Patent Application
20100059380
The present invention relates to apparatus and methods for conducting electrophoretic separation concurrently in a plurality of gels. More specifically, the present invention relates to apparatus and methods for performing multiple concurrent electrophoresis experiments with increased reproducibility among the gels through incorporation in the apparatus of improved passive thermal management features and improved electric field geometries.
Gel electrophoresis is a common procedure for the separation of biological molecules, such as DNA, RNA, and proteins. In gel electrophoresis, the molecules are separated into bands according to the rate at which an imposed electric field causes them to migrate through a filtering gel.
The basic apparatus used in this technique consists of a gel enclosed in a glass tube or sandwiched as a slab between glass or plastic plates. The gel has an open molecular network structure, defining pores which are saturated with an electrically conductive buffered solution of salt. These pores through the gel are large enough to admit passage of the migrating molecules.
The gel is placed in contact with buffer solutions that make electrical contact between the gel and the cathode and anode of an electrical power supply. A sample containing the macromolecules and a tracking dye is placed on top of the gel. An electric potential is applied to the gel causing the sample macromolecules and tracking dye to migrate toward the bottom of the gel. The locations of the bands of separated macromolecules then are determined. By comparing the distance moved by particular bands in comparison to the tracking dye and macromolecules of known mobility, the mobility of sample macromolecules can be determined. Once the mobility of the sample macromolecules is determined, the size of the macromolecule can be calculated.
As electrophoresis is used with increasing frequency in basic research, quality control, and in forensic and clinical diagnoses, it is increasingly important to be able to replicate all experimental conditions in multiple locations and labs.
Among these experimental conditions, temperature is extremely important.
The application of an electrical field to a gel results in the generation of heat. In general, higher temperatures increase the molecular kinetics, which results in faster migration of macromolecules through the separating gel. Further, a temperature increase affects the electrical conductivity of an electrolyte solution and may cause dissociation.
Without temperature control or uniform electric field geometry, gels often exhibit uneven temperatures across the width of the gel resulting in “smile” or “frown” distortions. Smile distortions occur when bands migrate faster on the sides than in the middle of the gel; frown distortions occur when bands migrate faster in the middle than on the sides.
Often, even a small temperature differential between the front and rear plates of the gel, if not mitigated, can cause the resulting bands to slant front to back, depending on the thickness of the gel and the heat transfer properties of the cassette plates. This challenge is particularly acute in test runs where the molecular migration rates exhibit overly temperature sensitive characteristics, as in DNA sequencing. For such runs, even a slight temperature differential, e.g. of 0.1° C., can cause the slanted bands to appear overlapping.
Additionally, overheating of the gel (e.g., greater than 70° C.) can result in deleterious effects such as breakdown of the gel matrix resulting in poor resolution and band shape, alteration of the macromolecules including denaturation, alkylation or oxidation, and/or damage to the electrophoresis apparatus itself.
In DNA sequencing, electrophoresis is conducted at high voltage (1200-3000 volts, 55 watts) to maintain a gel temperature of 45°-50° C. for maximum resolution of the denatured DNA strands. The temperature is controlled by the amount of power applied to the gel. Gels that run too cool (e.g., <40° C.) will have bands that are blurred, perhaps due to incomplete denaturation. Gels that run too warm (e.g., >60° C.) will lose resolution, perhaps due to the breakdown of the polyacrylamide.
Precise temperature control is particularly critical in Single Stranded Conformational polymorphism (SSCP) analysis of DNA, where bands are extremely close together. The relative temperature differential between the front and the back surfaces of the gel therefore can have a critical effect on the resolution of the DNA bands.
Various means have been used to attempt to control the temperature of the gel during electrophoresis. These include applying active or passive heat sinks to one side of the gel, regulating power to the gel, employing an enclosed heat exchanger internal to one of the buffer chambers, immersing the gels in a buffer-filled tank containing a heater/circulator, circulating the buffer through tubing immersed in an ice water bath, circulating the buffer through an external metal heat exchanger, and use of piezo thermo-electric heater/cooler controls.
These means are limited in their ability to provide a compact apparatus for maintaining consistent and uniform thermal control across the area encompassing the front and back of the electrophoresis gels. The heat sinks exchange heat on only one side of the gel; the regulation of power to the gels cannot control regional hot spots and obviously limits the application of high wattage to the gels; the internal heat exchanger again exchanges heat on only one side of the gel and does not actively circulate buffer, resulting in vertical thermal gradients within the buffer chamber; immersing the gels in a heater tank is cumbersome, in that it requires a large volume of buffer and cannot cool the gels; and circulating the buffer through tubing immersed in an ice water bath is also cumbersome, and makes difficult fine control of temperature.
Circulating the buffer through an external metal heat exchanger provides the most satisfactory temperature control. However, with the current electrophoresis systems, two pumps and heat exchangers would be required to assure uniformity of temperature and separation of the buffer fluids between the cathode and anode chambers. Further, with current electrophoresis systems, circulation of buffer within the chambers and across the gels is random and undirected, which may result in vertical and horizontal thermal gradients.
