Nucleic acid amplification reaction apparatus

Claims

What is claimed is:

1. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture containing a target nucleic acid segment including denaturation, annealing and extension processes, said apparatus comprising:

a capillary tube reaction chamber having a portion adapted to contain the reaction mixture at a predetermined position;

first temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber for subjecting the reaction mixture at said position to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber at said position for subjecting the mixture to a second temperature suitable to cause the annealing and extension process to occur in the mixture;

sample handling means connected to the capillary tube reaction chamber for automatically moving the reaction mixture into and out of the capillary tube reaction chamber and to and from said predetermined position; and

positioning means for automatically positioning the first and second heat exchange means sequentially in contact with the portion of the capillary tube reaction chamber,

wherein each of the heating means is a segment of a cylindrical heat exchange drum.

2. The apparatus according to claim 1 further comprising a third temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube for subjecting the portion to a temperature above the first temperature prior to contacting said portion with said first temperature controlled heating means for a time sufficient to drive the temperature of the mixture up to said first temperature.

3. The apparatus according to claim 1 wherein the cylindrical heat exchange drum has at least two axially extending radial segments, each separated from each other by a layer of thermal insulation.

4. The apparatus according to claim 2 wherein each of the segments has an arcuate outer surface and a groove in the arcuate outer surface each adapted to receive the portion of the capillary tube reaction chamber.

5. The apparatus according to claim 4 wherein the drum is rotatable about its central axis between positions in which each of the segments contacts the portion of the capillary tube reaction chamber.

6. The apparatus according to claim 5 further comprising a programmable computer connected to the heating means and the rotatable drum operable to control the temperatures of the heat exchange means and the position of the drum in accordance with user-defined parameters.

7. The apparatus according to claim 1 further comprising a plurality of open capillary tube reaction chambers each having a portion adapted to receive a reaction mixture.

8. The apparatus according to claim 7 wherein each of the segments has an arcuate outer surface and a plurality of parallel grooves in the arcuate outer surface each adapted to receive one of said portions of the capillary tube reaction chambers.

9. The apparatus according to claim 8 wherein the drum is rotatable about its central axis between positions in which each of the segments contacts said portion of each of the capillary tube reaction chambers.

10. The apparatus according to claim 9 further comprising a programmable computer connected to the heating means and the rotatable drum and wherein the computer controls the rotatory position of the drum in accordance with user defined parameters.

11. An apparatus for performing a reaction in a reaction mixture containing a target nucleic acid segment including denaturation, annealing and extension processes in a capillary tube, the apparatus comprising:

a capillary tube reaction chamber for containing the reaction mixture;

first temperature controlled movable heat exchange means adapted to be coupled to the capillary tube for subjecting a portion of the capillary tube reaction chamber to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to be coupled to the capillary tube for subjecting said portion of the capillary tube to a second temperature suitable to cause the annealing and extension processes to occur in the reaction mixture;

means for automatically moving the reaction mixture into said portion of the capillary tube reaction chamber and discharging the reaction mixture from the capillary tube reaction chamber; and

control means for sequentially coupling said first and second heat exchange means to the capillary tube in accordance with a reaction protocol to cause said reaction to take place in said reaction mixture.

12. The apparatus according to claim 11 wherein the capillary tube reaction chamber has one open end.

13. The apparatus according to claim 11 wherein the means for automatically moving is a computer-controlled peristaltic pump.

14. An apparatus for simultaneously performing a nucleic acid amplification reaction including denaturation, annealing and extension processes in reaction mixtures in a plurality of capillary tubes, the apparatus comprising:

a plurality of open ended capillary tubes each having a portion adapted to contain a reaction mixture suitable for causing the nucleic amplification reaction to create copies of a target nucleic acid segment;

first temperature controlled movable heat exchange means for simultaneously subjecting said portion of each of the capillary tubes to a first temperature suitable to cause the denaturation process in the nucleic acid amplification reaction to occur in the mixtures;

second temperature controlled movable heat exchange means for simultaneously subjecting said portion of each of the capillary tubes to a second temperature suitable to cause the annealing and extension processes in the nucleic acid amplification reaction to occur in the mixtures;

positioning means for causing said temperature controlled movable heat exchange means to subject the reaction mixtures to the first and second temperatures in accordance with a predetermined sequence of steps;

plurality of reaction tubes corresponding to said capillary tubes; and

handling means for operatively connecting an open end of each of the capillary tubes to a corresponding reaction tube containing the reaction mixture and for cleansing the capillary tubes.

15. The apparatus according to claim 14 wherein the handling means includes a reaction tube array translation and elevation system.

