JP4121698B2 - Automated parallel capillary electrophoresis system - Google Patents

Description

[Technical Field of the Invention]
The present invention relates to an apparatus for performing electrophoresis. More specifically, the present invention relates to an automated electrophoresis system using capillary cartridges formed so that commercially available standard size microtiter trays can be used. A carousel device, a multi-wavelength beam generator, a gel transfer system, and an off-line regenerator that avoids cross-contamination of samples, improves system performance, and increases throughput. Become.
[0001]
BACKGROUND OF THE INVENTION
Electrophoresis is a well-known technique for separating macromolecules. In electrophoresis applications, the sample molecules to be analyzed move in a medium to which a certain voltage is applied. Often, the sample is propagated through a gel that acts as a sieving matrix to help delay or separate individual molecules as they move.
[0002]
One application of gel electrophoresis is in DNA sequencing. Prior to the electrophoretic analysis, a DNA sample is prepared by a well-known technique. The result is a solution of DNA fragments of any possible length corresponding to the same total sequence order, each fragment ending with a label corresponding to the identification of a certain terminal base.
[0003]
This separation method uses a capillary filled with a conductive gel. To introduce the sample, one end of this capillary is inserted into a DNA reaction bottle. After introducing a small sample into the end of the capillary, both ends of the capillary are placed in a separate buffer solution. A certain voltage is then applied across the capillary. Due to the voltage drop at this time, the DNA sample is moved from one end of the capillary toward the other end. Due to the difference in the movement speed of the DNA fragments, the fragments are separated into bands of similar lengths. As these bands cross the capillary tube, the bands are typically read using one of several sensing techniques at any point along the capillary.
[0004]
The most widely used fluorescent dyes for labeling DNA samples have a maximum absorption wavelength in the range of 490-580 nm. Basic detection techniques include a CCD camera with a wide-angle lens, a capillary tube array disposed below the camera lens, the plane of which is disposed in parallel to the CCD imaging chip, and the capillary array. And a laser beam that is irradiated so as to cross. However, the single laser line used in this basic detection technique cannot conveniently accept all labels simultaneously. Therefore, multiple lasers or optical filters are used to compensate for this drawback.
[0005]
Usually, multiple DNA preparation reactions are performed using commercially available microtiter trays, which have a large number of small volume wells each capable of holding a sample on the order of 200-1,000 microliters. This microtiter tray is standardized in size. The microtiter tray currently preferred in the biotechnology industry has a rectangular array of 8 columns × 12 rows of wells. The center-to-center spacing between adjacent wells in a single row is about 0.9 cm, but this number may vary from 1/10 to 2/10 mm. The same applies to the spacing between adjacent wells in a single row. This 96-well rectangular array is designed to fit within a predetermined area (footprint) of less than 7.5 × 11 cm.
[0006]
The reduction allows a larger number of wells to be accommodated in a microtiter tray with the same footprint. New trays have already been introduced with wells arranged in the same predetermined footprint at a density four times that of the conventional, and this is rapidly becoming the industry standard. That is, this new tray is composed of wells of 16 columns × 24 rows, and the interval between wells is about 0.45 cm.
[0007]
It is not unusual to analyze thousands of DNA samples in a DNA sequencing project. Needless to say, it takes time to perform thousands of experiments using a single capillary.
[0008]
In the conventional apparatus, means for simultaneously analyzing DNA bands in a number of capillaries has been proposed. Such a device is described in Yeung et al. No. 5,498,324, the entire contents of which are incorporated herein by reference. This US patent teaches a means for detecting DNA bands separated in a number of capillary tubes arranged parallel to each other. However, in such a system, it is necessary to fill each capillary tube with a gel and introduce a sample into each capillary tube.
[0009]
Therefore, a considerable amount of time is required to fill each capillary tube with the gel. Furthermore, considerable effort is required to reliably and reliably introduce the reaction sample into one end of each tube.
[0010]
It is not uncommon to perform several consecutive sample analyzes using the same capillary. However, this obviously carries the risk of cross-contamination of the sample, which can also be a disadvantage in the conventional system.
[Summary of the Invention]
One object of the present invention is to provide an apparatus capable of simultaneously introducing samples directly from a standard size microtiter tray into multiple capillary tubes.
[0011]
Another object of the present invention is to provide a stacked dual carousel device, thereby avoiding cross-contamination of DNA samples without compromising system performance.
[0012]
Another object of the present invention is to provide a gel transfer module, whereby the gel is uniformly and rapidly distributed in the capillary tube.
[0013]
Another object of the present invention is to provide an off-line capillary regenerator, thereby completely cleaning the capillary cartridge off-line and improving system throughput while minimizing cost increases.
[0014]
Another object of the present invention is to provide an apparatus capable of generating a multi-wavelength beam. This multi-wavelength beam device allows simultaneous detection of multiple DNA samples labeled with different fluorescently labeled dyes.
[0015]
These objects are achieved by providing a disposable capillary cartridge that can be cleaned during electrophoretic analysis and having a plurality of capillary tubes. In this case, the first end of each capillary tube is held by the mounting plate, and these first ends together form an array in the mounting plate. The spacing between these first ends corresponds to the spacing between the centers of the wells of a standard size microtiter tray. Accordingly, the first ends of these capillaries can be simultaneously immersed in the sample in the well of the microtiter tray. The cartridge is provided with a second mounting plate, which holds the second end of the capillary. In another embodiment, instead of using this second mounting plate, the second end of the capillary tube is bundled together and received by a liquid transfer chamber, preferably a high pressure T-shaped mounting member. ing.