Moreover, for electrophoretic separation, the first and second buffer solutions must be isolated from one another. To provide isolation, prior art electrophoresis systems use various methods, among which is use of a buffer core to which the gel cassettes are secured during electrophoresis. Previously known electrophoresis systems using a buffer core commonly use a buffer core subassembly containing clamps or latches that secure the gel cassettes to the buffer core. Once the cassettes are secured, the buffer core subassembly must then be loaded in the container prior to electrophoretic separation. For example, in prior art systems that use a clamping mechanism, a user generally must first construct a clamping subassembly that is then loaded into the container prior to performing electrophoresis. It would be desirable to provide a clamping device that is easier to use and does not require additional or moving parts. For example, there would be no need to configure, assemble, or adjust a clamp or other adjustable part.
Various prior art patents have proposed apparatus and methods for simultaneously running multiple gels, but many potential problems exist, including ineffective temperature control on both sides of the gel cassettes, ineffective or inconvenient clamping of gel cassettes, and inability to apply a uniform electrical field to all of the gels.
For example, U.S. Pat. No. 6,451,193 to Fernwood et al. (Fernwood) describes a single cell configured to receive multiple slab gels for conducting simultaneous electrophoresis experiments. The multitude of slab gels are supported vertically and parallel to one another while immersed in a buffer solution. A voltage is applied to all gels simultaneously while temperature control is achieved by circulating the buffer solution upward through the cell and cooling the circulating buffer solution with a tube heat exchanger positioned on the floor of the cell.
There are several drawbacks associated with the electrophoresis system described in Fernwood, and in particular, the relative complexity of the buffer circulation and cooling mechanisms that are employed. For example, with respect to the cooling mechanism, circulation is effected by a coolant pump and chilling of the coolant prior to its return to the tank requires an external chilling or refrigeration unit. With respect to the buffer circulation mechanism, an external pump and an external circulation line are required. All of these external components make the device more cumbersome, and proper circulation of the buffer and coolant depend on proper and consistent operation of several external components.
Another drawback associated with the device described in Fernwood is that the coolant is only circulated in tubing at the bottom of the tank, which may result in inconsistent cooling of a vertically upright gel cassette. Moreover, the coolant traverses the floor of the tank four times before further chilling of the coolant occurs. Therefore, coolant properties may vary at different locations that the coolant traverses the floor of the tank.
In view of these drawbacks of previously known systems, it would be desirable to provide apparatus and methods for conducting multiple electrophoresis experiments that employ a passive cooling mechanism to avoid the need for complex, ineffective or cumbersome active cooling mechanisms.
It also would be desirable to provide apparatus and methods for conducting multiple electrophoresis experiments that uses a simple clamping mechanism, without moving parts, to secure the gel cassettes in place and provide an effective seal between anode and cathode buffer solutions.
It further would be desirable to provide apparatus and methods for conducting multiple electrophoresis experiments that employ one lower buffer chamber that is common to all gel cassettes in the container.
It still further would be desirable to provide apparatus and methods for conducting multiple electrophoresis experiments that consistently control the temperature of the electrophoresis gels, regardless of the number of gels being run at any given time, particularly while maintaining a uniform electric field across the width of the gel
In view of the foregoing, the present invention provides an apparatus and methods for conducting multiple electrophoresis experiments that consistently control the temperature of the electrophoresis gels, regardless of the number of gels being run at any given time. This temperature control is achieved for electrophoretic separation concurrently in a plurality of gels, by using passive thermal management to avoid the need for complex, ineffective or cumbersome active cooling mechanisms.
Furthermore, the apparatus and methods for conducting electrophoretic separation of the present invention provide homogeneous electric fields across the width of a gel. The temperature and electric field control of the present invention results in dye fronts that are within 10 mm from each other, within 5 mm from each other, or within 25%, 15%, 10%, or 5% of the length of a run.
In yet another embodiment, the present invention provides an apparatus and methods for conducting electrophoretic separation concurrently in a plurality of gels using a simple clamping mechanism, without moving parts, to secure the gel cassettes in place and provide an effective seal between anode and cathode buffer solutions.
In yet another embodiment, provided herein is an apparatus and methods for conducting multiple electrophoresis experiments that employ one lower buffer chamber that is common to all gel cassettes in the container.
Accordingly, provided herein in a first embodiment, is an apparatus or a system for removably positioning one or more gel cassettes for electrophoresis, each gel cassette having a first face, a second face, and a gel disposed therebetween. The apparatus comprises a fluid-retaining container and means for apportioning the interior of the container into a plurality of volumes upon the positioning of one or more gel cassettes within the container. Each of the volumes is proportionate to the number of positioned cassette faces with which it is in fluid contact. Accordingly, the upper buffer volumes within buffer cores of the container are within 75% of each other, and lower buffer volumes per gel are within 75% of each other when the chamber has different numbers of gels, for example from 3 to 50 gels.
