APPARATUS AND METHOD FOR THERMOCYCLIC BIOCHEMICAL OPERATIONS

Abstract

Process and apparatus for the optimization of DNA detection and comprising: charging a plurality of reaction vessels with reagents and primers suspected of being suitable for the particular sample, in various quantities; placing in each reaction vessel a sample of the target DNA; subjecting each vessel concurrently to PCR; simultaneously observing optically the whole PCR process in each reaction vessel.

Claims

1.-36. (canceled)

37. A process for the optimization of DNA detection comprising: charging a plurality of reaction vessels with reagents and primers suspected of being suitable for the particular sample, in various quantities; placing in each reaction vessel a sample of the target DNA; subjecting each vessel concurrently to PCR; concurrently observing optically the whole PCR process in each reaction vessel.

38. A process as claimed in claim 37 and arranged to examine at least several of the following parameters: anneal temperature; annealing time denaturation temperature; denaturation time; extension temperature; extension time; temperature at which fluorescence readings are taken; ramping rates (for all steps); magnesium chloride concentration; dNTP concentration; primer concentration; target concentration.

39. A process as claimed in claim 37 and comprising spectrographic interrogation of the emitted fluorescence from both an intercalating dye and a sequence specific probe at the same time and temperature, thus measuring the FRET and hence providing information about the hybridisation state of the target.

40. A process as claimed in claim 37 and comprising spectral deconvolution to separate the individual component dyes and comparing their total fluorescent output.

41. A process as claimed in claim 37 and arranged to discriminate between highly similar sequences and comprising designing a pair of primers to cover the region of interest, and placing these primers in the same reaction vessel, the primers differing in both melt point and fluorescent label and thus determining the actual temperature at which annealing occurs and enabling the required discrimination.

42. A process as claimed in claim 37 and wherein optical means are arranged to capture the full visible spectrum from each of the reaction vessels.

43. A process as claimed in claim 42 and further comprising separating the fluorescence arising from each of the reaction vessels, plotting the fluorescence values against time, temperature and concentration of each assay and indicating an ideal optimised PCR.

44. A process as claimed in claim 37 and wherein the reaction vessels are in an 812 microtitre vessel array.

45. A process as claimed in claim 37 and comprising determining automatically, from the results in each reaction vessel, the most rapid and efficient identification process for a given DNA target, and indicating same.

46. Apparatus for carrying out the process of claim 37, the apparatus comprising an array of icrotiter reaction vessels, means for performing polymerase chain reaction each in each reaction vessel concurrently on an individual basis, a light source, a multi-channel imaging spectrograph, means for controlling the time of the PCR, a multi-fiber probe bundle arranged for excitation and the reception of a collimated output of the light source and terminating above at least eight reaction vessels, each fiber probe actually comprising a plurality of excitation fibers and at least one collector fiber, the said at least one collection fibre being arranged to be focused onto a large area detector.

47. Apparatus as claimed in claim 46 and wherein the at least one collection fiber is focused onto the detector via a diffraction grating,

48. Apparatus as claimed in claim 46 and wherein the means for performing polymerase chain reaction on the contents of the reaction vessels comprises a heater, a heat removal module, a heat sink coolant reservoir and a pump.

49. Apparatus as claimed in claim 46 and wherein the light source is a laser or laser diode.

50. Apparatus as claimed in claim 46 and employing an optical multiplexer.

51. Apparatus as claimed in claims 46 and wherein the spectrograph is arranged to capture the full visible spectrum from the wells.

52. Apparatus as claimed in claim 46 and comprising an array of 96n, where n is an integer, microtitre reaction vessels in 128 array, at least a plurality of which are arranged for individual control and further comprising an eight well scanning head having a single detector and two diffraction gratings to focus eight spectra onto the one sensor.

53. Apparatus as claimed in claim 46 and wherein the optical means comprises a single detector and rotary distribution wheel, an eight well scanning head, a spectral photometer capable of reading one to eight reaction vessels, preferably without moving, or an imaging spectrograph which can view all the reaction vessels at the same time, as described above.

54. Apparatus as claimed in claim 46 and further comprising a shuttle arranged to center the spectrograph over each column of wells in turn.

