktionsgemisches ein Volumen von 0,05 bis 5 µl aufweist.
  6. Verfahren nach einem der Ansprüche 1 bis 5, wobei die erste Anzahl von Thermozyklen kleiner als die zweite Anzahl von Thermozyklen ist.
  7. Verfahren nach einem der Ansprüche 1 bis 6, wobei die Teilmenge des ersten Reaktionsgemisches physikalisch aus dem Rest des ersten Reaktionsgemisches entfernt wird.
  8. Verfahren nach einem der Ansprüche 1 bis 7, wobei eine oder mehrere zusätzliche Teilmengen des ersten Reaktionsgemisches in Schritt b) Thermozyklen unterzogen werden.
  9. Verfahren nach einem der Ansprüche 1 bis 8, wobei die erste Amplifikationskammer zur Reinigung der Nucleinsäuren verwendet wird, die vor dem Durchführen der ersten Anzahl von Thermozyklen in der ungereinigten Probe vorhanden sind.
  10. Verfahren nach einem der Ansprüche 1 bis 9, zusätzlich umfassend:
    c) Bestimmen der Bildung von Nucleinsäuren als ein Maß für das Vorhandensein oder Nichtvorhandensein oder die Menge von Nucleinsäuren, die bestimmt werden sollen,
    wobei das Volumen der zweiten Amplifikationskammer kleiner als das Volumen der ersten Amplifikationskammer ist, und die Bildung von Nucleinsäuren entweder während oder nach Abschluss von Schritt a) und b) bestimmt wird.
  11. Diagnosegerät zum Herstellen von Nucleinsäuren aus einer Matrize, umfassend:
    a. eine erste Amplifikationskammer, und
    b. eine zweite Amplifikationskammer,
    c. Mittel zum Beheizen und Kühlen der Kammern,
    d. Mittel zum Regeln der Temperatur der Amplifikationszyklen während der Thermozyklen,
    wobei das Volumen der zweiten Amplifikationskammer kleiner als das Volumen der ersten Amplifikationskammer ist.
  12. Gerät nach Anspruch 11, wobei das Volumen der ersten Menge der ersten Amplifikationskammer ein Volumen von 5 bis 200 µl aufweist.
  13. Gerät nach Anspruch 11 oder 12, wobei das Volumen der zweiten Amplifikationskammer ein Volumen von 0,05 bis 5 µl aufweist.
  14. Gerät nach einem der Ansprüche 11 bis 13 mit Mitteln zum Befördern von Flüssigkeiten aus der ersten Amplifikationskammer zur zweiten Amplifikationskammer.
  15. Computerprogramm zum Steuern eines Verfahrens zur Herstellung von Nucleinsäuren aus einer Matrizennucleinsäure unter Verwendung von Thermozyklen, dadurch gekennzeichnet, dass das Computerprogramm so festgelegt ist, dass es eine erste Anzahl von Thermozyklen auf die Probe und anschließend eine zweite Anzahl von Thermozyklen mit einer kürzeren Zykluszeit auf ein kleineres Volumen eines aus der gleichen Probe stammenden Reaktionsgemisches anwendet.
  16. Computerprogrammprodukt, umfassend ein Programm nach Anspruch 15 auf einem physikalischen Speichermittel.
  17. Vorrichtung zum Herstellen von Nucleinsäuren, umfassend,
    a. ein Diagnosegerät nach einem der Ansprüche 11 bis 14, und
    b. eine Einheit zum Steuern des Diagnosegeräts,
    wobei die Einheit zum Steuern des Diagnosegeräts mit einem Computerprogramm nach einem der Ansprüche 15 bis 16 geladen ist.
EP20050024993 2004-11-20 2005-11-16 Präparieren von Nucleinsäuren Revoked EP1666150B1 (de)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK171161B1 (da) 1985-03-28 1996-07-08 Hoffmann La Roche Fremgangsmåde til påvisning af forekomst eller fravær af mindst én specifik nukleinsyresekvens i en prøve eller til skelnen mellem to forskellige nukleinsyresekvenser i denne prøve
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5185243A (en) 1988-08-25 1993-02-09 Syntex (U.S.A.) Inc. Method for detection of specific nucleic acid sequences
US5573907A (en) 1990-01-26 1996-11-12 Abbott Laboratories Detecting and amplifying target nucleic acids using exonucleolytic activity
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5639423A (en) 1992-08-31 1997-06-17 The Regents Of The University Of Calfornia Microfabricated reactor
WO1995021271A1 (en) 1994-02-07 1995-08-10 Molecular Tool, Inc. Ligase/polymerase-mediated genetic bit analysistm of single nucleotide polymorphisms and its use in genetic analysis
US8293064B2 (en) 1998-03-02 2012-10-23 Cepheid Method for fabricating a reaction vessel
US6429007B1 (en) * 1997-05-02 2002-08-06 BIOMéRIEUX, INC. Nucleic acid amplification reaction station for disposable test devices
US20020031777A1 (en) 2000-09-08 2002-03-14 Linda Starr-Spires Ultra yield amplification reaction
EP1371419A1 (de) 2002-06-12 2003-12-17 F. Hoffmann-La Roche AG Verfahren und Vorrichtung zur Detektion eines Analyten in einer Probe
AU2003298706A1 (en) 2002-12-04 2004-06-23 Applera Corporation Multiplex amplification of polynucleotides

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