RAPID THERMAL CYCLING FOR SAMPLE ANALYSES AND PROCESSING

Abstract

Method and apparatus for thermal processing of nucleic acid in a thermal profile is provided. The method employs at least a first bath and a second bath, the method further employing a reactor holder for holding reactor(s) accommodating reaction material containing the nucleic acid. The method comprises maintaining bath mediums in the baths at two different temperatures; and alternately allowing the reactor(s) to be in the two baths in a plurality of thermal cycles to alternately attain a predetermined high target temperature THT, and a predetermined low target temperature TLT, wherein the bath medium in at least one of the baths is a high thermal conductivity powder.

Claims

1. Method for thermal processing of nucleic acid in a thermal profile, the method employing at least a first bath and a second bath, the method further employing a reactor holder for holding reactor(s) accommodating reaction material containing the nucleic acid and the reactor(s) being in any form such as tubes or wellplates or chips or cartridges, the method comprising: maintaining bath mediums in the baths at two different temperatures; and alternately allowing the reactor(s) to be in the two baths in a plurality of thermal cycles to alternately attain: a predetermined high target temperature T.sub.HT, and a predetermined low target temperature T.sub.LT, wherein the bath medium in at least one of the baths is a high thermal conductivity powder.

2. The method according to claim 1 further comprise: maintaining the T.sub.HT in the region 85-99 degree Celsius for denaturation of the nucleic acid; and maintaining the T.sub.LT 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 method according to claim 2 further employing a third bath, the method comprising: allowing the reactor(s) to be in the third bath to attain a predetermined medium target temperature T.sub.MT corresponding to the extension of primers in nucleic acid or the annealing of primers or probes onto nucleic acid.

4. (canceled)

5. The method according to claim 1 further employing a fourth bath, the method comprising: before the thermal cycling, allowing the reactor(s) to be in the fourth bath for an additional process from the group consisting: a) reverse transcription-polymerase chain reaction (RT-PCR), b) hot start process, and c) isothermal amplification reaction.

6. (canceled)

7. The method according to claim 1 further comprising: during the thermal cycling, allowing the reactor(s) to be in an air zone for conducting fluorescence imaging or electrochemical detection of the nucleic acid.

8. (canceled)

9. The method according to claim 1 wherein the high thermal conductivity powder is metal powder or metal powder dispersed in a liquid.

10. (canceled)

11. The method according to claim 9 wherein the powder comprises metal particles of substantially spherical shape.

12. The method according to claim 11 wherein the powder is copper powder of particle size: 1 m-5 mm.

13. (canceled)

14. (canceled)

15. The method according to claim 1 further comprising: employing a reactor guard to partially confine the reactor(s) to prevent the reactor(s) from getting deformed under resistive forces of the bath medium and the T.sub.HT when the reactor(s) is/are received in the powder.

16. The method according to claim 1 further comprising: attaining the target temperatures by a temperature guided motion controlling means (TeGMCM) that is operable based on the real-time temperature as sensed by a reactor temperature sensor during thermal cycling.

17. The method according to claim 1 further comprising: attaining the target temperatures by a time guided motion controlling means (TiGMCM) that is operable based on the time-periods for which the reactor(s) are allowed to be in the baths.

18. (canceled)

19. The method according to claim 1 further comprising: calibrating a transfer means to initiate lift-off of the reactor(s) from the bath(s) when the reactor(s) reach a first lift-off temperature that is lower than the T.sub.HT and a second lift-off temperature that is higher than the T.sub.LT, in order to compensate for operational electro-mechanical delays that unwantedly cause over heating or over cooling of the reactor(s).

20. 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 and the reactor(s) being in any form such as tube(s) or wellplate(s) or chip(s) or cartridge(s), the apparatus comprising: a first bath; a second bath, bath mediums in the baths being respectively maintainable at two different temperatures, and a transfer means for allowing the reactor(s) to be in the two baths in a plurality of thermal cycles to alternately attain: a predetermined high target temperature T.sub.HT, and a predetermined low target temperature T.sub.LT; and an amount of high thermal conductivity powder to serve as the bath medium in at least one of the baths.