In one series of embodiments, the apportioning means include means that are integral to the container and at least one means that is removably engageable within the container.
In some embodiments, the apparatus further comprises means for concurrently establishing an electric field within the gel of each positioned cassette, wherein the field is substantially uniform among all of the positioned gels and substantially homogeneous across the width of each gel. Substantially uniform means that the field is within 10% among all positioned gels. In certain of these embodiments, the field establishing means include means integral to the container and at least one means removably engageable within the container. The combination of field uniformity and temperature regulation of apparatuses and methods of the present invention results in dye fronts that are within 15 mm from each other, within 10 mm from each other, within 5 mm from each other, or a traveled distance difference that is no more than 25%, 15%, 10%, 5%, 4%, 3%, or 2% of the length of a run at the end of an electrophoretic separation run. Therefore, for a 10% distance difference for a 65 mm gel length electrophoretic run, the dye fronts between different gels in the container at the end of the run are within 7 mm of each other.
In certain embodiments, each of the apportioning means and the field-establishing means includes both means that are integral to the container and means that are removably engageable therein. In particularly useful embodiments, each one of the removably engageable field establishing means is integrated into one of the at least one removably engageable apportioning means to form a buffer core body.
Typically, the apparatus is configured so that the plurality of apportioned volumes includes at least one first volume and a single second volume; the positioned cassettes render each of the at least one first volumes fluidly noncommunicating with the single second volume. In embodiments that include at least one buffer core, each of the at least one first volumes is internal to a buffer core.
In various embodiments, the integral apportioning means include, for each buffer core potentially engageable within the container, a set of opposing first and second bulkheads.
The opposing bulkheads of each set are typically configured to provide an inward pressure upon gel cassettes assembled to the buffer core body engaged therebetween.
For example, in certain embodiments the bulkheads of each opposing set each comprises at least one upper protrusion, the protrusions configured to apply an inward pressure upon gel cassettes assembled to the buffer core engaged therebetween. In some embodiments, the bulkheads of each opposing set each further comprises at least one lower protrusion, the lower protrusions configured to apply an inward pressure upon gel cassettes assembled to the buffer core engaged therebetween.
In embodiments particularly useful in establishing a uniform field across each of the gels within positioned cassettes, at least one of the opposing bulkheads of each set includes a plurality of lower wedge-shaped protrusions, the plurality of wedge-shaped protrusions collectively making discontinuous contact to the cassette assembled to the buffer core engaged therebetween.
In typical embodiments, each of the bulkheads includes an aperture disposed through the bulkhead between its upper and lower protrusions.
In some embodiments, the thickness of each of the end walls of the container is greater than that of each of the side walls of the container.
In another aspect, the invention provides a container having a removable lid and a plurality of communicating chambers. Each of the plurality of chambers is configured to receive and engage a buffer core assembly. Each buffer core assembly preferably comprises a buffer core body and first and second cassettes securely coupled to front and back sides of the buffer core body. A space between the buffer core body and the first and second cassettes forms an upper buffer chamber, which is configured to receive a first buffer.
Each chamber in the container preferably is formed using first and second opposing bulkheads. The first and second bulkheads each have a laterally protruding upper region, recessed central region, and an aperture disposed through the recessed central region. Further, at least one wedge-shaped member is disposed beneath the aperture in the first bulkhead, and at least one wedge-shaped member is disposed beneath the aperture in the second bulkhead.
In application, each buffer core assembly is configured to be inserted between the first and second bulkheads of a desired chamber. As the buffer core assembly is inserted, the first and second gel cassettes contact the wedge-shaped members of the first and second bulkheads, respectively. This causes the first and second cassettes to be pressed inward towards the buffer core body. The pressure applied by the wedge-shaped members, along with the pressure applied by the laterally protruding upper regions of the bulkheads, provides an effective seal for the upper buffer chamber. Advantageously, since the wedge-shaped members are an integral component of the container, no moving clamping mechanisms are required to secure the gel cassettes in place and provide an effective seal between anode and cathode buffers.
In accordance with one aspect of the present invention, a common lower buffer chamber is formed when a plurality of buffer core assemblies are placed in adjacent chambers of the container. Specifically, the common lower buffer chamber is formed as a space between a second cassette of a first buffer core assembly and a first cassette of a second buffer core assembly, a second cassette of a second buffer assembly and a first cassette of a third buffer assembly, and so forth. Therefore, when a second buffer is poured into the common lower buffer chamber, the second buffer may be placed in fluid communication with each of the gel cassettes, regardless of the number of cassettes employed.
