Abstract
An apparatus for thermal processing of nucleic acid in a thermal profile. The apparatus employs a reactor holder for holding reactors to accommodate reaction material containing nucleic acid. The apparatus includes at least two baths separated by thermally insulating partition plate(s) where bath mediums are each maintainable at a predetermined temperature; and a transfer means for allowing the reactors to change position once or plurality of times between any two adjacent baths by selectively opening the partition plate(s) and without lifting the reactors out of the baths.
Claims
1. An apparatus for thermal processing of nucleic acid in a thermal profile, the apparatus employing a reactor holder for holding reactor(s) to accommodate reaction material containing nucleic acid, the apparatus comprising: at least two baths separated by thermally insulating partition plate(s) where bath mediums in use are each maintainable at a predetermined temperature; and a transfer means for allowing the reactor(s) to change position once or plurality of times between any two adjacent baths of the at least two baths by selectively opening the partition plate(s) and without lifting the reactor(s) out of the baths.
2. The apparatus according to claim 1, wherein the at least two baths comprise: a first bath where the reactor(s) is/are allowed to attain a predetermined high target temperature T.sub.HT, wherein the T.sub.HT is in the region 85-99 degree Celsius for pre-denaturation and denaturation of the nucleic acid; and a second bath where the reactor(s) is/are allowed to attain a predetermined low target temperature T.sub.LT, wherein the T.sub.LT is in the region 45-75 degree Celsius for annealing of primers or probes onto nucleic acid or for primer extension, the first and the second baths being for thermal cycling the reactor(s) to attain polymerase chain reaction (PCR) amplification or primer extension.
3. The apparatus according to claim 2, further comprising: a third bath where the reactor(s) is/are allowed to attain a predetermined medium target temperature T.sub.MT, wherein the T.sub.MT is for annealing of primers or probes onto nucleic acid.
4. The apparatus according to claim 2, further comprising: a fourth bath where the reactor(s) is/are allowed to attain a predetermined medium target temperature T.sub.MT, wherein the T.sub.MT is for extension of primers on nucleic acid.
5. The apparatus according to claim 2, further comprising: a fifth bath where the reactor(s) is/are allowed to attain a temperature T.sub.AP for an additional process for the reactor(s) before thermal cycling, the additional process being one selected from the group consisting of a) reverse transcription-polymerase chain reaction (RT-PCR), b) hot start process, and c) isothermal amplification reaction.
6. The apparatus according to claim 2, further comprising: a sixth bath that can be progressively heated while conducting melt curve analysis after thermal cycling.
7. The apparatus according to claim 6, wherein the reactor(s) is/are allowed to stabilize in the first bath, the second bath, the third bath, the fourth bath, the fifth bath or the sixth bath.
8. A method of using the apparatus of claim 3, the method comprising: setting the T.sub.LT equal to the T.sub.MT.
9. A method of using the apparatus of claim 1, the method comprising: using the bath medium in at least one phase selected from the group consisting of air, liquid, solid, and powder.
10. The method according to claim 9, further comprising: orienting the reactor(s) held by the reactor holder to allow minimum surface area of the reactor(s) to be in a direction of movement of the reactor(s) between the baths.
11. A method of using the apparatus of claim 4, the method comprising: setting the T.sub.LT equal to the T.sub.MT.
12. The apparatus according to claim 1, wherein in operation, the partition plates open along a horizontal direction.
13. The apparatus according to claim 1, wherein in operation the partition plates open along a vertical direction.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In the following drawings, same reference numbers generally refer to the same parts throughout. The drawings are not to scale, instead the emphasis is on describing the concept.
[0020] FIG. 1A is a cross-sectional elevation view of a typical set up in the art for thermal cycling a reaction material containing nucleic acid;
[0021] FIG. 1B is a cross-sectional elevation view of a set up for thermal cycling a reaction material containing nucleic acid according to an embodiment of the invention;
[0022] FIG. 2A to FIG. 2F are plan views of the movable partition plate separating the high temperature and the low temperature baths in a 2 step PCR process, according to an embodiment of the invention;
[0023] FIG. 2A shows the plan view of the movable partition plate separating the high temperature and the low temperature baths when the reactors are in the high temperature bath and the partition plate is at a close position, separating the hot and cold baths;
[0024] FIG. 2B shows the plan view of the movable partition plate separating the high temperature and the low temperature baths when the partition plate moves to an open position after the reactors have been in bath for a predetermined time;
[0025] FIG. 2C shows the plan view of the movable partition plate separating the high temperature and the low temperature baths when the reactors speedily move to the low temperature bath.