21. (canceled)

22. (canceled)

23. (canceled)

24. The apparatus according to claim 20 further comprising: a third bath wherein the transfer means allows the reactor to attain a predetermined medium target temperature T.sub.MT during the thermal cycling of the reactor(s), wherein the T.sub.MT corresponds to the extension of primers in nucleic acid or the annealing of primers or probes onto nucleic acid

25. (canceled)

26. The apparatus according to claim 20 further comprising: a fourth bath wherein before the thermal cycling the transfer means allows the reactor(s) to attain a temperature T.sub.AP for an additional process from the group consisting a) reverse transcription-polymerase chain reaction (RT-PCR), b) hot start process, and c) isothermal amplification reaction.

27. (canceled)

28. The apparatus according to claim 20 further comprising: an air zone wherein the transfer means places the reactor(s) for conducting fluorescence imaging or electrochemical detection of the nucleic acid during the thermal cycling.

29. (canceled)

30. The apparatus according to claim 20 wherein the powder is metal powder or metal powder dispersable in a liquid.

31. (canceled)

32. The apparatus according to claim 20 wherein the powder comprises metal particles of substantially spherical shape.

33. The apparatus according to claim 32 wherein the powder is copper powder of particle size: 1 m-5 mm.

34. The apparatus according to claim 20 wherein the T.sub.HT is in the range 85-99 degree Celsius for denaturation of the nucleic acid and the T.sub.LT is in the range 45-75 degree Celsius for annealing of primers or probes onto nucleic acid or for primer extension for thermal cycling the reactor(s) to attain polymerase chain reaction (PCR) amplification or primer extension.

35. (canceled)

36. (canceled)

37. The apparatus according to claim 20 further comprising: a reactor temperature sensor; and at least one means from the group consisting: a temperature guided motion controlling means (TeGMCM) that is operable based on the real-time temperature as sensed by the reactor temperature sensor during thermal cycling, and a time guided motion controlling means (TiGMCM) that is operable based on the time-periods for which the reactor(s) are allowed to be in the baths, wherein the transfer means is operable by the at least one means.

38. The apparatus according to claim 37 wherein the TiGMCM can be user calibrated for the time-periods.

39. The apparatus according to claim 20 where in use the transfer means can be calibrated to initiate lift-off of the reactor(s) from the bath(s) when the reactor(s) reach a first lift-off temperature that is lower than the T.sub.HT and a second lift-off temperature that is higher than the T.sub.LT, in order to compensate for operational electro-mechanical delays that unwantedly cause over heating or over cooling of the reactor(s).

40. The apparatus according to claim 20 further comprising: shaking means for shaking at least one from the group consisting: a) the bath(s), b) the reactor(s), and c) both a) and b), during reactor insertion in the bath(s).

41. Reactor guard for the apparatus according to claim 20, the reactor guard comprising: confining means to partially confine the reactor(s) to prevent the reactor(s) from getting deformed under resistive forces of the bath medium and the T.sub.HT when the reactor(s) is/are received in the bath medium comprising high thermal conductivity powder.

42. The reactor guard according to claim 41 being made up of materials comprising metal or glass or high temperature plastics or ceramics.

43. The reactor guard according to claim 41 being an extension of the reactor holder.

44. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] FIG. 1 is an schematic view of a set up for thermal cycling of a reaction material containing nucleic acid using copper powder as the bath medium, according to an embodiment of the invention.

[0025] FIG. 2 is an isometric view of an embodiment of the apparatus for the process of thermal cycling as at FIG. 1.

[0026] FIG. 3 is an isometric view of an embodiment of the reactor array and baths with optics modules of the apparatus in FIG. 2.