In a preferred method, each buffer core assembly to be used is inserted into a respective chamber of the container, then secured using the clamping force applied by the wedge-shaped members of the bulkheads, as described above. A predetermined volume of a first buffer then is poured into each upper buffer chamber, one at a time. In a next step, a corresponding predetermined volume of a second buffer is poured into the common lower buffer chamber at one location, then flows through various open spaces in the container to contact the outer surfaces of the gel cassettes in the container. In effect, the inner surfaces of each gel cassette are in contact with the first buffer in the upper buffer chamber, while the outer surfaces are in contact with the second buffer filling in the common lower buffer chamber.
In a next step, the removable lid is placed on top of the container. The removable lid is coupled to first and second cables, which are adapted to be coupled to a power supply or charging means. The removable lid also is electrically coupled to negative and positive wires that are in electrical contact with each of the first and second buffers, respectively.
When an electrical potential is applied across each of the negative and positive wires, an electric field on each of the gels in the container is developed. The electrical fields in the gels effect molecular separation of the electrophoresis samples in the gels The electrical fields in the gels effect molecular separation of the electrophoresis samples in the gels since the gels act as the only conductive path between the buffer solutions which are charged at opposite polarities.
In accordance with one aspect of the present invention, passive thermal management techniques are used to control the temperatures of the gels in the cassettes. The passive thermal management techniques rely on the heat sinking capabilities of the first and second buffers to maintain a relatively equal temperature on the outer and inner plates of the cassette. According to passive thermal management provided herein, the temperature between upper buffers in separate buffer cores within a container at the end of an electrophoretic separation is within 25, 20, 15, 10, 5, 4, 3, 2, or 1° C. Furthermore, the temperature difference between an upper buffer and a lower buffer is within 25, 20, 15, 10, 5, 4, 3, 2, or 1° C. at the end of an electrophoretic separation performed using the apparatus or methods. Furthermore, according to passive thermal management provided herein, the temperature between gels at the end of an electrophoretic separation is within 25, 20, 15, 10, 5, 4, 3, 2, or 1° C. In certain illustrative examples, the temperature between upper buffer cores, between upper and lower buffers, and between gels is within 10° C. at the end of an electrophoretic separation.
The heat sink principles that are used in conjunction with the present invention take into account several variables, including the specific heat of the buffers, the mass of the buffers added, the change in temperature, the current and voltage applied to the gels, and other variables. By knowing the voltage and current applied, knowing the time duration required to complete separation, knowing the specific heat of the buffer, and by calculating the mass of buffer to be added, the temperature increase of the gels can be kept below a predetermined threshold (for example, 60° C.). Furthermore, the apparatus and methods of the present invention ensure that the same temperature is maintained on the outer and inner surfaces of each gel cassette to avoid slanting of the migrating bands in a sample. The present invention also ensures that each gel in the apparatus is exposed to the same thermal environment as each of the other gels.
If desired, a dam system may be used in conjunction with the apparatus of the present invention to run fewer than the maximum number of gels that the container can run. The dam interrupts flow to certain areas of the common lower buffer chamber, based on its placement in the container. For example, if the container has the capacity to run six gels simultaneously, but a user only wishes to run two gels, the dam is positioned such that flow in the lower buffer chamber is interrupted to the other four regions of the container.
The dam system, which preferably is adapted to be coupled to the buffer core assembly in lieu of one of the cassettes, is configured to displace half the volume of an upper buffer chamber. Therefore, when an odd number of gels are being run, only one-half of buffer is poured into the upper buffer chamber, relative to when two cassettes are used in a buffer core assembly. Accordingly, a proportional amount of buffer is used, regardless of whether an even or odd number of gels are being run, thereby ensuring that the temperatures on the outer and inner surfaces of the cassettes will remain the same during electrophoresis.
Further features of the invention, its nature and various advantages will be more apparent from the accompanying drawings and the following detailed description of the preferred embodiments, in which:
Referring now to
As shown in
Container 20 preferably comprises first and second side walls 21 and 22, closed bottom 23, and first and second end walls 24 and 26, as shown in
Container 20 further comprises negative bus bar 44 and positive bus bar 45. Negative and positive bus bars 44 and 45 preferably are disposed atop first and second side walls 21 and 22, respectively, as shown in
Negative bus bar 44 is electrically coupled to pole conductor 48, and further coupled to plurality of sockets 46a-46c, which correspond to chambers 30a-30c of container 20. Positive bus bar 45 is electrically coupled to pole conductor 49, and further coupled to plurality of sockets 47a-47c, which correspond to chambers 30a-30c, respectively, as depicted in
In a particularly useful embodiment of the present invention, black and red polarity tabs 37 and 38 are affixed to container 20 on opposing lateral sides of the container, as depicted in
Referring now to
Container 20 has a plurality of chambers 30a-30c, which are adapted to receive buffer core assemblies 60a-60c, respectively. In a preferred embodiment, each chamber 30 is formed by first and second opposing bulkheads 110a and 110b. Each bulkhead 110 preferably comprises a laterally protruding (i.e., protruding along the X axis, see
Each bulkhead 110 further preferably comprises at least one wedge-shaped member 115 disposed beneath apertures 113. The wedge-shaped member preferably is manufactured using a suitable substantially noncompliant compound, such as plastic.