[0026] FIG. 2D shows the plan view of the movable partition plate separating the high temperature and the low temperature baths when the partition plate moves to the close position after the reactors moved to the low temperature bath;
[0027] FIG. 2E shows the plan view of the movable partition plate separating the high temperature and the low temperature baths when the partition plate moves to the open position again after the reactors have been in low temperature bath for another predetermined time;
[0028] FIG. 2F shows the plan view of the movable partition plate separating the high temperature and the low temperature baths when the reactors move back to the hot bath and the partition plate closes thereafter (not shown) to complete one thermal cycle of a 2-step PCR thermal cycling process;
[0029] FIGS. 3A to 3D are plan views of the movable partition plates separating the high, the medium and the low temperature baths for a 3 step PCR process, according to an embodiment of the invention;
[0030] FIG. 3A shows plan view of the movable partition plates separating the high, the medium and the low temperature baths for a 3 step PCR process when the reactors are in the high temperature bath;
[0031] FIG. 3B shows plan view of the movable partition plates separating the high, the medium and the low temperature baths for a 3 step PCR process when the reactors are in the medium temperature bath;
[0032] FIG. 3C shows plan view of the movable partition plates separating the high, the medium and the low temperature baths for a 3 step PCR process when the reactors are in the low temperature bath;
[0033] FIG. 3D shows plan view of the movable partition plates separating the high, the medium and the low temperature baths for a 3 step PCR process when the reactors are back to the high temperature bath;
[0034] FIG. 4 is a plan view of the movable partition plates separating five baths, according to an embodiment of the invention:
[0035] FIG. 5A illustrates an exemplary thermal profile wherein the profile for thermal cycling is achievable by the configuration at FIG. 2:
[0036] FIG. 5B illustrates an exemplary thermal profile wherein the profile for thermal cycling is achievable by the configuration at FIG. 3;
[0037] FIG. 6A is a perspective view of an exemplary reactor in the form of chips that is also usable with the apparatus under the present invention;
[0038] FIG. 6B is a perspective view of another exemplary reactor in the form of chips that is also usable with the apparatus under the present invention; and
[0039] FIG. 7 is a cross-sectional elevation view of a reactor with optical fibres for illuminating the reaction material and collecting the emitted light for analyses.
DETAILED DESCRIPTION
[0040] The following description presents several preferred embodiments of the present invention in sufficient detail such that those skilled in the art can make and use the invention.
[0041] FIG. 1B shows a schematic view of an embodiment of a portion of the thermal cycling apparatus for thermal cycling of nucleic acid such as for PCR, primer extension or other enzymatic reactions. The apparatus has two baths 50 and 51 each containing the bath medium 75, a bath heater 17 and a bath temperature sensor 39 mounted along the bath surface to enable control of the temperature of the bath medium 75. The bath temperature sensors 39 may be positioned inside the baths 50, 51 as shown. In this embodiment, the bath 50 is suitable for the step of denaturation and the bath 51 is suitable for the step of annealing and/or extension. The cooler 16 is useful when the bath 51 needs to be actively cooled to below room temperature. Active cooling device such as a thermoelectrical cooler or a fan can also be installed. The bath medium 75 shown here is liquid, however any other type of fluid or powder or solid bath medium 75 may also be used. For some embodiments, the bath heater 17 on the low temperature bath 51 is optional, if bath 51 does not have to be heated over room temperature. For the thermal cycling, the reactor 15 is alternately transferred between the baths 50, 51 multiple times. To enable fast movement of the reactor 15 in the bath medium 75, slim reactor 15 is preferable such as glass capillaries. The reactor 15 is sealed with a sealant or a cap 77 and a portion of the reactor 15 herein is transparent (not shown) to allow light to pass through for dye or probe excitation and fluorescence imaging. A temperature monitoring unit 34 is installed on the reactor holder 33 that moves along with the