[0027] FIGS. 4a and 4b are exemplary graphical representations of typical 2-step and 3-step PCR processes along with melt curve analysis.

[0028] FIG. 5 is a cross-sectional diagram of an array of tubular reactors held by the reactor holder and with the bottom tips of the reactors being coated by a metallic layer as according to an embodiment of the invention.

[0029] FIG. 6 is a cross-sectional diagram of an array of tubular reactors partially confined within reactor guards.

[0030] FIG. 7 is a top view of one reactor of FIG. 6, along with the reactor guard and reactor holder.

[0031] FIGS. 8a and 8b are perspective views of the reactors being accommodated in a biochip for use with the powder bath medium.

[0032] FIG. 9 is a cross-sectional view to illustrate the illumination and fluorescence emission detection module with an opaque tubular reactor that is particularly suited for powder bath medium as according to an embodiment.

[0033] FIG. 10 is a side elevation view to illustrate the illumination and fluorescence emission detection module with a powder bath as according to an embodiment.

DETAILED DESCRIPTION

[0034] 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.

[0035] FIG. 1 shows a schematic view of an embodiment of a portion of the thermal cycling apparatus for PCR or other enzymatic reactions to amplify nucleic acids or a fragment of nucleic acid. The apparatus has two baths 50 and 51 containing copper powder as the bath medium 75. Each bath 50 or 51 has 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. According to another embodiment, the bath temperature sensors 39 may be positioned inside the baths 50, 51. Bath 50 is set at a temperature that is suitable for the step of denaturation and the bath 51 is set at temperature that 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. The copper powder bath medium 75 enables faster and more accurate ramp-up and ramp down of the temperatures of the reactor 15 than that achievable with liquid bath medium. For some embodiments, the bath heater 17 on the low temperature bath 51 is optional, if bath 51 does not have to be heated. For the thermal cycling, the reactor 15 is alternately transferred between the baths 50, 51 multiple times. To enable fast plunging of the reactor 15 into 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 is transparent to allow light to pass through for dye or probe excitation and fluorescence imaging when the reactor is not in the copper powder bath medium 75. A temperature monitoring unit 34 is installed on the reactor holder 33 and 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, for the temperature reading and thermal response to be 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. Although only one reactor 15 is shown, according to other embodiments the reactor holder 33 may accommodate a plurality of reactors 15. The reactor 15 may be in the form of tube(s) as shown or as wellplate or chip(s) or cartridge(s). 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. There are many possible designs of the reactor transfer mechanism 85. One such mechanism is comprised of 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 reactors 15 to move them down to enter the bath medium 75 or to be withdrawn from the bath medium 75. Such a transfer mechanism 85 can also consist of a rotary arm (not shown) that moves the reactor 15 and the temperature monitoring unit 34 in an angular direction along with the Z stage 88 moving along the Z axis linear guide 89. The reactors 15 have an opening for loading and retrieving reaction material 21 from inside, 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 reactors 15, it is able to block the vapor generated from the reaction material 21 from leaking out.

[0036] In another embodiment (not shown) of thermal cycling, in between the baths 50 and 51, the reactor 15 with the monitoring unit 34 may be inserted into a 3rd bath containing bath medium at a medium target temperature or positioned in hot air for a period of time required for annealing and or extension. Herein the thermal cycling is performed in three-steps by inserting the reactor 15 into the three baths within each thermal cycle.

[0037] According to yet another embodiment (not shown) a fourth bath is maintained at a predetermined temperature T.sub.AP and before the thermal cycling, the reactor 15 with the temperature monitoring unit 34 is inserted in the fourth bath to allow at least one additional process from the group consisting reverse transcription-polymerase chain reaction (RT-PCR) and isothermal amplification reaction. The RT-PCR is carried out prior to the thermal cycling for nucleic acid amplification.