First and second bulkheads 110a and 110b have substantially identical configurations, with the main exception that laterally protruding upper region 112a of first bulkhead 110a is situated slightly higher with respect to the side walls of container 20 than laterally protruding upper region 112b of second bulkhead 110b. The slight height differential facilitates insertion of buffer core assemblies 60a-60c, because the buffer assemblies may be initially inserted at a slight vertical angle. The slight vertical angle allows the buffer core assemblies to slide into their respective chambers with little or no frictional interference, until each buffer core assembly contacts the wedge-shaped members at the bottom of the chamber. When each buffer core assembly contacts the wedge-shaped members, the wedge-shaped members force a vertical positioning of the buffer core assembly, as described in greater detail hereinbelow with respect to
Referring now to
Buffer core body 61 comprises upraised side walls 62 and 63, and lower base 64 disposed between the side walls, as shown in
First male conductor 102 is coupled to wire 104. A portion of wire 104 runs in groove 75, which is formed in a lateral surface of side wall 62, as shown in
Second male conductor 103 is coupled to wire 105. Wire 105 runs through first aperture 69a of horizontal beam 67, and continues to extend through second aperture 69b at the other end of beam 67, as depicted in
Buffer core assembly 60 further comprises first and second recesses 73a and 73b, which are disposed in side walls 62 and 63, respectively. Recesses 73a and 73b are disposed on front side 71 of buffer core body 61, as shown in
In application, first gel cassette 80a is placed in the recesses that are disposed on back side 72 of buffer core body 61, as depicted in
Upper buffer chamber 130 is formed between first gel cassette 80a, second gel cassette 80b, and side walls 62 and 63 of buffer core body 61, as depicted in
Referring back to
Referring now to
Divider 82 is affixed to peripheral ridge 86 along the lateral sides and the bottom periphery of retainer 84 to define an internal gel compartment 88 for holding an electrophoresis gel 90. As shown in
Located along the lower portion of divider plate 82 and traversing the width of cassette 80 is a slot or opening 96 that opens gel compartment 88 to the exterior of cassette 80 and hence allows a direct electrical coupling with the charged buffer solution.
Gel cassettes suitable for the present invention are known in the art. In a typical gel cassette, the gel is pre-filled within the internal gel compartment for ease of handling. Top opening 92 is closed with a comb (not shown), and slot 96 is masked closed with a removable tape (not shown). An example of the gel cassettes that are suitable for this application are the 12% Tris-glycine gels sold by INVITROGEN CORPORATION of Carlsbad, Calif., under catalog No. EC6005. Gel cassettes of similar types also are commercially available from other firms.
Prior to use of cassette 80, the comb (not shown) and the tape (not shown) disposed over top opening 92 and slot 96, respectively, are removed. The sample to be analyzed is introduced into gel compartment 88 through comb opening 92 by an appropriate means, such as a pipette. The cassettes with their retainer plates 84 proximal to buffer core body 61 are held to rest within side recesses 73 and base supports 79, as described hereinabove with respect to
Referring now to
As each buffer core assembly further is inserted into its respective chamber 30, each gel cassette 80 is urged in an inward direction, i.e., towards buffer core body 61, by a force applied by wedge-shaped members 115, as shown in
The forces applied by wedge-shaped members 115 against gel cassettes 80a and 80b ensure fluidic and electrical isolation between a second buffer present in common lower buffer chamber 140 and a first buffer present in each of the individual upper buffer chambers 130a-130c. Fluidic and electrical isolation of first and second buffers reduces the risk of electrical grounding of the power supply or other sensitive instruments used in connection with the electrophoresis.
At about the same time that each buffer core assembly is securely wedged into its chamber, male conductors 102 of buffer core assemblies 60a-60c engage respective sockets 47a-47c (see
It should be noted that both male conductors 102 and 103 are disposed on front portion 119 of buffer core body 61, as depicted in
Referring to
In accordance with one aspect of the present invention, lower buffer chamber 140 allows a second buffer (not shown) to be placed in contact with each buffer core assembly 60a-60c. When the second buffer is poured into any region of lower buffer chamber 140, the second buffer will be distributed in a substantially equal fashion to the other regions of lower buffer chamber 140. Specifically, the second buffer will flow through apertures 113 in bulkheads 110a and 110b (see
Referring still to
A corresponding, predetermined volume of a second buffer (not shown) then is introduced into lower buffer chamber 140 of container 20. Pouring the predetermined volume of the second buffer into any region of lower buffer chamber 140 will cause the second buffer to be distributed substantially equally throughout chamber 140. It should be noted that, in alternative embodiments, the second buffer may be added before the first buffer is added.