reactor 15 between the baths 50, 51. The temperature monitoring unit 34 contains a fast response temperature sensor 38 inside. The temperature monitoring unit 34 has a shape similar to that of the reactor 15 and is constructed to have a similar or the same steady state and transient thermal characteristics as those of the reactor 15, so that the temperature reading and thermal response is similar or same as those of the reactor 15 unless another reactor 15 itself is used for the purpose. For example, the temperature monitoring unit 34 may have the fast response temperature sensor 38 inserted into water or oil or a layer of oil over water 22 and sealed. The reactor transfer mechanism 85 transfers the reactor 15 and the temperature monitoring unit 34 at high speed among the baths 50 and 51 to expose them alternately to the different temperatures in the baths 50 and 51 as required for the thermal cycling. The reactor transfer mechanism 85 comprises an X stage 86 moving along an X axis linear guide 87 for the reactor 15 and the temperature monitoring unit 34 to reach to a region above the baths 50 and 51, and a Z stage 88 moving along a Z axis linear guide 89 for the reactor 15 to move them down to enter the bath medium 75 or to be withdrawn from the bath medium 75. The reactor 15 has an opening for loading the reaction material 21 and the openings are sealable. The sealant 77 may be made of a silicone rubber or UV cured polymer, hot melt and/or wax and/or gel which is in solid phase during thermal cycling. The sealing can also be achieved using liquid such as oil, viscous polymer, and gel. The highly viscous liquid can be applied to the opening and/or top section of the reactor 15 to block the vapor generated from the reaction material 21 from leaking out. The arrangement for the fluorescent imaging may be in any form as in the art. Herein, the bath 51 has a transparent window 25 so that illumination from the illumination source 44 reaches the reactor 15 inside and the emitted beam is received by the receiver 43. The partition plate 316 within the two baths 50, 51 opens for a minimal duration that is just adequate for the reactor transfer mechanism 85 to operate the X stage 86 for enabling the reactors 15 to change position between the baths 50, 51, without operating the Z stage 88 as illustrated by the double headed dashed arrow 200. On both sides of the partition plate 316, the levels of the bath mediums 75 are maintained substantially the same, hence the flow of the bath mediums 75 between the baths 50, 51 is negligible and the heat diffusion between the two baths 50, 51 is also minimized. The partition plate 316 being thermally insulating, the temperatures of the baths 50, 51 are better maintained. When the bath medium 75 is a high thermal conductivity metallic powder, the flow of the bath mediums 75 between the baths 50, 51 is even lesser as compared to the case of liquid.
[0042] FIGS. 2A-2F show plan views of the high temperature bath 50 and the low temperature bath 51 for a 2-step thermal cycling process. The baths 50, 51 are separated by a partition plate 316 that is made of a thermally insulating material. At (a), the reactors 15 are in the high temperature bath 50 and the partition plate 316 is at close a position, separating the hot and cold baths 50, 51. At (b), the partition plate 316 moves to an open position after the reactors 15 have been in bath 50 for a predetermined time. At (c), the reactors 15 speedily move to the low temperature bath 51 and promptly the partition plate 316 moves to the close position as shown at (d). At (e) the partition plate 316 moves to the open position again after the reactors 15 have been in bath 51 for another predetermined time. At (f) the reactors 15 move back to the hot bath 50 and the partition plate 316 closes thereafter (not shown) to complete one thermal cycle of a 2-step PCR thermal cycling process. The line arrows illustrate the movement of the partition plate 316 and the block arrows illustrate the movement of the reactors 15. In this embodiment, in the bath 50 the reactors 15 are allowed to attain a predetermined high target temperature T.sub.HT, wherein the T.sub.HT is in the region 85-99 degree Celsius for pre-denaturation and denaturation of the nucleic acid. In the bath 51, the reactors 15 are allowed to attain a predetermined low target temperature T.sub.LT, wherein the T.sub.LT is in the region 45-75 degree Celsius for annealing the nucleic acid. These are typical temperature ranges for PCR thermal cycling.