55. Apparatus as claimed in claim 46 and wherein each fibre bundle comprises a single central core collector fiber surrounded by six excitation fibres.

56. Apparatus as claimed in claim 46 and wherein the large area detector is a CCD or a CMOS.

57. Apparatus as claimed in claim 53 and wherein the eight well scanning head comprises a single detector and two diffraction gratings to focus eight spectra onto the one sensor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Embodiments of the invention will now be described by way of example with reference to the accompanying drawings, of which:

[0053] FIGS. 1 to 4 illustrate a 96 microtitre reaction vessel array with individual PCR control.

[0054] FIG. 5 is a schematic drawing of an array of fibre optic bundles;

[0055] FIG. 6 is a sectional view of one fibre optic bundle;

[0056] FIGS. 7 and 8 are graphs illustrating the advantage of continuous reading; and

[0057] FIGS. 9 to 16 are plan views of examples of plate layouts for factorial optimisation;

SPECIFIC DESCRIPTION

[0058] A 96 microtitre reaction vessel PCR apparatus in a standard 128 array is described in, among other patent specifications, those of UK Patent 2404883 and co-pending UK Patent Application 1401584.6, both of which describe individual well control. A resume of the latter is described below with reference to FIGS. 1 to 4.

[0059] The apparatus comprises twelve heat removal module slices 10 sandwiched between two end plates 51 having coolant liquid inlet and outlet necks 52, 53. Each slice has eight reaction stations 11 at a top edge, coolant liquid entry 12 and exit 13 manifold bores therethrough at each end, and a series of grooves 14 extending along one face from the top to the bottom edge thereof. A heat exchanger liquid hollow extends between the manifold bores 12 and 13.

[0060] The reaction stations 11 are circular hollows sized for the bases of reaction vessel holders 40 to be an interference fit therein. A small hole 16 leads from the base of each station 11 to the groove 14 and acts in use to permit the escape of gases (air) from the stations 11 when the vessel holders are driven in.

[0061] Around each manifold on one face of the slice are grooves 17 for an O-ring seal and further out are slide attachment holes 18 of which one has a locating hush 19.

[0062] At each bottom corner on one face is a separation rebate 20 arranged to assist in separating the slices when required. Between each station 11 there is a cut 21 arranged to maximise thermal isolation between each station 11. Rebates 22 on one side of each slice 10 are formed for a like purpose.

[0063] A printed circuit board (PCB) 30 clips into the grooves 14 and projects above and below the slice 10. The PCB 30 carries heater and sensor electrical conduits which terminate in connectors 31 at the top and 32 at the bottom thereof. The thickness of the PCB 30 is the depth of the grooves 14.

[0064] A reaction vessel holder 40 fits into each of the reaction stations 11. The reaction vessel holder 40 comprises a reaction vessel receiving portion 41; a heater portion 42 and a cooling portion 43, the latter being arranged to anchor the station in a heat removal module. The vessel receiving portion 41 is shaped to receive snugly a microtitre reaction vessel and in the wall thereof is located a temperature sensor 44. The heater portion 42 has a helical groove therearound into which is wound a heater coil 45. Flexible tubing (not shown) connects the necks 52, 53 with a heat sink coolant reservoir (not shown) via a pump (not shown).

[0065] The reaction vessel 61 is a microtitre vessel formed of a carbon loaded plastics material and is 2 cm overall length. It comprises, in descending order, a cap receiving rim, a filler portion and a reaction chamber with a base thereto. The filler portion has a maximum outer diameter of 7 mm and a depth of 5 mm. The reaction chamber tapers down from 3 mm to 2.5 mm, the whole having a wall thickness of 0.8 mm. Accordingly the reaction chamber is of substantially capillary dimensions.

[0066] The array of holders 40 is adapted to accept snugly a 128 standard microtitre well tray 60

[0067] During a reaction electrical supply via the conduits is arranged to heat the wells 61 according to a predetermined program, while other of the conduits convey signals relating to the temperature in the wells. This program is predetermined for each well, as the apparatus is particularly suited for performing totally independent reactions in each well 61. Thus, where the reactions comprises a heating-cooling cycle, as is the case for example in PCR, one well 61 may be in a heating phase and another in a cooling phase, one at rest and another complete.