[0016]
A plurality of plate holes may be provided in each mounting plate, and capillary tubes may be inserted into these plate holes. In such a case, the plate holes are hermetically sealed to allow the side of the mounting plate having the exposed capillary end to be pressurized. By applying a positive pressure in the vicinity of the capillary opening of the mounting plate, air and fluid can be introduced during the electrophoresis operation, and the gel and the fluid can be removed from the capillary tube during regeneration. It can also be used to extrude other materials. When these capillary tubes are inserted into these plate holes, it is possible to prevent the capillary tubes from being damaged by using a needle made of a cannula and / or a plastic tube. Also, if metal cannulas or the like are used, they can serve as electrical contacts for current during electrophoresis.
[0017]
In a preferred embodiment, the stacked dual carousel device can avoid the problem of cross-contamination of DNA samples without compromising system performance. This system uses a buffer with a gel, which provides a medium for moving DNA from one end of the capillary tube to the other during electrophoresis. Since this buffer also moves through the capillary tube during electrophoresis, one end of the capillary tube must be immersed in the buffer solution so that the buffer solution is continuously replenished in the capillary tube. is there. Therefore, this buffer solution may be contaminated with the DNA sample during electrophoresis. The DNA in the buffer can then move into the capillary tube during subsequent runs if electrophoresis is performed continuously using the same buffer. The stacked dual carousel apparatus avoids the problem of cross-contamination of DNA samples by providing a buffer tray for each DNA sample tray and avoiding reusing the same buffer tray. In addition, since the stacked dual carousel apparatus has an additional carousel for holding the buffer tray, there is no need to replace the sample tray in order to provide a space for the additional buffer tray. Therefore, this dual carousel device can avoid the problem of contamination without impairing the system capability.
[0018]
In another preferred embodiment of the invention, the detection system uses both a multi-wavelength beam generator and a multi-wavelength detector so that they are labeled with different labeling dyes in the same instrument without switching the laser or optical filter. In addition, a plurality of DNA base sequence samples can be detected simultaneously.
[0019]
The multi-wavelength beam generator is provided by an argon ion laser that can generate multi-wavelength beams with wavelengths of 457 nm, 476 nm, 488 nm, 496 nm, 502 nm, and 514 nm. This multi-wavelength beam generator compensates for different absorption spectra in different labeled dyes, improves the uniformity of the peak detection signal in the DNA fragment and improves the signal / noise ratio of the detection signal.
[0020]
In another preferred embodiment of the invention, the gel transfer module moves the gel through the capillary quickly and uniformly. This gel transfer module uses a gel syringe to move the gel because the viscosity of the gel is too high to be moved by the pump. The gel transfer module includes a gel carriage for holding a disposable gel cartridge. The stepper motor / linear actuator has a movable actuator shaft, which is arranged to drive a Teflon plunger located at one end of the gel syringe so that the gel substance is provided at the other end of the gel syringe. It is designed to flow rapidly through the member. Further, the gel transfer module relaxes the gel in the capillary tube using the same member as used in electrophoresis, and achieves a uniform distribution of the gel.
[0021]
In another embodiment of the gel transfer module, a compressible gel bag is utilized. In this embodiment, the gel bag is disposed inside a high-pressure chamber, and the high-pressure chamber comprises a hollow cylinder whose top is open and whose bottom is closed, and a cap that is detachably attached to the top of the cylinder. An outlet assembly comprising an inner end detachably attached to the gel bag and an outer end connected to the T-shaped attachment member is attached to the high pressure chamber. This high pressure chamber is also connected to a pressure regulation assembly that can increase or decrease its internal pressure. When the pressure inside the high pressure chamber increases, the gel is squeezed outside through the outlet assembly and delivered to the T-shaped mounting member.
[0022]
In another preferred embodiment of the present invention, a simplified off-line capillary regenerator is able to achieve an increased system throughput while completely purifying the capillary off-line and suppressing an increase in system cost. . The operator can perform electrophoresis while cleaning the previously used capillary cartridge with an off-line capillary regenerator. Since it generally takes about 20 minutes to clean completely, it is not necessary to have the system wait for complete cleaning of the capillary cartridge 909 between successive runs of electrophoresis, so this Offline capillary regenerators can improve system throughput.
[0023]
This off-line capillary regenerator comprises a few low cost components including a solvent container for holding the cleaning fluid, a manifold for selecting the cleaning fluid, and a simple controller for managing the cleaning. . This simplification of the off-line capillary regenerator has the advantage that an increase in system throughput can be achieved while an increase in system cost is suppressed.
Detailed Description of Preferred Embodiments
FIG. 1 schematically illustrates a usage pattern of the apparatus of the present invention. The cartridge 30 of the present invention includes a plurality of capillary tubes 32 having substantially the same length. These capillary tubes 32 extend between the sample side connection array (array) 34 and the gel side connection array 36. The capillary tube 32 ends with an array of first capillary ends 38 on the sample side and ends with an array of second capillary ends 40 on the gel side.