[0038] A temperature guided motion controlling means (TeGMCM) (not shown) may preferably be employed in the apparatus for allowing the reactor(s) 15 to remain in the bath(s) 50 to 54 until the corresponding target temperature is attained, in order to maintain better accuracy of the predetermined target temperatures attained by the reactor(s) 15. The TeGMCM may be provided with advance signals when the reactor(s) 15 are about to reach the target temperatures as sensed by the temperature monitoring unit 34 in order to avoid over heating or over cooling of the reactor(s) 15. The thermal cycling with this method provides higher accuracy and needs no user calibration though is at a higher cost and complexity of the apparatus due to requirement of very fast temperature sampling and signal processing electronics, fast data communication with the reactor transfer mechanism, and very responsive mechanical motion components such as motors and actuators in the reactor transfer mechanism. Alternately, a time guided motion controlling means (TiGMCM) may be used that is operable based on the time-periods for which the reactor(s) are allowed to be in the baths. The TiGMCM may be user calibrated for the time-periods. This embodiment requires lower complexity of the apparatus as it operates by time-duration hence does not require highly responsive set-up though needs user calibration.

[0039] FIG. 2 shows an isometric view of the apparatus with the reactor transfer mechanism 85, a bath module 204, an optical module 206, 205, 207 and 208. The bath module 204 is shown to comprise five baths 50, 51, 52, 53, 54 placed next to each other and each maintained at a predetermined temperature. An optics module 206 carries out fluorescent detection of nucleic acid inside the reactors 15 when the reactors 15 are situated in the low target temperature bath 51 having air or transparent liquid as the bath medium 75 in this embodiment. A temperature controller module 205 controls the bath temperatures. A motion controller module 207 controls all motions and a system controller module 208 controls the system, with data communication and processing, image processing and data analysis. The additional processes like reverse transcription-polymerase chain reaction (RT-PCR), hot start process, and isothermal amplification reaction can also be carried out in any of the baths for thermal cycling which can be set at the required temperature before the thermal cycling and reset to the temperature for thermal cycling after the completion of the above additional processes.

[0040] FIG. 3 shows a blown up illustration of the bath module 204 and the optical module 206. The optical module 206 comprises an illuminator 44 and a fluorescence detector 43. The incident beam 46 to reaches the reaction material 21 within the reactor 15. The reflected beam 47 from the reaction material 21 is captured thereafter. The array of reactors 15 with the temperature monitoring unit 34 are transferred among the baths 50 to 54. In this embodiment, the bath 51 has air or transparent liquid as the bath medium 75 along with a transparent window 25 at the bottom to pass the illumination light and emission light to and from the reactor 15. The bath heaters 17 or cooler 16 are connected to the sidewalls of the baths. The apparatus may may further comprise a hot air zone (not shown) for placing the reactors 15 particularly during imaging. This simplifies the apparatus. The method may employ an electrical heater or an infrared heater for forming the hot air zone above a bath or inside a bath. The heater may be installed with the reactor holder 33 so that only the air in the vicinity of the reactors 15 is heated while the reactor(s) 15 is/are moving between the baths. This feature saves energy and saves the other parts of the apparatus from getting undesirably heated.

[0041] FIGS. 4a and 4b are exemplary time-temperature graphical representations of typical 2-step and 3-step thermal cycling processes (for PCR, primer extension and the kind), followed by a melt curve analysis. Only three cycles are shown over the processes of denaturation at the temperature T.sub.HT and annealing at the temperature T.sub.LT employing two baths in FIG. 4a and employing three baths in FIG. 4b for an additional step for extension at the temperature T.sub.MT. After the thermal cycling, the reactors 15 are placed in at least a partially transparent bath medium that is progressively heated while melt curve analysis is conducted. The fluorescence signals from the reactors 15 are acquired at multiple temperatures to form a fluorescence-temperature curve for the melt curve analysis.

