Inline Thermo-Cycler

This invention relates to cycling biological materials through desired sequences of temperatures. More particularly, it relates to apparatus for thermo-cycling fluid while the fluid flows through the apparatus and to methods of using such apparatus.

Genetic testing of DNA and related materials is an integral part of clinical, commercial and experimental biology. In the medical field, for example, genetic tests are critical for effective treatment of cancer and inherited diseases. Oncologists use genetic tests to obtain the cytogenetic signature of a malignancy which in turn guides the choice of therapy and improves the accuracy of prognoses. Similarly, monitoring frequency and type of mutations persisting after chemotherapy or radiation therapy provides a quick and accurate assessment of the impact of the therapy. Perhaps the most important application of molecular diagnosis in oncology is the emerging possibility of using anti-sense genetic therapy to fight tumor growth.

Inherited diseases occur when a person inherits two copies of a defective (referred to hereinafter as an allele) version of a gene. Genetic tests can determine which genes and alleles are responsible for a given disease. Once the gene is identified, further testing can identify carriers of the allele and aid researchers in designing treatments for the disease.

Most genetic tests begin by amplifying a portion of the DNA molecule within a sample of biological material. Amplification is made practical by the polymerase chain reaction (PCR) wherein a DNA synthesizing enzyme (polymerase) is used to make multiple copies of a targeted segment of DNA. By repeating the polymerase copying process, many copies of the targeted segment are produced. For example, thirty (30) repetitions can produce over one million (1,000,000) copies from a single molecule.

Thermo-cycling typically begins with heating the sample to about 95.degree. C. to separate the double strands and make them accessible as templates for polymerase replication. Cooling to about 55.degree. C. allows the polymerase initiators (primers) to hybridize with their target DNA segments. Control of the temperature during the hybridization process is critical for accurate hybridization of the primer to the DNA. Heating from about 55.degree. C. to about 72.degree. C. is necessary for efficient performance of the polymerase enzyme. At the appropriate temperature, the polymerase reaction catalyzes the elongation of new DNA complementary in nucleotide sequence to the target DNA. At the end of the elongation reaction, heating the solution to about 95.degree. C. causes the newly-formed double-stranded DNA to separate into single strands, thus providing templates for another round of PCR amplification.

Current thermo-cycling methods are complex, time consuming and expensive. One thermo-cycling device (known as the MJ Research DNA engine) comprises a surface upon which are placed micro-wells and under which rests a thermoelectric block for heating and cooling biological material placed in the wells. However, this device takes about one and one-half (1.5) minutes to perform each cycle, even when using a simplified two temperature format. The device thus requires approximately forty-five (45) minutes to perform a thirty cycle run.

Various devices using capillary tubes can perform a thirty cycle run in from ten to thirty minutes. These devices require loading and unloading samples to and from the tubes, sealing the tubes and then exposing the tubes to forced air heating. When the loading and unloading steps are included, these procedures may consume as much as two hours of laboratory time. These procedures also require relatively skilled technicians who can handle microliter volumes of reagents.

One conventional type of thermo-cycler uses forced water circulation to heat and cool vessels immersed in a water bath. Three or more reservoirs hold water at different temperatures and rapid pumps and valves bring water from the reservoirs into the bath to produce a huge thermal mass which heats or cools the material in the vessels. Another device used in PCR processes (See European Patent No. 381501) utilizes a flexible bag-like structure with an inner system of chambers. The DNA sample and reagent fluids are loaded into the chambers and the bag is placed on a hot plate for thermo-cycling. After thermo-cycling, the bag is squeezed with external rollers to move the fluid into chambers containing detection reagents.

