APPARATUS AND METHOD FOR THERMOCYCLIC BIOCHEMICAL OPERATIONS

Abstract

An amplicon separation apparatus and process for enabling the identification of a plurality of target DNA molecules in a sample such as blood, the apparatus including a consumable and a docking instrument for receiving the consumable. The consumable incorporates a reaction vessel heating station, a reaction vessel content transfer station, an amplicon size separation station, and a reader station. The instrument controls and monitors the consumable and the operation thereof.

Claims

1-61. (canceled)

62. An amplicon separation consumable incorporating the following stations: a heating station adapted to receive and hold a microtitre reaction vessel having a reaction chamber and a base; a reaction vessel content transfer station; an amplicon size separation station; and a reader station.

63. A consumable as claimed in claim 62 and wherein the heating station is constructed to receive withdrawably a thermocycler device, the thermocycler device being constructed to envelop the reaction chamber of a reaction vessel.

64. A consumable as claimed in claim 62 and incorporating a shuttle device operable to transfer the reaction vessel from the heating station to the reaction vessel content transfer station.

65. A consumable as claimed in claim 62 and wherein the consumable content transfer station has piercing means arranged for piercing the reaction vessel base and allowing vessel content to pass into the size separation station.

66. A consumable as claimed in claim 62 and wherein the amplicon size separation station includes a size separation microfluidic chip.

67. A consumable as claimed in claim 66 and wherein the reaction vessel transfer station comprises means for wicking reaction fluid into a collection well on the chip and thus to transfer fluid from a completed reaction to the size separation chip.

68. A consumable as claimed in claim 62 and wherein the amplicon size separation station contains a sieving matrix.

69. An amplicon separation consumable incorporating the following stations: a heating station wherein is held a reaction vessel; a reaction vessel content transfer station; an amplicon size separation station; and a reader station, the heating station being constructed to receive withdrawably a thermocycler device offered into the heater station from below the consumable and arranged to nestle snugly a reaction chamber part of the reaction vessel; the reaction vessel being formed of a carbon loaded plastics material and being of microtitre proportions with a length of 2 cm a filler portion with a maximum outer diameter of 7-8 mm and a depth of about 4-5 mm, the reaction chamber tapering down from 3 mm to 2.5 mm, the whole having a wall thickness of the order of 0.8 mm, the reaction vessel thus being of substantially capillary dimensions and comprising in descending order, a cap receiving rim and a transparent cap therefor, a filler portion, a reaction chamber with a base and containing required reagents and fluorescently labelled primers, freeze dried, for a PCR process; the consumable having a shuttle device operable to transfer the reaction vessel from the heating station to the content transfer station; the content transfer station having piercing means arranged for piercing a base of the reaction vessel and allowing the vessel content to pass into the size separation station; the amplicon size separation station including a size separation glass microfluidic chip, arranged for operation by electrophoresis and containing a sieving matrix; electrical contacts, connected to the size separation station wherethrough an electrical current may be supplied to drive amplicon size separation; the consumable comprising a substantially rigid case and having a removable film over top and bottom surfaces thereof, covering reaction vessel and heater accesses; and the reader station being accessible by optical interrogation means.

70. An amplicon separation instrument in the form of a docking station constructed to receive and hold a consumable as claimed in claim 62, the docking station incorporating a thermocycler device.

71. An instrument as claimed in claim 70 and wherein the thermocycler device comprises a metal sleeve adapted snugly to surround the reaction vessel reaction chamber in such a manner as to be contiguous therewith throughout the length thereof and, integral with the sleeve at the base thereof, a heat transfer module.

72. An instrument as claimed in claim 71 and wherein the heat transfer module is a peltier cell attached to a heat reference module and arranged for operation around a median temperature.

73. An instrument as claimed in claim 70 and incorporating monitoring means operable to monitor progress of PCR within the reaction vessel.

74. An instrument as claimed in claim 70 and incorporating a device arranged to determine that the reaction vessel can be moved and to cause the reaction vessel to be moved in the consumable from the heating station to the reaction vessel contents transfer station.

75. An instrument as claimed in claim 70 and incorporating reader means arranged to interrogate the consumable reader station.

76. An instrument as claimed in claim 70 and incorporating temperature control means operable to control the temperature of the consumable amplicon size separation station.

77. An instrument as claimed in claim 70 and having an automated opening drawer arranged to receive and eject a consumable.

78. An instrument as claimed in claim 70 and constructed to receive and process a plurality of consumables simultaneously.

79. An instrument as claimed in claim 70 and arranged to provide a result of comparing observed amplicon sizes and spectra to a known database.

80. An instrument as claimed in claim 70 and having an optical facility which incorporates a spectrophotometer relying on LED excitation and CCD detection.