[0024]
That is, both ends of the capillary tube 32 of FIG. 1 extend through individual plate holes in the base member 42. The base member 42 is preferably made of polycarbonate or acrylic resin. In addition, each array at the capillary end may be held by another mounting plate having a plate hole, and then each mounting plate may be fixed to the base member. Furthermore, instead of penetrating each capillary through an individual plate hole, one or more capillary tubes may be joined together to penetrate a common hole or no hole at all.
[0025]
Between these two arrays, the capillary tube 32 penetrates the thermoelectric member 44 mounted on the base member 42. The thermoelectric member 44 is disposed on both sides of the window region 46. This thermoelectric member is used to control the capillary tube to a temperature within a predetermined range. The thermoelectric member 44 may be composed of two or more members as will be apparent to those skilled in the art. Further, the temperature may be controlled using another temperature control means such as a fluid circulation system or air convection.
[0026]
The capillary tubes 32 are arranged in parallel in the window region 46 adjacent to each other. The length of each capillary tube from the end of the first capillary to the window region 46 is substantially the same in all the capillary tubes 32. This length is determined taking into account the optimization of the analysis time of the sample (ie the minimum allowable time) and the minimum allowable analytical power of the separated sample. Nominally, this length is in the range of approximately 50-70 cm. The window region 46 represents a region in which incident excitation light can access a parallel capillary tube, and is a region in which fluorescence emitted from the capillary tube can be accessed. That is, this window region 46 allows detection of bands 50 in various capillary tubes.
[0027]
As shown in FIG. 1, the excitation light source comprises a laser 52 and a prism 54 is used to focus the light beam 56 through the window region 46 and onto the capillary tube 32. Next, the fluorescent beam 58 is sensed by the CCD camera 60 and the band 50 is captured. It should be noted that other illumination and detection means may be used as is known to those skilled in the art.
[0028]
The configuration shown in FIG. 1 allows samples to be introduced substantially simultaneously into the first capillary ends 38 of all capillary tubes 32. In particular, according to this configuration, various samples can be introduced by simultaneously immersing the array of first capillary end portions 38 in the plurality of wells 62 of the microtiter tray 64 of the standard size as described above. .
[0029]
To enable this, the individual capillary ends are spaced apart from each other, and the spacing is substantially the same as that of the array of wells of the standard size microtiter tray 64. That is, the spacing between adjacent first capillary ends is about 0.9 cm, and the overall footprint of this first capillary end array is less than 7.5 cm × 11 cm, and thus a standard size microtiter It corresponds to the tray.
[0030]
The array of second capillary ends 40 is inserted into the wells 66 of the second microtiter tray 68 where they come into contact with the buffer 70 as is known to those skilled in the art. Since the wells 66 of the second microtiter tray 68 are separated from each other, there is little possibility of mutual contamination between the second capillary ends 40.
[0031]
A voltage source 72 is used to provide a potential difference between the two capillary end arrays. As shown in FIG. 1, one voltage level is applied to each well 62 of the first microtiter tray 64 via individual leads 74, and a second voltage level is approximately the same via individual leads 76. Applied to each well 66 of the second microtiter tray 68. That is, current is passed through the individual samples through leads 74 and in the wells 66 of the second microtiter tray 68 through the first capillary end 38, the capillary tube 32 and the second capillary end 40. The buffer solution 70 is energized and finally passes through the lead 76.
[0032]
2A and 2B show a plan view and a side view, respectively, of one example of a cartridge 80 of the present invention. The cartridge 80 includes a base member 82 made of, for example, polycarbonate or acrylic. The base member 82 is provided with first and second mounting plates 84 and 86, respectively. These plates are preferably formed from an electrically insulating material.
[0033]
The array of first capillary ends 88 protrudes from the bottom surface 90 of the first mounting plate 84, and the array of second capillary ends 92 protrudes from the bottom surface 94 of the second mounting plate 86. The capillary tube 96 passes through and is fixed to the plate holes formed in the mounting plates 84 and 86, and protrudes from the upper surfaces of the mounting plates 84 and 86. Preferably, each capillary tube 96 is protected by a tube assembly (assembly) 102 fixed to a plate hole of these mounting plates at a penetration portion with respect to the mounting plates 84 and 86.
[0034]
As clearly shown in FIG. 2A, the tube assemblies protrude from the mounting plates 84, 86 to form an 8-row, 12-column rectangular array with the associated capillary tubes, respectively. The distance between adjacent plate holes to which the tube assemblies 102 are fixed and the distance between adjacent capillary ends 88 and 92 are the distance between adjacent wells of a standard size microtiter tray 64. And correspond. As a preferred example, the spacing between adjacent capillary ends is about 0.9 cm, and the entire array of capillary ends, and therefore the footprint of the array of plate holes through which the capillary 96 passes, is no more than about 7.5 cm × 11 cm. .
[0035]
First and second enclosures shown as 104 and 106 are provided on the upper surfaces 98 and 100 of the mounting plates 84 and 86, respectively. In a preferred embodiment, each of these enclosures is provided with an inlet 108 and an outlet 110. The first enclosure outlet 110 is connected to the second enclosure inlet 108 via a plastic tube 112. The first enclosure inlet 108 is connected to a first plastic shut-off valve 114, and the second enclosure outlet 110 is connected to a second plastic shut-off valve 116. Conversely, these plastic shut-off valves 114 and 116 are connected to the first and second quick disconnects 118 and 120, respectively.