In the prior art methods, a significant amount of thermo-cycle time is consumed by ramp periods wherein the temperature of the biological material is changed from one target temperature to the next. The length of each ramp period is a function of both the thermo-cycling equipment and the volume of material heated. The prior art methods generally require first heating the test chamber which then transfers heat to the material contained therein. The thermal mass of the test chamber and volume of material in the test chamber produces high thermal inertia and poor heat-loss surface area to volume ratios. Furthermore, the reagents used in the procedure are expensive and their volume (even if only in the fifty microliter range) can make the procedure prohibitively expensive.

Prior methods pose further time limitations by typically generating only one thermo-cycle at a time. Thus, processing multiple samples at the optimum thermo-cycle of each requires processing the samples one after the other (serial processing). Serial processing may be accelerated by using multiple thermo-cyclers, but this approach consumes capital, energy and laboratory space.

Multiple DNA samples can be processed simultaneously using parallel processing. The most common parallel processing technique involves grouping several DNA samples together and subjecting them to a common thermo-cycle. However, the common cycle is necessarily a compromise among the optimum cycles and time savings are thus achieved at the expense of quality of results.

In accordance with the present invention, thermo-cycling apparatus is provided which has a channel extending through a plurality of temperature control units. The channel has a fluid inlet at one end and a fluid outlet at the opposite end. A fluid sample of biological material introduced into the channel through the inlet passes each of temperature control units while flowing to the outlet. Each temperature control unit maintains a portion of the channel at a desired temperature. Thus the fluid is thermo-cycled through the desired temperature sequence while flowing through the channel.

The channel is preferably designed to handle small fluid volumes. For example, a capillary tube with its inlet equipped with a conventional fitting to facilitate automated loading of unprocessed fluids is suitable. The outlet may be equipped with a conventional fitting for connecting to a storage or analysis channel or tube for collecting the processed fluid. The temperature control units are preferably thermoelectric modules arranged in a linear array and positioned in thermal contact with the fluid channel. Because of the size of the tube used, the quantity of expensive reagents needed can be minimized. The ramp periods between each thermo-cycle target temperature are substantially reduced because of the small fluid volume. Direct discharge into the analysis chamber reduces the number and complexity of steps which must be performed by technicians. Since the apparatus may include multiple, independently controllable, inline thermo-cycling devices, multiple material samples can be processed simultaneously, all at their optimum thermo-cycles. Various other features and advantages of the invention will become more readily understood from the following detailed description taken in connection with the appended claims and attached drawing in which:

FIG. 1 is a perspective view of apparatus employing the preferred embodiment of the invention;

FIG. 2 is an enlarged perspective view of one temperature control unit as shown FIG. 1;

FIG. 3 is a sectional view of a thermoelectric module taken along line 3--3 of FIG. 2;

FIG. 4 is a sectional view of a thermoelectric module taken along line 4--4 of FIG. 2; and

FIG. 5 is a sectional view of a large inline apparatus employing the invention.

The invention is disclosed herein by showing various examples of how the invention can be made and used. Like reference characters are used throughout the several views of the drawing to indicate like or corresponding parts.

In FIG. 1 the reference character 10 generally refers to apparatus comprising three inline thermo-cycling devices 12, 14 and 16, all of which are secured together in a base 18. The base is preferably large enough to hold from one (1) to two hundred and fifty six (256) devices but may be large enough to hold a greater number of devices if desired.

Inline device 12 is generally illustrative and comprises an elongated fluid channel or capillary tube 20 extending through three temperature control units 22, 24 and 26. The number of control units used will depend, of course, on the desired thermo-cycle sequence. The device 12 also has an inlet fitting 28, outlet fitting 29, and a storage or analysis channel or tube 30. Alternatively, the fluid channel may be positioned adjacent or only partially enclosed by the temperature control units.

Control unit 22 is generally representative of control units 24 and 26 and is shown in greater detail in FIGS. 2 and 4. Control unit 22 (as illustrated) comprises seven (7) thermoelectric modules 32a-32g. Each module has a thickness 40 and a diameter 42. The thickness and diameter will typically be the same for each module but may vary if desired. The modules are positioned adjacent each other in a linear fashion to form the control unit which has a length 44. Any desired number of modules can be used to form control units of desired lengths.