16. The apparatus according to claim 14 wherein the handling means includes a system for inserting and removing each of the capillary tubes from each of the reaction tubes.

17. The apparatus according to claim 14 wherein the positioning means comprises a programmable computer controlling the handling means and the first and second temperature controlled heat exchange means in accordance with a user defined set of parameters.

18. An apparatus for performing a nucleic acid amplification reaction on a plurality of reaction mixtures contained in a plurality of capillary tubes simultaneously, the reaction including denaturation, annealing and extension processes to generate a nucleic acid amplification reaction product, the apparatus comprising:

a first heat exchanger including a thermoregulating system for stabilizing the temperature of the first heat exchanger at a temperature in a range of temperatures suitable to cause the denaturation process to occur in the nucleic acid amplification reaction in the plurality of reaction mixtures;

a second heat exchanger including another thermoregulating system for stabilizing the temperature of the second heat exchanger at a temperature in a range of temperatures suitable for causing the annealing and extension processes in the nucleic acid amplification reaction to occur in the mixtures;

a plurality of capillary tubes each routed so that each has a portion in thermal contact with at least one of the first and second heat exchangers;

sample handling means coupled to each of the capillary tubes for drawing the reaction mixture into the tube and positioning said mixtures in said portions, and for discharging finished reaction product from each of the tubes; and

user controllable means for sequentially moving the first and second heat exchangers into thermal contact with the portions of the capillary tubes containing the reaction mixtures so as to cause the nucleic acid amplification reaction to take place in the mixtures in the capillary tubes.

19. The apparatus of claim 18 further comprising a detector means optically coupled to the capillary tubes for detecting optical changes in the mixtures.

20. The apparatus according to claim 19 wherein the detector means includes a photodiode array.

21. The apparatus according to claim 19 wherein the detector means is coupled to a spectrophotometer.

22. The apparatus according to claim 19 wherein the detector means detects a fluorescence change occurring in the mixture during a portion of the nucleic acid amplification reaction.

23. The apparatus according to claim 22 wherein the detector means includes a photodiode device or CCD detector coupled to a computer.

24. The apparatus according to claim 18 wherein the first and second heat exchangers are each portions of an elongated rotatable drum.

25. The apparatus according to claim 24 wherein each of the heat exchangers is an arcuate segment of the cylindrical drum, each segment having an arcuate outer surface.

26. The apparatus according to claim 25 wherein the surface has a plurality of parallel grooves receiving the portions of the plurality of capillary tubes.

27. The apparatus according to claim 26 wherein the heat exchanger segments are separated by an insulating layer.

28. The apparatus according to claim 27 wherein the grooves are circumferential.

29. The apparatus according to claim 28 wherein the means for positioning is a computer-controlled stepper motor coupled to the rotatable cylindrical drum, the motor rotating the drum between a first angular position in which the first heat exchanger is in thermal contact with each of the portions of the capillary tubes and a second angular position in which the second heat exchanger is in thermal contact with each of the portions of the capillary tubes.

30. The apparatus according to claim 28 wherein the motor is further operable to rotate the cylindrical drum to a third angular position wherein a third heat exchanger segment having a temperature greater than that of the first and second heat exchanger segments contacts the portions of the tubes.

31. The apparatus according to claim 29 wherein the motor is further operable to rotate the cylindrical drum to a fourth angular position wherein a fourth heat exchanger segment having a temperature less than that of the first, second or third heat exchanger segments contacts the portions of the tubes.

32. The apparatus according to claim 19 wherein the means for drawing has at least one syringe pump connected to the capillary tubes.

33. The apparatus according to claim 32 wherein the means for drawing further comprises a lift mechanism coupled to an open end of each of the capillary tubes for inserting and withdrawing the one end of each tube into and out of at least one sample container.

34. The apparatus according to claim 33 wherein the means for drawing further comprises a computer controlled stepper motor mechanically connected to an elevating clamping bar holding the ends of the tubes in a spaced arrangement.

35. The apparatus according to claim 34 further comprising a translation mechanism coupled to a stage operable to index the sample containers to and from a position in registry with at least one of the ends of the capillary tubes.

36. The apparatus according to claim 35 wherein the stage contains a cleaning solution container and the translation mechanism is further operable to position the stage controllably in a clean and rinse position wherein the tube ends are in registry with the cleaning solution container.

37. The apparatus according to claim 36 further comprising a programmable computer operably coupled to the first and second heat exchangers, the lift mechanism, the translation mechanism, the means for positioning, and the thermoregulating systems to automatically handle the reaction mixture and perform the nucleic acid amplification reaction on the reaction mixture in accordance with user defined parameters to generate the reaction product.