[0036]
During operation, the cartridge 80 can be connected to the pump assembly 122. The pump assembly 122 is configured to circulate a temperature-controlled liquid refrigerant through the enclosures 104 and 106. In this case, the first quick disconnect 118 of the cartridge is connected to the output 124 of the pump assembly 122 and the second quick disconnect 120 is connected to the input 126 of the pump assembly 122. With this configuration, the temperature of the portion of the capillary tube 96 that protrudes from the top surfaces 98 and 100 of the mounting plate and is located in the enclosures 104 and 106 can be maintained. In order to do this, the mounting plates 84, 86 need to be liquid tightly sealed to the base member 82. This liquid-tight seal needs to be formed between the plate hole and the tube assembly 102 and the capillary tube 96 or in the capillary tube 96 itself.
[0037]
Capillary tubes 96 lead between the two arrays of tube assemblies 102 in the area of the cartridge not covered by the enclosures 104,106. As described above, thermoelectric temperature control means 128 or the like is disposed on either side of the window section 130 of the capillary tube 96 to control the temperature of the capillary tube not within the enclosures 104,106.
[0038]
Capillaries 96 are arranged parallel to each other within at least a portion of the window area 130 and are readable by the sensing means. Preferably, the base member 82 is provided with an opening 132 on which the window area 130 is disposed. As a result, at least a part of the irradiation means or the detection means can be disposed below the base member, and the capillary tube 96 exposed therefrom can be seen linearly.
[0039]
FIG. 3A shows the needle 140 used to form the tube assembly 160, which can be inserted directly into the mounting plate 162 as shown in FIG. 3B. The needle 140 includes a metal cannula 142. In a preferred embodiment, cannula 142 comprises a stainless steel tube having an inner diameter of 0.064 inches and an outer diameter of 0.072 inches. The cannula 142 is provided with a bevel 144 at the end immersed in the well.
[0040]
Within the cannula 142, a polyetheretherketone (PEEK) polymer tube 146 is coaxially disposed, which acts as a sleeve. The polymer tube 146 has an inner diameter of about 0.006 inches and an outer diameter of 0.0625 inches. Therefore, the polymer tube 146 can be inserted into the cannula 142 without difficulty.
[0041]
A capillary tube 148 associated with the needle 140 passes through the center of the polymer tube 146 along the longitudinal axis of the needle 140. The capillary tube 148 is made of fused silica and has an inner diameter of about 0.003 inches and an outer diameter of about 0.006 inches. That is, the capillary tube 148 is fitted inside the polymer tube 146. The capillary tube 148 terminates at an end 150 that is substantially flush with the tip 152 of the cannula 142. That is, the gap between the two ends 150, 152 is about 0.035 inches.
[0042]
A UV curable, medical grade epoxy sealant 154 is used at both ends of the polymer tube 146 to secure the polymer tube 146 and capillary tube 148 to the cannula 142. Preferably, epoxy sealant 154 forms an air and liquid tight seal through cannula 142. This epoxy sealant prevents the polymer tube 146 from being exposed to the atmosphere and prevents the capillary tube 148 from contacting the cannula 142 directly.
[0043]
It should be noted that the needle can be formed in a form other than that depicted in FIG. 3A. For example, instead of a tubular cannula, the needle may simply consist of a capillary tube, covered with a plastic material that has been injected or coextruded, and this covering fixed to a metal strip. Other configurations are possible.
[0044]
FIG. 3B shows a hollow high pressure compression mounting member 164 made of nylon, into which the needle 140 is inserted to complete the tube assembly 160. The needle 140 can also be fixed to the cylindrical inner wall of the compression mounting member using an epoxy sealant. Each tube assembly 160 is then inserted into a plate hole 166 drilled in the mounting plate 162, and the plate hole 166 can also be sealed using an epoxy sealant. As a result, an airtight and liquid tight seal is formed between the bottom surface 168 and the surface 170 of the mounting plate 162, and even if positive pressure is applied to the bottom surface of the area where the plate hole for fixing the tube assembly is provided, The mounting plate can withstand.
[0045]
The compression mounting member 164 may be completely omitted, and a plate hole corresponding to the outer diameter and size of the needle 140 may be formed in the mounting plate 162. In that case, the needle is directly inserted into each plate hole of the mounting plate 162 and fixed thereto using an epoxy resin. Such an attachmentless approach can improve the structural integrity of the mounting plate 162 due to the reduced size of the plate holes. Further, since the interface where the epoxy sealant is used is reduced, it is possible to improve the airtight and liquid tight seals. Further, it should be understood that the capillary 148 encased in the capillary tube 148 or the polymer tube 146 can be held directly in an appropriately sized plate hole formed in the mounting plate 162.
[0046]
It should be understood that in a preferred embodiment, one capillary tube is held in each plate hole, whether or not a compression mounting member is used or a cannula and / or polymer tube is used. It is. The spacing of the array of plate holes preferably corresponds to that of a well of a standard size microtiter tray. However, it is also possible to form the plate hole off-center (center deviation) and tilt the end of the capillary.
[0047]
Furthermore, it should be understood that the array of capillary ends can be fixed in a desired shape without drilling holes in the mounting plate 162. For example, individual capillary ends can be arranged and fixed in a desired shape on the mounting plate 162 by bonding or tightening. In addition, it is also possible to hold a plurality of capillaries together and hold their ends while maintaining a desired shape with acrylic or the like. What is important is that the array has a capillary end spacing corresponding to the well spacing of a standard size microtiter tray.