The construction of thermoelectric module 37f is illustrative of the other modules and is shown in greater detail in FIGS. 3 and 4. Module 32f preferably comprises N-type and P-type semiconductor materials but may be suitable dissimilar metallic materials. The thermoelectric materials are formed into blocks which are circularly arranged around a tube 20 constructed from electrically insulating material such as glass. The tube 20 has an exterior surface 50 and an interior surface 52 defining a tube wall thickness 54 therebetween. The interior surface defines an internal fluid passage 56 having a diameter 58 (FIG. 3). While the cross-sectional configuration shown is circular, the fluid passage may have other configurations if desired. For example, a channel having a thin rectangular cross-section (not shown) may be used to increase the heat transfer surface area.

Module 32f comprises at least one circular row 59 (FIG. 3) of N-type blocks 60-66 and P-type blocks 70-75 sandwiched between upper and lower plates 80 and 82 (FIG. 4). Block 61 is illustrative of the other N-type blocks and has an internal surface 61a, external surface 61b, side surfaces 61c and 61d, and upper and lower surfaces 61e and 61f. Block 74 is illustrative of the other P-type blocks and has internal surface 74a, external surface 74b, side surfaces 74c and 74d, and upper and lower surfaces 74e and 74f.

Plates 80 and 82 are preferably electrically insulating, thermally conductive material such as polyamide or the like. As shown in FIG. 3, plate 82 has an outer perimeter 82a and upper and lower surfaces 82b and 82c. The plate also has a centrally located hole 84 with a radius 84a as measured from axis A1 to accommodate tube 20.

The N-type and P-type blocks are arranged in sequentially alternating order on upper surface 82b of plate 82 with their internal surfaces facing the capillary tube 20. The number of blocks shown is for illustrative purposes only. Module 32f may have a greater or fewer number of blocks if desired. Each block is electrically connected to the adjacent blocks by conductive traces. The traces are preferably metallic material such as copper and may be formed by any suitable process. As shown in FIG. 3 external traces 86a-86f interconnect the external surfaces of blocks 60 and 70, 61 and 71, 62 and 72, 63 and 73, 64 and 74, 65 and 75, respectively. Internal traces 88a-88f interconnect the internal surfaces of blocks 70 and 61, 71 and 62, 72 and 63, 73 and 64, 74 and 65, 75 and 76, respectively. The traces may be positioned in alternative arrangements provided the resulting current flow through the blocks results in heat being pumped either toward or away from the capillary tube 20.

The internal traces are positioned adjacent the exterior surface 50 of tube 20 and provide a thermally conductive path between the tube and the row of blocks 59. The external traces are surrounded by a spacer ring 90 which has internal and external surfaces 90a and 90b and upper and lower surfaces 90c and 90d. Internal surface 90a is positioned adjacent the external traces and the thickness 92 of the ring extends radially to position exterior surface 90b flush with outer perimeter 82a of lower plate 82. The spacer ring 90 includes a gap 94 through which terminals 96 and 98 extend. An external power source (not shown) supplies current to the row of blocks through the terminals.

The blocks are connected in series so that current will flow in either direction through the row of blocks depending on the polarity of the current at terminals 96 and 98. Since the traces alternate between the external and internal surfaces on the blocks, the direction of the current flow in each block will be either toward or away from the tube 20 depending upon the direction of the current flow. The module will thus pump heat either toward or away from the tube 20, depending on polarity.

Modules 32a-32e and 32g are constructed similarly to module 32f and are arranged with their centrally-located holes in alignment along axis A1. The modules are preferably secured together to form temperature control unit 22. The aligned holes define a cylindrical opening 106 which accommodates the length portion of the tube 20 equal to the length 44 of the control unit.