38. The apparatus according to claim 18 wherein the first and second heat exchangers are each metal blocks.

39. The apparatus according to claim 38 wherein each of the heat exchangers is a generally rectangular block.

40. The apparatus according to claim 39 wherein each of the heat exchangers has a flat surface having a plurality of parallel grooves receiving the portions of the plurality of capillary tubes.

41. The apparatus according to claim 40 wherein the heat exchangers are separated by an insulating layer.

42. The apparatus according to claim 41 wherein the insulating layer is air.

43. The apparatus according to claim 42 wherein the means for positioning is a computer controlled syringe pump coupled to each of the tubes, the pump moving the reaction mixtures between the heat exchangers in accordance with a user-defined sequence of steps to perform the nucleic acid amplification reaction.

44. The apparatus according to claim 1, wherein temperature of the segments is thermostatically controlled.

45. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture containing a target nucleic acid segment including denaturation, annealing and extension processes, said apparatus comprising:

a capillary tube reaction chamber having a portion adapted to contain the reaction mixture at a predetermined position;

first temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber for subjecting the reaction mixture at said position to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber at said position for subjecting the mixture to a second temperature suitable to cause the annealing and extension process to occur in the mixture;

sample handling means connected to the capillary tube reaction chamber for automatically moving the reaction mixture into and out of the capillary tube reaction chamber and to and from said predetermined position;

positioning means for automatically positioning the first and second heat exchange means sequentially in contact with the portion of the capillary tube reaction chamber; and

a third heating means adapted to contact the portion of the capillary tube for subjecting the portion to a temperature above the first temperature.

46. An apparatus for performing a nucleic acid amplification reaction on a plurality of reaction mixtures contained in a plurality of capillary tubes simultaneously, the reaction including denaturation, annealing and extension processes to generate a nucleic acid amplification reaction product, the apparatus comprising:

a first heat exchanger including a thermoregulating system for stabilizing the temperature of the first exchanger at a temperature in a range of temperatures suitable to cause the denaturation process to occur in the nucleic acid amplification reaction in the plurality of reaction mixtures;

a second heat exchanger including another thermoregulating system for stabilizing the temperature of the second heat exchanger at a temperature in a range of temperatures suitable for causing the annealing and extension processes in the nucleic acid amplification reaction to occur in the mixtures;

a plurality of capillary tubes each routed so that each has a portion in thermal contact with at least one of the first and second heat exchangers;

sample handling means coupled to each of the capillary tubes for drawing the reaction mixture into the tube and positioning said mixtures in said portions, and for discharging finished reaction product from each of the tubes; and

user controllable means for sequentially causing relative motion between the first and second heat exchangers and the portions of the capillary tubes containing the reaction mixture so as to cause the nucleic acid amplification reaction to take place in the mixtures in the capillary tubes.

47. The apparatus according to claim 46 wherein the first and second heat exchangers are each stationary metal blocks.

48. An apparatus for performing a nucleic acid amplification reaction in a reaction mixture containing a target nucleic acid segment including denaturation, annealing and extension processes, said apparatus comprising:

a capillary tube reaction chamber having a portion adapted to contain the reaction mixture at a predetermined position;

first temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber for subjecting the reaction mixture at said position to a first temperature suitable to cause the denaturation process to occur in the reaction mixture;

second temperature controlled movable heat exchange means adapted to contact said portion of the capillary tube reaction chamber at said position for subjecting the mixture to a second temperature suitable to cause the annealing and extension process to occur in the mixture;

sample handling means connected to the capillary tube reaction chamber for automatically moving the reaction mixture into and out of the capillary tube reaction chamber and to and from said predetermined position; and

positioning means for automatically positioning the first and second heat exchange means sequentially in contact with the portion of the capillary tube reaction chamber; and

a plurality of open capillary tube reaction chambers each having a portion adapted to receive a reaction mixture;

wherein each of the heating means is a thermostatically controlled segment of a cylindrical heat exchange drum.

49. The apparatus according to claim 1, wherein the segments are radial segments.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to amplifying nucleic acids by thermal cycling and, more particularly, to automated machines for performing amplification reactions such as a polymerase chain reaction (PCR).

2. Description of the Related Art

DNA (Deoxyribonucleic acid) may be amplified by thermally cycling a specially constituted liquid reaction mixture according to protocol such as a polymerase chain reaction (PCR) protocol which includes several incubations at different temperatures. The reaction mixture is comprised of various components such as the DNA to be amplified (the target) and at least two oligonucleotide primers selected in a predetermined way so as to be complementary to a portion of the target DNA. The reaction mixture also includes various buffers, enzymes, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dGTP, and dTTP. The duplex DNA molecule is denatured into two complementary single strands. The primers then anneal to the strands, and, in PCR, nucleoside monophosphate residues are then linked to the primers in the presence of an enzyme such as a thermostable DNA polymerase to create a primer extension product. After primer extension, twice as many duplex DNA molecules exist. This process is repeated, each time approximately doubling the amount of DNA present. The result is an exponential increase in the concentration of target DNA, known as "amplification" of the target DNA.