81. An amplicon separation instrument in the form of a docking station constructed to receive and hold a consumable as claimed in claim 1, the docking station comprising: a thermocycler device, the thermocycler device including a metal sleeve adapted snugly to surround the reaction vessel reaction chamber in such a manner as to be contiguous therewith throughout the length thereof and, integral with the sleeve at the base thereof, a heat transfer module, the heat transfer module being a peltier cell attached to a heat reference module and arranged for operation around a median temperature; offering means arranged to insert the thermocycler into the consumable so that the sleeve surrounds the reaction vessel; monitoring means operable to monitor the progress of PCR within the reaction vessel, the monitoring means being operable to determine that the PCR is complete; a device arranged to determine that the reaction vessel can be moved and to move the reaction vessel in the consumable from the heating station to the contents transfer station; reader means arranged to interrogate the consumable reader station, the reader means comprising an optical facility incorporating an LED excitation source and a spectrophotometer, the reader means further incorporating means to maintain a time base in order that fluorescence can be plotted against time; and temperature control means operable to control the temperature of the consumable size separation station.

Description

[0055] Embodiments of the invention will now be described by way of example, with reference to the accompanying drawings, of which:

[0056] FIG. 1 is a sectioned side elevation of a consumable in a first configuration;

[0057] FIG. 2 is a sectioned side elevation of a PCR heater engaging a microtitre reaction vessel;

[0058] FIG. 3 is a sectioned side elevation of a consumable in a second configuration;

[0059] FIG. 4 is a sectioned plan view of a consumable in the first configuration;

[0060] FIG. 5 is a sectioned plan view of a consumable in the second configuration;

[0061] FIG. 6 illustrates detail of a reaction vessel content transfer station;

[0062] FIG. 7 is an insertion end view of a consumable;

[0063] FIG. 8 is an isometric view of a chip;

[0064] FIG. 9 is a schematic plan view of a chip;

[0065] FIG. 10 illustrates the exterior of a docking station;

[0066] FIG. 11 depicts the content of a docking station;

[0067] FIG. 12 is a sectional view of a PCR heater;

[0068] FIG. 13 is a detail view of the docking station table area;

[0069] FIG. 14 illustrates a display on the docking station screen.

[0070] Depicted in FIGS. 1 to 9 is an amplicon separation consumable comprising a case 10 and incorporating the following stations: [0071] a heating station 20 holding a reaction vessel 30; [0072] a reaction vessel content transfer station 40; [0073] an amplicon size separation station 50; and [0074] a reader station 60.

[0075] At the heating station 20 is a mitrotitre reaction vessel 30. The reaction vessel 30 is formed of a carbon loaded plastics material and is 2 cm overall length. It comprises, in descending order, a cap receiving rim 31 with a lid 32 flexibly attached thereto, a filler portion 33, a reaction chamber 34 with a base 35 thereto. The filler portion 33 has a maximum outer diameter of 7-8 mm and a depth of 5 mm. The reaction chamber 34 tapers down from 3 mm to 2.5 mm diameter. The reaction chamber 34 has a wall thickness of the order of 0.8 mm. Accordingly the reaction vessel is of substantially capillary dimensions in order to maximize the rates of heat transfer to and from the vessel contents. The lid 32 fits sealedly to the vessel rim 31.

[0076] The reaction vessel 30 is held in the heating station 20 in a configuration rendering the vessel 30 accessible to a lid heater and an optical reader above and a heater below. The reaction vessel 30 is actually held in a shuttle 11 in the consumable 10 accessible via an aperture 10a to be driven from outside the consumable and thereby movable from the heating station 20 to the contents transfer station 40. The consumable case 10 also has formed thereon a probe 10b by which it the consumable can be removably locked into the docking station. The consumable case 10 is also constructed so that it can be offered to the docking station in only one orientation.

[0077] At the reaction vessel content transfer station 40 on the floor of the consumable is a well 41 sized to receive sealedly the base 35 of the reaction vessel 30. In the centre of the well 41 is a needle 42 operable to pierce the base 35 and allow the vessel contents to flow into the well 41. At the station 40 the ceiling of the consumable 10 allows access to a solenoid device operable to push the reaction vessel 30 downwards into the well 41 and onto the needle 42. The needle 42 is one which upon piercing the base 35 permits liquid to flow thereby. The needle 42 is a back-to-back C.

[0078] The well 41 is formed in a glass electrophoresis chip 12 and communicates with a first capillary 13 formed therein. The capillary 13 terminates in a well 41a in the chip 12. Midway along the first capillary 13 is a junction 14 with a second capillary 15. The second capillary 15 extends between a power source well 16 and a terminal well 17. The consumable is arranged so that the reading station is just ahead of the terminal well 17. Electrodes 18 associated with each of the four wells 41, 41a, 16 and 17 serve to drive the electrophoresis. Within the capillaries 13, 15 is a sieving matrix.

[0079] The docking station illustrated in FIGS. 10 to 14 has a consumable reception table 100 with a docking lock 101. Above the table 100, opposite the consumable heating station 20 is an optical reader 102 and an associated vessel lid heater 102a, while opposite the fluid transfer station 40 is a device in the form of a screw operated pusher 103 for pushing the vessel down onto the needle 42.