Container 20 is configured such that the volumes between assemblies 60a and 60b, and between 60b and 60c are approximately twice as great as the volumes between cassette 80a of assembly 60a and end wall 26, and between cassette 80b of assembly 60c and end wall 24. Therefore, when the second buffer poured into lower buffer chamber 140 settles to a height h, approximately twice as much second buffer will settle between the adjacent buffer core assemblies as will settle between assembly 60a and end wall 26, and assembly 60c and end wall 24.
For example, if 600 mL of the second buffer is poured into lower buffer chamber 140, then after the buffer settles in container 20, approximately 100 mL of the second buffer will settle between first cassette 80a of assembly 60a and end wall 26, approximately 200 mL of the second buffer will settle between assemblies 60a and 60b, approximately 200 mL will settle between assemblies 60b and 60c, and approximately 100 mL will settle between second cassette 80b of assembly 60c and end wall 24. Therefore, each outer surface of each cassette 80 will have approximately 100 mL of second buffer devoted as a heat sink disposed adjacent the outer surface.
In a preferred embodiment of this aspect of the present invention, components of container 20 are dimensioned so that equal volumes of second and first buffers are devoted as heat sinks for the outer and inner surfaces 81a and 81b of each gel cassette 80a. Therefore, as an example, if 600 mL of second buffer is poured into common lower buffer chamber 140, as described above, then 200 mL of first buffer should be poured into each upper buffer chamber 60a-60c. Since there are six gel cassettes in container 20, and two cassettes per upper buffer chamber, then the inner surfaces of each of the six cassettes will have approximately 100 mL of first buffer devoted as a heat sink to the inner surfaces of the cassettes.
As will be described in greater detail hereinbelow, the actual volumes of first and second buffers may be selected to ensure adequate heat sinking during electrophoresis to keep the temperature of gel 90 below a predetermined threshold.
Referring now to
Asymmetric mating of removable lid 50 with container 20 preferably is employed to ensure a proper electrical connection. Specifically, in one embodiment, lid 50 will only fit onto container 20 when slot 53 can fit over short tab 25, and slot 54 can fit over long tab 27, as illustrated in
After lid 50 is seated, conductor cables 57 and 59 are coupled to a power supply system or charging means for delivering an appropriate electrical potential to the electrophoresis system. In one embodiment of the present invention, cable 57 is coupled to the power supply to deliver a negative potential, and cable 59 to deliver a positive potential. In practice, the polarity of the electrical potential can be reversibly applied to the buffers, as a matter of choice.
As a negative electrical potential is applied across pole conductor 48, the electrical charge also is applied across each wire 105 (see
This in turn imposes an electrical potential difference between the first buffer, which is in contact with wire 105, and the second buffer, which is in contact with wire 104. Accordingly, the first buffer is negatively charged, while the second buffer is positively charged.
As discussed hereinabove, gel 90 of cassettes 80a and 80b is in contact with the first buffers (in upper buffer chambers 130a-130c), and gel 90 is also in contact with the second buffer in common lower buffer chamber 140. Therefore, the electrically charged buffers will result in an electrical field in gel 90 between top opening 92 and slot 96 to effect molecular separation of analytes in the sample.
For optimally reproducible results among gels run concurrently, the electric field provided to each gel should be substantially identical; and for optimal separation within a gel, the electric field should be homogeneous across the gel (i.e., in the direction perpendicular to the direction of analyte migration).
The apparatus of the present invention provides advantages with respect to both of these parameters in part by the design of container 20, and in part by the placement of wires 105, which span the length of the underside of buffer core body 61 (in direction y; as described hereinabove).
In particular embodiments of container 20, at least one of opposing bulkheads 112a and 112b of each set includes a plurality of lower wedge-shaped protrusions 115, rather than a single wedge-shaped protrusion 115 that extends across the width of bulkhead 112. The plurality of wedge-shaped protrusions 115 collectively make discontinuous contact with the cassette assembled to the buffer core engaged between the bulkheads, creating channels 116 (see
By spanning the underside of buffer core body 61, wires 105 provide a uniform electric field across the gel cassettes in direction y. Moreover, wires 105 are situated within container 20 such that they provide a substantially uniform electric field to all gel cassettes.
As mentioned hereinabove, heat is generated during electrophoretic molecular separation within gel 90, thus creating uneven temperature gradients on the surfaces of the gel, as well as across its thickness. Such problem is effectively mitigated by controlling the surface temperature of the gel cassettes.