[0042] FIG. 5 is a cross-sectional diagram of an array of tubular reactors 15 held by a reactor holder 33 and with the bottom tips of the reactors 15 being coated by a metallic layer 5 as according to an embodiment of the invention. When the bath medium 75 in at least one bath is metal powder, in order to enhance the insertion capability of the reactors 15 into the metal powder the reactors 15 are provided with metallic layer 5 at the bottom tips and the bottom tips are sharper with lesser cross-sectional area than the rest of the reactors 15.

[0043] FIG. 6 is a cross-sectional diagram of an array of tubular reactors 15 partially confined within a reactor guard 7 that may be optionally used. The reactor guard 7 provides physical support to prevent the plastic reactors 15 from getting deformed under the resistive forces and the T.sub.HT or the glass reactors 15 from breaking under the resistive forces when the reactors 15 are inserted into the powder bath medium 75. Typically, such force is maximum on the reactor tip when the reactor 15 is inserted into the powder bath medium 75. The reactor guard 7 may be made up of materials comprising metal or glass or high temperature plastics or ceramics. FIG. 7 shows a top view of one reactor 15 with the reactor guard 7 and the reactor holder 33. As shown, the reactor guard 7 covers only a small portion of the reactors 15 hence does not obstruct fluorescent imaging.

[0044] FIG. 8 (a) is a biochip 31 consisting of only reactors 15 in the form of wells with no channel 315. The reaction material 21 is dispensed from the opening of the reactors 15 and sealed by a cover or sealing fluid 30. Then, the biochip 31 is mounted onto the reactor holder 33. FIG. 8b is a perspective view of the reactors 15 being accommodated in a biochip 31 for use with the baths. Herein, a plurality of the reactors 15 are arranged in the biochip 31. The edges of the biochip 31 may be disposed with a material (not shown) that retains rigidity at high T.sub.HT and when used with the powder bath medium 75. 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. For moving inside the powder bath medium 75.

[0045] FIG. 9 shows an embodiment where the reactor 15 is made of a metal tubing which is suitable for metal powder as the bath mediums 75. For the fluorescent imaging, an optical fiber 309 transmits light 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). The sealant or cap 77 holds the optical fibers 309, 310. This facilitates the optical detection for the non-transparent reactor 15.

[0046] FIG. 10 is a side elevation view to illustrate the illumination and fluorescence emission detection module 206 with the opaque powder bath medium 75 as according to an embodiment. The optical module 206 comprises an illuminator 44, a fluorescence detector 43, optical filters 81 and a dichroic mirror 82 generate the incident beam 46 to reach the reaction material 21 within the reactor 15 and capture the reflected beam 47 from the reaction material 21 thereafter. The imaging mechanism is evident from the optical arrangement. Fluorescence imaging of the reactors 15 while the reactors 15 are inside the bath medium 75 is important since the reactors 15 may have to be maintained at the annealing or extension temperature for a prolonged period of time when multiple images of different wavelengths are taken for multiplex detection or to acquire fluorescence images of control genes. The reactor caps 32 are transparent to the lights for the imaging.

[0047] Different bath may contain different bath medium 75 for specific advantages as desired. The reactors 15 may be made up of 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. In another embodiment, the reactors 15 can be in a form of a reactor array chip or a microfluidic reactor chip or arrayed chip. For example, the reactors 15 can be in a form of wells or channels of a substrate plate and optionally covered with a solid layer of material to form closed reaction chambers, in which the reaction fluid or reaction system is situated. The reaction material 21 in all the reactors 15 in the reactor holder 33 may not be identical. Simultaneous PCR can be advantageously conducted for different materials 21 if the bath temperatures are suitable. At least part of the reactor wall may be made of metal sheet of thickness 1 m-2 mm. This feature enhances the rate of heat transfer between the bath and the reaction material 21. At least part of the reactor wall may be made of plastic or glass sheet of thickness 0.5 m-500 m. At least a part of the reactor wall is made of transparent material so as to enable the imaging and detection process. 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.

[0048] 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.