[0048]
The conductive plate 172 is fixed to the mounting plate 162 with screws, an adhesive, or other known means. The conductive plate 172 has an array of conductive holes 174 corresponding to the plate holes 166 in the mounting plate. Each conductive hole 174 is formed by an H-shaped slit, and a pair of tabs 176 and 178 are formed between the legs of the H. When the needle 140 is inserted into the conductive hole 174, these tabs 176, 178 will retract and contact both sides of the needle 140.
[0049]
Since this conductive plate 172 is entirely conductive, all needles 140 in the array will share a common electrical connection. The voltage applied to the conductive plate 172 appears outside each needle 140 in the array. During electrophoresis application, this voltage appears in the buffer in each well in which the capillary end 150 is inserted.
[0050]
As known to those skilled in the art, the voltage difference can be applied to the first capillary end by other means as well. For example, instead of contacting the common plate to which the needle is connected, the voltage lead may be connected directly to each needle. In addition, each lead may be immersed in the liquid in each well. Another alternative is to apply a voltage through a metal coating, such as gold. In this case, the metal coating is deposited only on the outer surface of the end of each capillary that contacts the liquid in the well. In addition, a voltage may be applied directly to the well via one or more leads as described above. As will be apparent to those skilled in the art, any other approach can be selected to apply a voltage to the first capillary end.
[0051]
FIG. 4 shows a monitor plate 190 used with the cartridge illustrated in FIGS. 2A and 2B. In the cartridge of the present invention, the conductive plate 172 is provided on at least the needle of the first mounting plate 84 as described above. The second mounting plate 86 can be provided with a monitor plate 190.
[0052]
This monitor plate has an array of monitor holes 192. This array of monitor holes 192 is aligned with the second array of plate holes formed in the second mounting plate 86. Each monitor hole 192 comprises a separate electrical contact 194 that is electrically connected to the monitor plate connector 196 via individual leads 198. Each needle in the second mounting plate 86 contacts a corresponding electrical contact 194 in the monitor plate.
[0053]
The purpose of this monitor plate is to provide a means for measuring the presence of electrical continuity between any needle in the second mounting plate 86 and the needle in the first mounting plate 84. To this end, it will be appreciated that the monitor plate 190 can be secured to the mounting plate 86 in much the same manner as the conductive plate 172. What is important is that each electrical contact 194 is connected only to one needle in the second mounting plate.
[0054]
FIG. 5 shows a cartridge 200 in which a first array 202 and a second array 204 are partially disposed on a first carousel 206 and a second carousel 208, respectively. The CCD camera 210 is disposed on the cartridge portion between the arrays 202 and 204, and detects a band (not shown in FIG. 5) in the capillary. Each carousel 206, 208 has eight platforms 212 on which standard size microtiter trays are placed.
[0055]
Wells in each titer tray hold one or more liquids such as samples, gels, buffers, acidic solutions, basic solutions and the like. As shown in FIG. 5, the first carousel holds six sample trays 214, one buffer tray, and one waste tray, one of which is below the first needle array 202. Is located. Further, as shown in FIG. 5, the second carousel has a pair of acidic solution trays 220, a pair of basic solution trays 222, and a pair of waste liquid trays 224 (one of which is located below the second needle array 204). One buffer tray 226 and one gel tray 228 are held. That is, the first carousel 206 is a sample-side carousel, and the second carousel 208 is a gel-side carousel.
[0056]
The cartridge is detachably attached to the automatic electrophoresis apparatus. During operation, the platform below either of the two needle arrays 202, 204 is moved up and down by the lifting means. When the microtiter tray is brought close to one of the needle arrays 202, 204, the needles and associated capillary ends of these arrays are immersed in the contents of each well of the microtiter tray. When the platform underneath any of these needle arrays is lowered, the carousel associated with this platform is rotated so that different platforms 212 holding different microtiter trays can be raised.
[0057]
When the platform 212 is raised, the peripheral surface of this platform contacts the opposing surface, thereby sealing the pressure chamber below the bottom surface of the needle array. When a pressurized inert gas, such as helium, is introduced into the pressure chamber at a pressure of approximately 30 psi, a uniform force is applied to the sample in the wells of the microtiter tray held on the platform. This introduces a portion of each sample into the corresponding array at the first capillary end.
[0058]
Applying pressurized helium to introduce the sample into the first capillary end, or instead of introducing a high voltage for a short time on the order of 20-40 seconds to apply the sample to the first capillary end It can also be moved to. The use of high voltages for this purpose, however, can be size-selective. That is, the larger the molecule, the easier it is to be introduced at the end of the first capillary, potentially causing distortion in subsequent electrophoretic analysis.
[0059]
Next, the operation of the automatic electrophoresis apparatus shown in FIG. 5 will be described. Initially, various microtiter trays are filled with designated buffers, gels, samples, and the like. Next, the gel tray 228 on the carousel 208 rises and goes through the second capillary end (hidden in FIG. 5) associated with the second needle array 204 (not shown in FIG. 5). Introduced in. Thereafter, the gel tray 228 is lowered. The sample tray 214 on the carousel 206 is then raised and the sample is introduced through the first capillary end (hidden in FIG. 5) associated with the first needle array 202. Subsequently, the sample tray 214 is lowered. The carousels 206, 208 are then rotated to place the buffer trays 216, 226 below the corresponding needle arrays 202, 204. A potential difference is then applied across the two needle arrays and an electrophoretic analysis is performed.