Unlike previously-known apparatus and methods that actively circulate a coolant to control temperature, the present invention employs passive thermal management techniques to effect temperature control of the surface temperatures of gel cassettes 80. In particular, the dimensions of the apparatus are configured to permit first and second buffers to serve as heat sinks during electrophoresis, when the first and second buffers are disposed in upper buffer chambers 130a-130c and common lower buffer chamber 140, respectively. This temperature control is achieved for electrophoretic separation concurrently in a plurality of gels, by using passive thermal management to avoid the need for complex, ineffective or cumbersome active cooling mechanisms. According to passive thermal management provided herein, the temperature between upper buffers in separate buffer cores within a container at the end of an electrophoretic separation is within 25, 20, 15, 10, 5, 4, 3, 2, or 1° C. Furthermore, the temperature difference between an upper buffer and a lower buffer is within 25, 20, 15, 10, 5, 4, 3, 2, or 1° C. at the end of an electrophoretic separation performed using the apparatus or methods provided herein. Since this is typically the maximum temperature difference, the difference during an electrophoresis run is not as great. In one illustrative example, the temperature difference between an upper buffer and a lower buffer, and the temperature between upper buffers of separate buffer cores in the same container, is within 10° C. at the end of an electrophoretic separation performed using the apparatus or methods provided herein. The temperature of the lower buffer can be measured between buffer cores, but in certain illustrative aspects is measured in front of, or in back of, the buffer cores. The front and back lower buffer regions are expected to have a greater temperature differential with the upper buffer than the lower buffer between buffer cores.
The heat sink principles that are used to select dimensions of the apparatus of the present invention rely primarily on the heat transfer principle that the amount of heat added (“Q”) is equal to the product of specific heat of a substance (“c”), the mass of the substance (“m”) and the change in temperature (“ΔT”, or “Tfinal−Tinitial”).
With respect to the present invention, the amount of heat added Q to the gels can be approximated by determining the product of the current (“i”) and voltage (“V”) that are applied. Therefore, since current i and voltage V are known quantities, the approximate amount of heat added Q to each of the gels can be determined.
The approximate amount of heat added Q then is set equal to the product of specific heat of the buffer c, mass of the buffer m, and change in temperature ΔT (Tfinal−Tinitial). Since the specific heat of the buffer c is known, and the change in temperature is ascertainable (i.e., the initial temperature is known, and the final temperature is selected by the user), then the mass of the buffer to be added can be calculated.
Therefore, a user can determine how much first and second buffer should be added to keep the temperature increase of gels 90 below a predetermined threshold (i.e., Tfinal, such as 60° C.). Accordingly, in an other embodiment of the present invention, a method is provided for determining a volume of buffer to add to a cathode buffer reservoir or upper buffer reservoir, and the volume of buffer to add to an anode buffer reservoir, or lower buffer reservoir. The method includes selecting a target final temperature for a buffer and identifying an initial temperature for the buffer, and calculating a volume of buffer to add using a change in temperature between the target final temperature and the initial temperature and a specific heat of the buffer.
In application, it is desirable to maintain approximately the same temperature on outer and inner surfaces 81a and 81b of cassettes 80 (+/−25, 20, 15, 10, or 5° C. during a run to avoid slanting of the migrating bands in a sample. In a preferred embodiment of the present invention, the specific heat of the first and second buffers are within 25%, 20%, 15%, 10%, 5%, substantially identical, or identical. Therefore, to maintain approximately the same temperature on both sides of the cassette, the volume of first buffer devoted as a heat sink to each inner surface 81b is 50% to 150%, 75% to 125%, 85% to 115%, or 90% to 110% of the volume of second buffer devoted as a heat sink to each outer surface 81a.
Also, since heat is transferred to the effective heat sinks through faces of the cassettes, the inner and outer faces of the cassettes preferably are equal in area. Therefore, the heat flux out of one face is equal to the heat flux out of the other face, so long as the heat sink temperatures are equal.
In a preferred embodiment of the present invention, end walls 24 and 26 of container 20 each comprise thickness t1, as depicted in
In particular, enhanced thickness t1 of end walls 24 and 26 serves to insulate the lower buffer present between end wall 26 and first cassette 80a of buffer core assembly 60a, and between end wall 24 and second cassette 80b of buffer core assembly 60c; these end volumes of buffer have greater exposure to a wall of container 20 than do volumes defined further internal to container 20. By appropriately increasing the thickness of the end walls, increasing their insulating capacity, the temperature of the lower buffer present in the vicinity of end walls 24 and 26 is within 25° C. to the temperature of the lower buffer present in interior regions of container 20, thereby facilitating consistent runs for all gels in the container.
Similarly, side walls 21 and 22 of container 20 may have a chosen thickness designed to reduce radiant or convective heat loss through the side walls. However, if desired, side walls 21 and 22 may have a reduced thickness that allows for some heat loss through the side walls. In such cases, the heat loss may be accounted for in thermal calculations to ensure that a desired buffer temperature is achieved. Because side walls 21 and 22 are common to all chambers (or apportioned volumes), the lower buffer present in lower buffer chamber 140 will still have a temperature throughout all regions of container 20 that is within 35° C., 25° C., 15° C., 10° C., or 5° C., thereby facilitating relatively consistent electrophoretic conditions regardless of the number of gels being run.