[0043] FIGS. 3A to 3D show plan views of the high 50, the low 51 and the medium 52 temperature baths for a 3-step thermal cycling process, with two partition plates 316, 317, the mechanism of operation of the partition plates being similar to the previous embodiment at FIGS. 2A-2F. The partition plates 316 and 317 open and close at the fastest possible speed that the electro-mechanics of the apparatus allows so that the bath mediums 75 from the either side do not substantially diffuse into the opposite sides. Any small diffusion alters the temperatures of the adjacent baths slightly which are compensated by the heaters 17 and the coolers 16 as applicable. The figures show the reactors 15 in the bath 50, followed by bath 52, followed by bath 51 and then back to bath 50 in one thermal cycle. The transfer mechanism 85 may be appropriately utilized for allowing the reactors 15 to change position between any two adjacent baths for thermal cycling without lifting the reactors 15 out of the baths when the partition plate 316, 317 between the adjacent baths is in an open position. In (a) to (d) respectively the reactors 15 are in the high temperature bath 50, in the medium temperature bath 52, in the low temperature bath 51 and then back to the high temperature bath 50 when both the partition plates open for short durations for the reactor(s) 15 to move to the high temperature bath 50 by speedily crossing through the medium temperature bath 52. In this embodiment, in the bath 52 the reactors 15 are allowed to attain a predetermined medium target temperature T.sub.MT, wherein the T.sub.MT is for annealing of primers or probes onto nucleic acid. Alternately in the bath 52 the reactors 15 are allowed to attain a predetermined medium target temperature T.sub.MT, wherein the T.sub.MT is for extension of primers on nucleic acid.
[0044] FIG. 4 shows a plan view of the high 50, the low 51 and the medium 52 temperature baths for a 3-step thermal cycling process, with two partition plates 316, 317, the mechanism of operation of the partition plates being similar to the previous embodiment at FIGS. 3A-3D. In this embodiment, an additional process bath 53 and a melt curve analysis bath 54 are also shown along with the respective partition plates 318 and 319. The additional process bath 53 allow the reactors 15 to attain a temperature TA before the thermal cycling, the additional process being one from the group consisting reverse transcription-polymerase chain reaction (RT-PCR), hot start process and isothermal amplification reaction. The melt curve analysis bath 54 can be progressively heated while conducting fluorescent imaging for the reaction material 21 in the reactors 15 while in the bath 54. As shown by the block arrow, the reactors 15 after being in the bath 53 for a predetermined time is moved to bath 50 in a single step by opening the partition plate 318 for a short duration. Thereafter, the reactors 15 are cycled between the baths 50, 52, 51 several times as shown by the multiple arrows and as described under FIGS. 3A-3D. Once the thermal cycling is completed, the reactors 15 are moved to the bath 54 as shown by the dashed arrow by opening the partition plate 319 for a short duration, for the melt curve analysis while progressively heating the bath. All these movements for the reactors 15 to change the baths are conducted without lifting-up the reactors 15 from the baths. The process of thermal cycling may thus be preceded by any additional thermal process and may be followed by melt curve analysis in an integrated apparatus while advantageously using the inventive concept of employing the partition plates 316 to 319, to reduce the time of thermal processing. The invention may be advantageously used with any number of baths with the required number of partition plates by positioning the baths and the partition plates in an appropriate configuration so that the transfer means 85 allows the reactors 15 to change the baths only by the X-stage 86.