As shown in FIG. 4 inlet fitting 28 is secured to module first end 102. The fitting includes a first major face 112 adapted for use with automated loading apparatus and a second major face 114. The first major face 112 has a centrally located counter-bore 116 having a diameter 118 and a depth 120. The bottom surface 122 of the counter-bore has a centrally located orifice 124 with a diameter 126 the same size as the internal passage 56 of tube 20.

FIG. 5 shows the temperature control units 22, 24 and 26 aligned along longitudinal axis A1 and tube 20 extending through their centers. Control unit 22 has seven (7) modules 32a-32g as previously described and a length 44. Unit 24 has eight (8) 32h-32o modules and a length 128, and unit 26 has five (5) modules 32p-32t and a length 130.

Each pair of terminals extending from the modules is interconnected so that the modules within a control unit are commonly controlled. The connected pairs of terminals 96a-96g and 98a-98g, 96h-96o and 98l-98o, 96p-96t and 98p-98t are represented schematically in FIG. 5. Since the terminal pairs are connected together, control units 22, 24, 26 are operable via connections 134a-134b, 138-138b and 140a-140b, respectively, to power distributor 142. The power distributor 142 may be any combination of conventional power supplies, polarity switches, programmable controllers, etc.

Conventional loading apparatus 144, having a fitting 146 designed to mate with inlet fitting 28, is used to load unprocessed fluid into the tube 20. The tube 20 is preferably sized to provide a capillary effect for drawing fluid from the inlet fitting 28 to the outlet fitting 29. However, larger tubes or channels may be used and/or the device may rely on pumps or gravity to move the fluid through the tube.

The power distributor 142 maintains control units 22, 24 and 26 at different temperatures. For example, if the fluid is a DNA sample to be amplified using PCR, control unit 22 can be maintained at 95.degree. C., unit 24 at 55.degree. C. and unit 26 at 72.degree. C. Since the control units are in thermal contact with the fluid passage 56, the control units thus maintain a portion of tube 20 extending through their centers at the same temperatures.

Unprocessed fluid is introduced into tube 20 through inlet fitting 28 and drawn toward the outlet fitting 29. As the fluid flows through the length 44 of control unit 22 it is heated to 95.degree. C. As the fluid moves into the length 128 of control unit 24 it is cooled to 55.degree. C. Finally, as the fluid moves into the length 130 of control unit 26 it is heated to 72.degree. C. When the fluid passes through the outlet fitting 29 it has been thermo-cycled through one PCR replication. Additional temperature control units may be provided to repeat the three temperature cycle or to heat and cool the liquid to more than three temperatures.

The length of time a fluid remains at each temperature is a function of both the fluid flow rate and the length of the control unit. The flow rate depends on such factors as the size of the tube, fluid viscosity, temperature and pressure. Apparatus may designed in which these factors cooperate to yield any desired flow rate. The control units must also be designed with the lengths necessary to maintain the fluid at the desired temperature for the desired time period.

Heat transfer between adjacent temperature regions may result in transition regions where the tube is maintained at temperatures other than those necessary to perform the desired reaction. This effect may be controlled by increasing the lengths of the control units by amounts related to the lengths of the transition regions. The transition regions may also be minimized by spacing the control units apart from each other (not shown) or by interposing a thermal insulator (not shown) between adjacent control units.

The processed fluid is discharged from the outlet fitting 29 and flows into outflow channel or capillary tube 12. The outflow capillary may store or transport the processed fluid and may also be used for electrophoretic, fluorimetric or absorptiometric analysis.

From the foregoing it will be recognized that the principles of the invention may be employed in various arrangements to obtain the benefits of the advantages and features disclosed. It is to be understood, therefore, that although numerous characteristics and advantages of the invention have been set forth, together with details of the structure and function of various embodiments of the invention, this disclosure is illustrative only. Various changes and modifications may be made in detail, especially in matters of shape, size and arrangement of parts, without departing from the spirit and scope of the invention as defined by the appended claims.