The polymerase chain reaction (PCR) has proven to be a phenomenal success for genetic analysis, largely because it is simple and very versatile, and requires relatively low cost instrumentation. A key to this success is the concept of thermal cycling: alternating steps of melting DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of the double stranded DNA.

The methodology of the polymerase chain reaction is more fully described in U.S. Pat. Nos. 4,683,202 and 4,683,195 which are hereby incorporated by reference.

The polymerase chain reaction (hereafter PCR) has been performed in disposable reaction tubes such as small, plastic microcentrifuge tubes or test tubes which are placed in an instrument containing a thermally controlled heat exchanger. Examples of these instruments are disclosed in U.S. Pat. No. 5,038,852, U.S. application Ser. No. 07/709,374, filed Jun. 3, 1991, and U.S. application Ser. No. 07/871,264, filed Apr. 20, 1992, all of which are hereby incorporated by reference in their entirety.

The heat exchanger in these instruments is typically a metal block; however, hot air ovens and water baths also have been used. The temperature of the reaction mixture in the reaction tubes is changed in a cyclical fashion to cause denaturation, annealing and extension reactions to occur in the mixture. Three separate incubation temperatures commonly were used in the first generation PCR thermal cycling applications. These were typically around 94.degree. C. for denaturation, around 55.degree. C. for annealing, and around 72.degree. C. for extension. More recently, the annealing and extension incubations have frequently been combined to yield a two temperature incubation process, typically around 94.degree. C. for denaturation, and around 50.degree.-65.degree. C. for an annealing and extension incubation. The optimal incubation temperatures and times differ, however, with different targets.

Rapid, small scale, PCR capillary tube instruments also have appeared. For example, Idaho Technology introduced an instrument wherein the reaction mixture is placed in capillary tubes which are then sealed and placed in a hot air oven which cycles the temperature of the reaction mixtures in the tubes. A similar system was described in a paper by Wittwer et al., "Minimizing the Time Required for DNA Amplification by Efficient Heat Transfer to Small Samples", Analytical Biochemistry 186, 328-331 (1990). There, 100 microliter samples placed in thin capillary tubes were placed in an oven with a heating coil, a solenoid activated door and a fan. Air was used as the heat transfer medium. A very similar system was also described by Wittwer et al in another paper entitled "Automated Polymerase Chain Reaction in Capillary Tubes with Hot Air," Nucleic Acids Research, Volume 17, Number 11, pp. 4353-57 (1989).

The PCR volume has been limited to a range of from about 10 microliters to 1.5 milliliters in conventional heat block or liquid bath heat exchanger PCR instrument designs where the reaction mixture has been stored in microcentrifuge tubes. It is hard to scale up these volumes. The difficulty resides in the fixed dimensions of the wells in the heat exchange block for the tubes and the escalating difficulty in achieving heat transfer uniformity among all wells as dimensions get larger and heat gradient problems become more pronounced. As the volume of prior art reaction vessels is increased, the surface/volume ratio decreases. This change reduces the ability to change quickly the temperature of the reaction mixture in each tube because most heat exchange occurs between the walls of the tubes and the walls of the wells in the sample block.

In prior art instruments, thermal ramps were long because there was substantial lag in the temperature of the sample relative to the block caused by poor convection and conduction. Substantial thermal ramp durations between incubation temperatures were often necessary to prevent significant temperature gradients from developing because of the large thermal mass of the metal blocks used in many instruments as well as nondiffuse heat sources and sinks. These temperature gradients can cause non-uniform amplifications in different samples located at diverse points along the temperature gradient. There is no chemical or biological reason for using temperature ramps.

A capillary tube PCR instrument has the advantage of rapid thermal incubation transitions because the reaction volume and sample containment thicknesses can be minimized. One such instrument is disclosed in U.S. Pat. No. 5,176,203, issued to D. M. Larzul. The Larzul patent discloses a wheel shaped apparatus for automatic thermal cycling of a fluid sample contained in a closed loop or spiral coil of continuous capillary tube. Each loop of the tube is routed through three thermostatted zones. The sample is pushed through the loops by a motorized magnetic system in which a magnet on the end of a rotating central arm magnetically pulls a slug of mineral oil containing suspended metallic particles through the capillary tube. Since the slug abuts the sample in the capillary tube, the slug pushes the sample through the loop. The motorized system may be micro-processor controlled to regulate the movement of the sample in accordance with a predetermined protocol.