[0060]
When this electrophoretic analysis is complete, the cartridge is regenerated. This is done by draining the gel and sample from the previous analysis under pressure and cleaning the capillary tube with the acidic liquid 220 and / or the basic liquid 222. The cartridge is thereby ready for reuse, allowing analysis of the samples in the other one sample tray 214.
[0061]
Of course, the number of platforms formed in the carousels 204, 206 can be arbitrarily selected. Furthermore, this platform can be configured in a linear, quadrangular or other arrangement. What is needed is storage and positioning that allows any first microtiter tray to be moved to the first needle array 206 and any second microtiter tray to be moved below the second needle array 208. It is a system.
[0062]
6A and 6B show the side and plane of a cartridge 280 having a first mounting plate 282, respectively, where the array of plate holes in the first mounting plate 282 has been rotated 90 degrees. In other respects, that is, the arrangement for connecting the capillary tube to the first mounting plate is substantially the same as in the previous example. A first capillary end formed in an array having a desired gap protrudes from the bottom surface of the first mounting plate 282 and is held in a plate hole formed in the first mounting plate.
[0063]
The second mounting plate 284, however, is not the same as the previous cartridge example. In the cartridge 280, the second mounting plate 282 acts as a pressure holding member for the pressure cell 286 having a substantially cylindrical outer wall surface. For the sake of simplicity, in FIG. 6A, the capillary tube on the first mounting plate is partially omitted. Further, the capillary tube on the second mounting plate 284 is completely omitted. However, in reality, all the capillary tubes are present.
[0064]
The second mounting plate has a bevel surface (slope) 288 that is symmetrical in the radial direction, and a plurality of plate holes 290 are formed therein. Each of these plate holes 290 is fitted with a PEEK polymer tube 292, and the capillary is fitted and fixed in the PEEK polymer tube 292 using UV curable epoxy resin as described above, and is airtight in the plate hole 290. And a liquid-tight seal is formed. Capillary tubes are inserted through the PEEK polymer tube and the second end of each capillary tube communicates with the internal cavity of the pressurized cell. In the preferred example of this cartridge, the second mounting plate provided with the PEEK polymer tube and the capillary tube has been described. However, the same needle as described above can be used. Further, a single capillary tube fixed with epoxy can be used in the same manner. What is important is that each capillary tube 294 is kept airtight and liquid tight in the plate hole 290 and that the second end of this capillary tube communicates with the internal cavity of the pressure cell 286. is there.
[0065]
Similar to the cartridge 80 of FIGS. 2A and 2B, the cartridge 280 is provided with thermoelectric control means 298 and enclosures 300, 302, whose capillary tubes are arranged in parallel along at least a portion of the window region 304. . Although not shown in FIGS. 6A and 6B, these enclosures 300 and 302 can be provided with an inlet and an outlet for circulating the refrigerant, as with the other cartridges 80.
[0066]
As shown in FIG. 6A, the second mounting plate 284 has a frustoconical upper portion, and the top portion is a flat top portion 310. The curved conical surface 288 provided with a number of plate holes 290 is advantageous in terms of structural integrity when a positive pressure is applied from below the second mounting plate. Furthermore, by providing the plate holes 290 on such a surface, the plate holes 290 can be provided farther apart, which also improves the structural integrity of the pressure cell 286.
[0067]
The pressure cell 286 is fixed to the base member of the cartridge 280 and protrudes through the bottom of the base member. With this configuration, the pressure cell 286 can include an inlet 312 and an outlet 314 on both sides of the cylindrical outer wall. It is also possible to easily provide this inlet on the flat top 310 of the second mounting plate 284 and provide the outlet on the bottom surface of the lower part 316 of the pressure cell 286. In such a case, the pressure cell can be placed on the base member rather than protruding through the bottom of the base member, and the pipe mounting member is connected to the outlet through a hole formed in the base member. Thereafter, the holes are sealed.
[0068]
FIG. 7 shows the valve arrangement for a pressure cell 320 having an inlet 322 at the top surface 324. The other points are substantially the same as the pressure cell 286. In addition to the inlet 322, the pressure cell 320 is provided with an outlet 326, which is connected to a waste liquid valve 328. The waste valve 328 is opened to discharge the contents of the internal cavity of the pressure cell 320.
[0069]
Access to this inlet 322 is controlled by a shut-off valve 330. Liquid is introduced into pressure cell 320 via inlet 322 using pump 332. Preferably, the pump is a high pressure liquid chromatography (HPLC) pump and the pumping capacity is 4-40 milliliters per minute at a pressure of about 2000 psi. The pump 332 is connected to a multi-valve manifold 334 so that one of the four liquids can be selectively fed into the pressure cell. These four liquids, that is, a gel, a buffer solution, an acidic solution, and a basic solution are held in separate containers 336, 338, 340, and 342, respectively. Additional containers holding similar liquids can be held for backup or connected in series with the containers to increase the total feed rate.
[0070]
The waste liquid valve 328, the shut-off valve 330, the pump 332, and the multiple valve manifold 334 are all under the command of a controller, preferably a microcomputer. That is, the contents of the internal cavity of the pressure cell are regulated by this controller. Such a controller can accept input from various pressure and temperature monitors and other sensors to prevent damage to the pressure cell 320.