[0080] Above the docking lock 101 and ahead of the consumable's aperture 10a is a solenoid operated plunger 104.

[0081] The table 100 incorporates a PCR thermocycling device 105. This comprises a HRM 106, a Peltier cell 107 having a base face 108 and a working face 109, a heat exchanger comprising a heat transfer base 110 and a sleeve 111, and a drive 112. The HRM 106, the Peltier cell 107 and the heat transfer base 110 are attached one to another with a flexible solder. The sleeve 111 is formed to engage snugly but removably the reaction vessel 30 in the consumable 10. The HRM is in a heat transfer liquid circuit comprising a radiator 113 and fan 114 and a pump 115.

[0082] Upon the table 100, appropriately placed, are contacts 116 for the electrophoresis electrodes 18. Outward of the table 100 and below the level thereof is a camera 117. The table also incorporates, below and throughout the separation station 50, a heater 118. The optical reader 102 and the camera 117 feed a spectrograph 119.

[0083] The docking station also incorporates appropriate software.

[0084] The docking station has a case 120 incorporating a consumable reception hatch 121 and a touch control display screen 122.

[0085] To use the apparatus the vessel reaction chamber 34 is charged with appropriate reagents and fluorescent labelled primers. If these are freeze dried an appropriate amount of pure water is added before the sample to be investigated is added and the transparent lid 32 emplaced.

[0086] The docking station is switched on, when the liquid in the HRM circuit will warm up to a selected temperature just above the annealing temperature of the target DNA. Electrical circuitry will supply the touch screen with a signal to indicate the liquid temperature.

[0087] The consumable is then pushed into the docking device 120 via the hatch 121 until the probe 10b is engaged by the docking lock 101 and locked in place on the table 100, when the electrical contacts 116 engage the electrophoresis electrodes 18. The hatch 121 also serves to hold the consumable to the table 100.

[0088] The optical reader 102 and the lid heater are switched on and the thermocycler 105 moved up by the drive 112 so that the sleeve 111 engages snugly the reaction vessel 30. If required the thermocycler 105 is first arranged to perform a cell disruption cycle before a PCR cycle, which latter is observed by the optical reader 102. A report from the reader 102 enables the screen 122 to indicate that the PCR is complete and to switch off and withdraw the thermocycler 105. The thermocycler 105 is switched off at a little below its higher temperature in order to ensure pressure within the reaction vessel 30, whilst not encouraging a further DNA separation.

[0089] The plunger 104 pushes the consumable shuttle 11 from the heater station 20 to the contents transfer station 40. The pusher 103 imposes downward pressure upon the shuttle 11 and pushes the reaction vessel down into the well 41 and onto the needle 42, which penetrates the base 35 of the reaction vessel. Because of pressure retained in the reaction vessel 30 the contents thereof are readily driven out and into the well 41 where they dissipate into the sieving matrix in capillary 13 and migrate therealong. At junction 14 the amplicons are captured and driven along the second capillary 15 by electrophoresis. The smaller amplicons travel the fastest and accordingly all amplicons arrive at the reading station at different times. Both the size and the colour of each amplicon is detected by the camera and a spectral profile of each amplicon generated. The associated software in the docking station will from this information identify the DNA of the amplicon and the identity is displayed on the screen.

[0090] The consumable of this embodiment is a rectangular box of 12 cm total length, 23 mm breadth and 28 mm depth. The glass chip 12 is 7.5 cm long by 5 mm broad and 1.5 mm deep. The overall length of the capillary 15 is 7.0 cm. The consumer case 10 has a 1 mm recess in its base to receive and protect the chip. The chip has, as a result, about the minimum possible size consistent with relatively assured integrity and efficient operation, thus allowing construction of a consumable with an economy to be expected of a disposable item.

[0091] The docking station 120 has overall dimensions 20 cm length and breadth and 15 cm height.

[0092] Parts List

[0093] Consumable

[0094] Stations:

[0095] heating station 20; reaction vessel 30; vessel content transfer station 40; amplicon size separation station 50; reader station 60.

[0096] Parts:

[0097] consumable case 10; shuttle 11; aperture 10a probe 10b; vessel cap receiving rim 31; lid 32; filler portion 33; reaction chamber 34; base 35; electrophoresis chip 12; first capillary 13; junction 14; second capillary 15; power source well 16; reading station well 17; electrodes 18; receiver well 41; needle 42; sink well 41a.

[0098] Docking Station

[0099] reception table 100; docking lock 101; optical reader 102; solenoid operated plunger 103; solenoid operated plunger 104; thermocycler 105; HRM 106; Peltier cell 107; base face 108; working face 109; heat exchanger heat transfer base 110; sleeve 111; drive 112; radiator 113; fan 114; pump 115; contacts 116; camera 117; case 118; hatch 119; screen 120.