SUMMARY OF THE INVENTION

A number of alternative embodiments of capillary tube PCR instruments are envisioned herein which share certain common advantages. All of the instruments disclosed herein use thin walled capillary tubes to hold the reaction mixture as opposed to microcentrifuge tubes. A capillary tube as used herein is a tube which has an internal diameter less than 3 mm and preferably on the order of about 1 mm to 2 mm in internal diameter. These capillary tubes are heated and cooled in the embodiments taught herein according to a user-defined PCR protocol required of a particular reaction mixture fed into a programmable computer which, in turn, automatically controls sample handling, flow, velocity, pressure, and temperature to implement the protocol via conventional control programming. The differences among the various embodiments of the invention arise out of the different means used to heat and cool the PCR reaction mixture and different tube and fluid handling means used to move the reaction mixture.

For example, a first embodiment of the invention automatically pumps the reaction mixture repetitively through a continuous loop of capillary tube which is routed through two different thermostatted fluid baths, one at a denaturation temperature and one at an anneal/extend temperature. Additional baths could be added to increase the number of incubation temperatures.

A second embodiment directs the reaction mixture through a capillary tube only once, i.e. in a single pass. The capillary tube is routed in an alternating fashion back and forth between a first thermostatted heat exchanger held at a denaturation temperature and a second heat exchanger held at an anneal/extend temperature. Alternatively, a third heat exchanger may also be used if the anneal and extend temperatures differ. A positive displacement or peristaltic pump or syringe is used to push the reaction mixture through the single pass tube out into a product collection vessel.

A third embodiment of the invention involves a stationary reaction mixture in a multiple fluid bath arrangement. This embodiment uses a single loop of capillary tube for each sample. The portions of the loops containing the samples are enclosed in a reaction chamber. A hot fluid at a denaturation temperature is pumped into this reaction chamber from a first thermostatted fluid bath and held there during the denaturation incubation. Fluid at an anneal/extend temperature is pumped into the chamber from a second temperature stable bath after removal of the hot fluid from the first temperature stable bath to implement the anneal/extend incubation. This process is repeated as necessary to complete the PCR protocol.

A fourth embodiment utilizes two or three temperature stable fluid baths, each of which is constantly circulating its fluid through a separate conduit. Each fluid stream is thermostatted at one of the necessary PCR incubation temperatures. A single cylindrical heat exchanger chamber with a wire mesh at the input end is connected through a valve system to the fluid streams. The wire mesh holds individual small capillary tube reaction mixture vessels, which are each sealed at both ends in place in the heat exchange chamber. The vessels are held in a spaced relationship or array by the mesh because the seal at one end of each capillary tube vessel is too large to fit through the mesh. The valve system, preferably under the control of a computer or other automated controller, is used to select one of the streams at a time to be routed through the cylindrical heat exchanger chamber while the other one or two streams are bypassed around it. For example, to carry out a denaturation incubation, a stream of 94.degree. C. fluid is routed through the heat exchanger chamber while an anneal stream at 55.degree. C. and an extend stream at 75.degree. C. are routed around the heat exchange chamber.

A fifth embodiment uses two metal blocks each of which has its temperature stabilized at one of two temperatures needed for the denaturation and anneal/extend incubations. An open-ended, thin-walled capillary tube is routed through and between these two metal blocks. A peristaltic pump or a plunger and seal arrangement, similar to a syringe, is connected to one end of the capillary tube, under control of a computer programmed to carry out the PCR protocol. The pump or plunger is activated back and forth to draw reaction mixture into the capillary tube, move it into the region surrounded by the denaturation block, i.e., the block held at the denaturation temperature, and then move the reaction mixture through the capillary tube into the region of the capillary tube surrounded by the block held at the anneal/extend temperature. This cycle is repeated the required number of times to complete the PCR protocol. The plunger is then moved to discharge the PCR product. Alternatively, the blocks themselves could be translated back and forth against a stationary capillary tube containing the mixture to thermally cycle the mixture.

A sixth embodiment of the capillary PCR instrument in accordance with the invention is somewhat similar to the fifth in that spaced metal heat exchanger blocks are used. This embodiment preferably has a pair of spaced heat exchanger blocks, a plurality of open-ended capillary tubes routed through each of the blocks, and an automated sample handling system. This handling system simultaneously inserts the end of each of the capillary tubes into reaction mixture containers, withdraws the reaction mixture into the capillary tubes, then translates t