[0071]
During operation, the internal cavity of pressure cell 320 is filled by pump 332. Thereby, the fed liquid is forcibly sent to the second capillary end communicating with the internal cavity of the pressure cell 320. The first capillary end array and pressure cell 320 can be filled with the appropriate fluid in the appropriate sequence to perform the electrophoretic operation described above with respect to the apparatus of FIG.
[0072]
After the analysis, the pressure cell and capillary tube are regenerated and ready for the next analysis. This is accomplished by flushing the gel and sample from all capillary tubes simultaneously. However, pressures on the order of several thousand psi are applied using the pressure cell 320. These increased pressures allow viscous gels to be expelled from capillary tubes more rapidly. This allows the cycle time between analyzes to be reduced to about 1 to 2 hours, including the regeneration operation.
[0073]
FIG. 8 shows an exploded cross-sectional view of a pressure cell 350 similar to the pressure cell 286 of the cartridge 280. Like the other pressure cells, this pressure cell 350 is preferably made from aluminum or stainless steel. The pressure cell 350 includes a threaded inlet 352 that is formed in the top surface 354 of the upper portion 356. This top includes a second mounting plate. The pressure cell 350 is further provided with a threaded outlet 358 on the bottom surface 360 of its lower part 362. A high pressure pipe attachment member can be threaded into these threaded inlet 352 and threaded outlet 358.
[0074]
These upper part 356 and lower part 362 are held together by a plurality of bolts (not shown). These bolts are inserted through bolt holes 368 formed along the periphery of the bottom surface 360 of the lower portion 362. These bolts are then screwed into corresponding threaded holes 370 formed in the bottom surface 372 of the upper portion 356. An O-ring 364 is partially inserted into a rectangular groove 366 formed in the second mounting plate 356. O-ring 364 acts as a seal between upper portion 356 and lower portion 362. Instead of the O-ring, a gasket or the like can be used to act as a seal.
[0075]
An internal cavity 374 is formed in the central portion formed between the upper portion 356 and the lower portion 362 of the pressure cell 350. A plurality of plate holes 376 are formed in the upper part (second mounting plate). For simplicity, a plurality of plate holes 376 are shown only on one side of the upper portion 356 in FIG. However, it should be understood that there is naturally also on the other side of the upper portion 356. The plate hole 376 extends from an inclined surface 378 formed in the upper portion 356 to the internal cavity 374.
[0076]
Capillary tube 380 is retained in these plate holes 376 and its second end 382 communicates with internal cavity 374. Each capillary is fitted into a PEEK polymer tube, which extends from the point in each plate hole near the internal cavity 374 to the outside of the bevel 378. However, for simplicity, this PEEK polymer tube is not shown in FIG. In addition, you may make it insert the needle which consists only of a capillary tube or a capillary tube, a PEEK tube, and a cannula into each plate hole 376 by adjusting the dimension.
[0077]
As described above, both end portions of the plate hole 376 are sealed (sealed) using a UV curable epoxy sealant, and the airtight and liquid tightness of the plate hole 376 is maintained. For pressure cells, the end 384 of each plate hole near the internal cavity 374 is tapped or roughened. This provides a surface to which the epoxy sealant easily adheres during assembly.
[0078]
The liquid held in the internal cavity 374 is in contact with the material forming the internal cavity. When this liquid further contacts the second capillary end 382, the electrical connection between the internal cavity 374 of the pressure cell 350 and the first capillary end secured to the first mounting plate is complete. That is, by grounding the pressure cell 350 via a contact provided thereon, the internal cavity 374 is grounded, and a circuit necessary for electrophoresis is completed. Alternatively, since the pressure cell 350 is electrically insulated from the base member on which it is mounted, the potential of the pressure cell 350 may be floated. This allows a high voltage to be applied to the pressure cell 350 rather than to the needle associated with the first capillary end.
[0079]
FIG. 9 shows another arrangement for the plate holes 390 in the second mounting plate 392 having a top surface 394 and an inlet 396. Each set 398 of three plate holes is offset at an angle relative to the center of the top surface 394. This provides the maximum spacing between the plate holes. From the viewpoint of structural integrity, such an arrangement is preferable to the arrangement in which the plate holes are radially arranged as shown in FIG. 6B.
[0080]
FIG. 10 shows an electrophoresis apparatus 400 designed to be used with a capillary cartridge formed as shown in FIGS. The device includes a user interface 402 (shown as a video display terminal and keyboard) that is in communication with a controller 404 (preferably a microprocessor-based computer or the like). The user interface 402 allows the user to input commands, receive status information, and observe collected data.
[0081]
The apparatus 400 further includes a data computing computer 406 that receives, stores, and processes video signals from the CCD camera 408. The data calculation computer 406 is provided with optical read / write data storage means 410 and / or magnetic read / write data storage means 412. The data calculation computer 406 is provided with signal and image processing software so that signal data from the CCD camera 408 can be analyzed. The data computing computer 406 is connected to the controller 404, and responds to requests from the controller 404, and exchanges data and control information as necessary.
[0082]
The apparatus 400 further comprises a high voltage electricity supply 414 that can apply the required voltage across the ends of the capillary tube. The operation of the high voltage electricity supply unit is instructed by the controller 404.