[0045] FIG. 5A is an exemplary time-temperature graphical representations of typical 2-step thermal cycling process followed by a melt curve analysis. Only three cycles are shown over the processes of denaturation and annealing employing two baths 50, 51. After the thermal cycling, the reactor 15 are placed in bath 54 with at least a partially transparent bath medium 75 that is progressively heated while melt curve analysis is conducted. The fluorescence signals from the reactor 15 are acquired at multiple temperatures to form a fluorescence-temperature curve for melt curve analysis. The bath medium 75 for the melt curve analysis may be air or transparent liquid and the bath needs to have a transparent window 25. Both the bath medium 75 and the window 25 are required to have low auto-fluorescence. In this embodiment, the temperature of the bath medium 75 in the high temperature bath 50 is maintained at a temperature T.sub.HT and the temperature of the bath medium 75 in the low temperature bath 51 is maintained at a temperature T.sub.LT. This kind of thermal profile for thermal cycling is achievable by the bath configuration shown at FIGS. 2A-2F. The bath for the melt curve analysis is not shown where the reactor(s) 15 may be placed by the transfer mechanism 85 and by using another partition plate. FIG. 5B is an exemplary time-temperature graphical representations of typical 3-step thermal cycling process followed by a melt curve analysis. This kind of thermal profile for thermal cycling is achievable by the bath configuration shown at FIG. 3A-3D or 4. Herein the profile in the bath 53 has not been shown which may typically be in the range of 40-75 degree Celsius. Only three cycles are shown over the processes of denaturation, annealing and extension employing the three baths 50, 51, 52. After the thermal cycling, the melt curve analysis is conducted as described under FIG. 5A. The reactors 15 are placed in the medium temperature bath 52 for a longer duration to stabilize at T.sub.MT. Likewise, any other kind of thermal profile can be achieved by appropriately operating the partition plates 316, 317 and the X stage 86. Stabilization at T.sub.HT and T.sub.LT may also be obtained (not shown) by placing the reactors 15 in the baths 50 and 51 respectively for longer durations.
[0046] FIG. 6A is a biocchip 31 consisting of reactors 15 in the form of wells. The reaction material 21 is dispensed from the opening of the reactors 15 and sealed by a cover or sealing fluid 30. The biochip 31 is then mounted onto the reactor holder 33. FIG. 6B is a perspective view of the reactors 15 being accommodated in a biochip 31 for use in the baths. Herein, the reactors 15 are arranged in the biochip 31. There is at least one inlet 313 which is in fluid communication with the reactors 15 via a network of channels 315. The reaction material 21 to be tested can be loaded into the inlet 315 that subsequently flow into the reactors 15.
[0047] FIG. 7 shows an embodiment where the reactor 15 is made of a metal tubing. It is provided with an optical fiber 309 for light transmission from an illumination light source such as an LED (not shown) into the reaction material 21 inside the reactor 15. Optical fiber 310 is for light transmission from the reaction material 21 to a photodetector (not shown). This facilitates the optical detection for the reactor body even when non-transparent. Besides, it also helps to conduct the imaging while the reactors 15 are in any of the baths with transparent or non-transparent medium. The reactors 15 with this arrangement need not be made stationary for imaging thereby increasing the speed of the process of the thermal cycling.
[0048] The bath medium 75 may comprise one or more selected from a group consisting of water, oil, glycerin, chemical liquid, liquid metal, gas, air, metal powder and silicon carbide powder and/or beads and their mixture. The materials used to construct the reactors 15 may be plastics, elastomer, glass, metal, ceramic and their combinations, in which the plastics include polypropylene and polycarbonate. The glass reactor 15 can be made in a form of a glass capillary of small diameters such as 0.1 mm-3 mm OD and 0.02 mm-2 mm ID, and the metal can be aluminum in form of thin film, thin cavity, and capillary. Reactor materials can be made from non-biological active substances with chemical or biological stability. At least a portion of the reactor 15 is preferred to be transparent. The volume of the at least one reactor 15 may be in the range 1 L to 500 L. Smaller the volume, faster is the heat transfer, higher is the speed of PCR, smaller are the required bath sizes and more compact is the apparatus. The reaction material in all the reactors 15 in the reactor holder 33 may not be identical. Simultaneous PCR can be advantageously conducted for different materials if the bath temperatures are suitable. The baths may be shared between different process steps by altering the temperatures. The embodiments described above may be suitable for one reactor 15 or a plurality reactors 15. The reactor 15 may be in the form of tube(s) as shown or as wellplate(s) or chip(s) or cartridge(s) and the like.