[0068] The heating cycle is arranged to take place against a coolant environment in the HRM 50 which is fixed at 40 C. which is usually above room temperature and is a mid-point for heating and cooling efficiency.

[0069] The progress of the process in each reaction vessel is monitored in the optics unit 62

[0070] FIG. 5 illustrates an array of fibre optic bundles used in a 812 microtitre plate. A bundle of excitation fibres 71 emanate from a CCD light source 72 and pass into a multiplex unit 73 wherefrom emerge 96 fibre optic bundles 74 each comprising excitation fibres and at least one collection fibre. The bundles 74 each terminate in probes 75 destined to be mounted appropriately one above each reaction chamber. The collection fibres are connected in the multiplex unit 73 to an output bundle 76 which is passed to a spectrograph 77.

[0071] FIG. 6 is a sectional view of one fibre optic bundle 74, that is, a bundle emanating from the multiplex unit 73 and terminating in a probe 75. Each bundle 74 comprises a collection fibre core 78 and six excitation fibres 79 surrounding the core fibre 78. A standard protective shield surrounds the fibres.

[0072] It is the probes 75 which are in the optics unit 62 shown in FIG. 1, mounted with one probe 75 facing each well 61.

[0073] FIGS. 7 and 8 are graphs of light emission (y axis) versus the number of cycles (x axis). The graphs illustrate the difference between traditional PCR optical observation and that of the present invention with FIG. 8 illustrating a detail (four cycles) from FIG. 7. In the traditional optical observation wherein filters or movable probes are employed, a single image capture is made at the end of each cycle, that is, after each extension, of necessity. This is at point 80 in FIGS. 8 and 9. In continuous capture, that is, an image every 25 ms, images are captured at points 81, enabling the construction of a real time line 82 representing the whole PCR process. In particular the moment of extension can be captured (point 83) and slope angle and time length of each step, cT and R observed and optimised. The dashed line 84 provides accordingly a measure of in-cycle efficiency. The dashed line 85 is the measurement of the point at which doubling has completed.

[0074] Accordingly, when viewed in real time the data obtained makes possible the measurement of the point when amplification has been observed to have been completed for the given cycle. Any additional time on this cycle is unnecessary. Furthermore it is possible to visualise the in-cycle efficiency by measuring the slope (line 83)of the fluorescence increase within each cycle. Differing fluorescent chemistries, for example intercalating dyes and the 3 hydrolysis assay, will give differing amounts of data on each of the segments of the reaction. The example shown is for a 3 hydrolysis assay. An intercalator will also show the melt points of the DNA products and this will be of benefit to the automated software. By interrogating the same DNA target with different probe systems it is possible to build up a picture of the reaction in its entirety; annealing temperature, the effect of different chemical constituents, optimised temperatures, and hold times at the same.

[0075] FIGS. 9 to 16 illustrate patterns of concurrent PCR operations in a standard 812 microtitre reaction vessel array, where the numbers cited represent one variable, e.g. annealing temperature; extension time; magnesium chloride concentration etc; Thus: [0076] FIG. 9 shows an array set up for 4432 concurrent tests; [0077] FIG. 10 shows an array set up for 6422 concurrent tests; [0078] FIG. 11 shows an array set up for 644 concurrent tests; [0079] FIG. 12 shows an array set up for 384 concurrent tests; [0080] FIG. 13 shows an array set up for 128 concurrent tests; [0081] FIG. 14 shows an array set up for 616 concurrent tests; [0082] FIG. 15 shows an array set up for 244 concurrent tests; and [0083] FIG. 16 shows an array set up for 33'33 concurrent tests.

[0084] By set up is meant that the array, in the art usually called a plate, is pre-prepared with the range of, for example, magnesium chloride, primer, enzyme and dNTP concentrations.

[0085] Then, in the course of the concurrent tests, time gradient can for example be varied on a column by column basis and temperature gradients can be varied on a row by row basis, as illustrated in FIG. 17.