As described hereinabove, container 20 is configured such that the volumes between assemblies 60a and 60b, and 60b and 60c are approximately twice as great as the volumes between cassette 80a of assembly 60a and end wall 26, and cassette 80b of assembly 60c and end wall 24. Therefore, when the second buffer poured into lower buffer chamber 140 settles to a height h, approximately twice as much second buffer volume will settle between the adjacent buffer core assemblies as will settle between assembly 60a and end wall 26, and assembly 60c and end wall 24. In the example described hereinabove, if 600 mL of the second buffer is poured into lower buffer chamber 140, then after the buffer settles in container 20, approximately 100 mL of the second buffer will settle between first cassette 80a of assembly 60a and end wall 26, approximately 200 mL of the second buffer will settle between assemblies 60a and 60b, approximately 200 mL will settle between assemblies 60b and 60c, and approximately 100 mL will settle between second cassette 80b of assembly 60c and end wall 24. Therefore, each outer surface 81a of each cassette 80 will have approximately 100 mL of second buffer devoted as a heat sink disposed adjacent the outer surface.
Since the apparatus of the present invention is configured to simultaneously run any number of gels, temperature control is scalable to the number of gels being run. Advantageously, by placing a dam into the system to seal off the unused regions, as described hereinbelow with respect to
In one example, if only two gels are being run, as described in
Referring now to
Referring now to
At this time, rear section 206 of dam 200 faces away from upper buffer chamber 230. Rear section 206 preferably has a U-shaped slot 210 configured to receive and hold resilient strip 211, as shown in
Red and black polarity tabs 37 and 38 may be disposed on opposing lateral sides of dam 200 to facilitate coupling of dam 200 to buffer core body 61 in a proper orientation, as depicted in
Referring now to
In
In a next step, a first buffer (not shown) then is poured into upper buffer chamber 230a, and a second buffer (not shown) is poured into lower buffer chamber 140. Since only one gel is being run in buffer core assembly 160, only one-half of the volume of the first buffer is required in upper buffer chamber 230a, relative to using two gel cassettes in the buffer core assembly. This is because front section 204 of dam 200 protrudes halfway into upper buffer chamber 230a, as shown in
For example, when 100 mL of first buffer is poured into upper buffer chamber 230a, 100 mL of second buffer is poured into lower buffer chamber 140 between the outer surface of cassette 80a and end wall 26. Therefore, 100 mL of first and second buffers are devoted as heat sinks for the inner and outer surfaces of cassette 80a. Accordingly, the temperature on outer and inner surfaces 81a and 81b of cassette 80a will be within 25° C., 15° C., 10° C., or 5° C. during electrophoresis.
Protruding front section 204 of dam 200 preferably is configured to reduce radiant or convective heat loss through the dam. For example, a sufficient thickness associated with protruding front section 204 may be selected to reduce heat loss through the dam. This approach is similar to the that described hereinabove for reducing heat loss through end walls 24 and 26 of container 20. Like the end walls, heat loss through dam 200 may be reduced by varying the thickness of section 204 to facilitate consistent temperature properties during electrophoresis runs, regardless of the number of gels being run.
Referring now to
In the arrangement of
A first buffer (not shown) is poured into upper buffer chamber 130c, and a proportional amount of a second buffer (not shown) is poured into lower buffer chamber 140. For example, when 200 mL of first buffer is poured into upper buffer chamber 130c, and 200 mL of second buffer is poured into lower buffer chamber 140, then 100 mL of first buffer is devoted as a heat sink for each of the inner surfaces of cassettes 80a and 80b, and 100 mL of second buffer is devoted as a heat sink for each of the outer surfaces of cassettes 80a and 80b. Accordingly, the temperature on the outer and inner surfaces 81a and 81b of cassette 80a will be approximately the same, assuming the specific heat of the buffers are substantially identical.
Referring now to
In the arrangement shown in
A first buffer (not shown) is poured into upper buffer chamber 130a. Then, one-half of the first buffer volume poured into chamber 130a is poured into chamber 230b. A proportional amount of a second buffer (not shown) then is poured into one of the regions of lower buffer chamber 140 shown in
As will be apparent to one skilled in the art, four or five gels also may be run simultaneously by further varying the location of dam 200 within container 20 and varying the number of cassettes employed. Moreover, it will be apparent to one skilled in the art that greater than six gels may be run simultaneously by providing additional chambers 30. Advantageously, dam 200 can block flow to regions of container 20 so that any number of gels can be run simultaneously. The user simply needs to adjust the volume of first and second buffers in a proportional manner, as illustratively described hereinabove, to maintain proper thermal management in the system.
All patents and publications cited in this specification are herein incorporated by reference as if each had specifically and individually been incorporated by reference herein. Although the foregoing invention has been described in some detail by way of illustration and example, it will be readily apparent to those of ordinary skill in the art, in light of the teachings herein, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims, which, along with their full range of equivalents, alone define the scope of invention.
This application claims the benefit of U.S. provisional patent application Ser. No. 60/505,051, filed Sep. 22, 2003, the disclosure of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60505051 | Sep 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10948453 | Sep 2004 | US |
| Child | 12533907 | US |