[0049] According to an embodiment, the apparatus facilitates DNA melt curve analysis at the end of a PCR thermal cycle. Herein, after the DNA amplification process using PCR thermal cycling or other techniques, the reactors 15 are transferred to a bath (not shown) containing a transparent liquid bath medium 75. Such a bath has at least a portion to allow light to pass for illumination of the reactors 15 inside the bath and fluorescent imaging of the reactors 15. The bath medium 75 is capable of being heated up progressively while imaging the reactors 15 from a low temperature to a high temperature covering all the melt temperatures of the amplicons in the reaction materials 21 in the reactors 15. In the entire heating process, fluorescence signals from the reactors 15 are acquired at multiple temperatures to form a fluorescence-temperature curve for the melt curve analysis.
[0050] When using the above described apparatus for nucleic acid analysis and processing, the reaction material 21 comprises reaction constituents including at least one enzyme, nucleic acid and/or particle containing at least one nucleic acid, primers for PCR, primers for isothermal amplifications, primers for other nucleic acid amplifications and processing, dNTP, Mg.sup.2+, fluorescent dyes and probes, control DNA, control RNA, control cells, control micro-organisms, and other reagents required for nucleic acid amplification, processing, and analysis. The particle containing nucleic acid mentioned above comprises at least one cell virus, white blood cell and stromal cell, circulating tumor cell, embryo cell. One application may be to use the apparatus to test different kinds of reaction materials 21 against the same set of primer and probes, such as test more than one sample. For such application, different kinds of reaction material 21 containing no target primers and/or probes are each loaded into one reactor 15 in a reactor array, with all the reactors 15 being pre-loaded with the same set or the same sets of PCR primers and/or probes. For the same application, different kinds of reaction materials 21 pre-mixed with respective PCR target primers and/or probes are each loaded into one reactor 15 in a reactor array, with all the reactors 15 being not pre-loaded with the same set of PCR primers and or probes. The reaction materials 21 can include control genes and/or cells and corresponding fluorescent dyes or probes. In the above situations, the different probes emit light of different wavelengths. Another application of the methods and devices are used to test the same reaction material 21 against different sets of primer and probes. One example of such an application is to test one type of sample for more than one purpose. For this application, a single reaction material 21 is added into the reactors 15 each loaded with at least one different set PCR primers and or probes. The reaction material 21 can include control genes and/or cells and corresponding fluorescent dyes or probes. In the above situations, the different probes emit light of different wavelengths. The above reaction material 21 is used in polymerase chain reaction, reverse transcription-PCR, end-point PCR, ligase chain reaction, pre-amplification or target enrichment of nucleic acid sequencing or variations of polymerase chain reaction (PCR), isothermal amplification, linear amplification, library preparations for sequencing, bridge amplification used in sequencing. The variation of the polymerase chain reaction mentioned above comprises reverse transcription-PCR, real-time fluorescent quantitative polymerase chain amplification reaction and real-time fluorescent quantitative reverse transcription polymerase chain amplification reaction, inverse polymerase chain amplification reaction, anchored polymerase chain amplification reaction, asymmetric polymerase chain amplification reaction, multiplex PCR, colour complementation polymerase chain amplification reaction, immune polymerase chain amplification reaction, nested polymerase chain amplification reaction, the target enrichment of pre-amplification or nucleic acid sequencing, ELISA-PCR.
[0051] When the apparatus is in operation, the partition plate may open in a horizontal direction or in a vertical direction. Any other directions is also possible. In embodiments where the partition plate opens and closes in the horizontal direction and needs to protrude out of the bath area through a slot or a narrow cavity, in operation the slot or the cavity tends to get partially clogged with powder when the powder is used as the bath medium. Such a scenario offers increased resistance to the movement of the partition plate through the slot or the cavity. This makes the movement of the partition plate difficult.
[0052] According to an alternate embodiment, the partition plate opens and closes in the vertical direction, by lifting the partition plate up in the air and placing it down back to the original position, without having to move it through any such powder filled slot or cavity. There is however no such issue of partially clogging expected with the slot or the cavity when the bath medium is a liquid instead of a powder.
[0053] Any other mechanism for opening and closing the partition plate is equally possible.
[0054] The reactors may be in any form, such as tubes or wellplates or chips or cartridges. The tubes include capillaries.
[0055] From the foregoing description it will be understood by those skilled in the art that many variations or modifications in details of design, construction and operation may be made without departing from the present invention as defined in the claims.