[0083]
The controller 404 directs the operation of a pump interface 416 that includes a number of electronic switches. This pump interface 416 regulates the operation of the solenoid valve 418. The solenoid valve 418 connects a gas inlet 420 connected to an inert gas source such as a pressurized helium tank to the chamber 422. The pump interface 416 further regulates the operation of the high pressure liquid chromatography (HPLC) pump 426. Under the direction of the controller, the HPLC pump 426 transfers the liquids in the containers 428, 430, 432, and 434, that is, gel, buffer, acid, and base, to the pressure cell 436 of the cartridge 438 through the multi-valve manifold 440. It is designed to supply selectively.
[0084]
A carousel 442 having a plurality of platforms is rotated by a rotor 444. A platform 448 positioned below the first mounting plate is moved up and down by an elevating means 446 such as a hydraulic pump. This moves the microtiter tray 450 on the platform 448 toward or away from the capillary end array as described above. The rotor 444 and the lifting / lowering means 446 are connected to the controller 404 and driven by the controller 404.
[0085]
The apparatus 400 further includes a light source 452 (preferably a laser), and irradiates the capillary tube 454 according to instructions from the controller 404. That is, the light source 452 irradiates the capillary tube 454 from below through the opening of the base member of the cartridge 438 as described above. Since the detection of the capillary band is performed in a dark place, the light shroud 456 covers the camera 408, the light source 452, and at least the window region of the capillary tube 454.
[0086]
During operation, capillary tube 454 is first cleaned and then filled with gel via pressure cell 436 by driving pump 452. This pump 452 is switched off. Next, the platform 448 that supports the microtiter tray 450 that holds the sample is raised by the elevating means 446. This creates a seal between the platform 448 and the lower surface of the chamber 422. Further, this immerses the first capillary end in the well of the microtiter tray 450. Chamber 422 is sealed, solenoid valve 418 is released, and pressurized helium gas is introduced through inlet 420. This applies a uniform positive pressure on the order of 30 psi into each well of the microtiter tray 450 to force at least a portion of the sample into the first capillary end. As mentioned above, a high voltage may be applied for this purpose for a short time. The platform 448 is lowered, the carousel 442 is rotated, and the microtiter tray 450 filled with buffer is moved under the first capillary end. The buffer tray is then raised, the first capillary end is immersed in the buffer, the pressure cell 436 is filled with the buffer, and the second capillary end is similarly contacted with the buffer. Thereafter, the high voltage source 414 is switched on and an electrophoretic test is performed. A light source 452 and a camera 408 are used, and band inspection in all capillary tubes 454 is performed simultaneously. Video signal data from the camera 408 is processed and stored in the computer 406. This processed data is then displayed on the user interface 402. After this inspection, the cartridge 438 is regenerated (i.e. cleaned) and is ready for the next inspection.
[0087]
The configuration of the carousel device shown in FIG. 5 has a problem of buffer contamination. As shown in FIG. 5, there are six sample trays 214 and one buffer tray 216 on the first carousel 206. After the sample is introduced from the sample tray 214 on the first carousel 206 through the first capillary end (hidden in FIG. 5), the sample tray 214 is lowered and both carousels 206, 207 rotate. Both buffer trays 216, 226 are placed under the corresponding needle arrays 202, 204, respectively, and a potential difference is applied between the two needle arrays 202, 204 to perform electrophoresis. While the first capillary end (hidden in FIG. 5) is in contact with the buffer tray 216, some of the DNA sample from the first capillary end is diffused into the buffer tray 216 and the buffer tray. 216 is contaminated. This contamination adversely affects the accuracy of electrophoretic testing after using DNA samples from other sample trays 214. This is because the same buffer tray 216 used in the previous test is used in the subsequent electrophoretic test.
[0088]
The problem with this contamination is that the sample tray 214 is replaced with a buffer tray 216 until a one-to-one match is obtained between the set of sample trays 214 and the set of buffer trays 216 on the first carousel 206. Can be avoided. With this arrangement, each sample tray 214 has a single corresponding buffer tray 216. Although this arrangement can avoid contamination problems, it adversely affects the capacity of the first carousel. Assuming that the first carousel 206 has eight platforms, there is a one-to-one correspondence between the set of sample trays 214 and the set of buffer trays 216 to avoid the contamination problem. Attempting to obtain this first carousel 206 can only have a maximum of three sample trays 214.
[0089]
FIG. 11 shows a stacked dual carousel device in the sequencer module 600, which achieves a one-to-one match between the set of sample trays 214 and the set of buffer trays 216 without compromising system capabilities. And avoid the problem of contamination. The stacked dual carousel device includes an upper carousel 601 and a lower carousel 602 that are aligned and spaced along a common axis.
[0090]
As a preferred example, both carousels 601, 602 have seven sites 618 for receiving microtiter trays 214, 216 and one large notch 620 for passing microtiter trays 214, 216. Have. The large notch 620 in the upper carousel 601 is adapted to allow the tray initially placed at the site 618 of the lower carousel 602 to pass through the upper carousel 601 to the needle array 603.
[0091]
As an example, a sample tray 214 is held in the upper carousel 601, and a buffer solution tray 216 corresponding to the sample tray 214 is held in the lower carousel 602. FIG. 12A shows the placement of this tray on the lower carousel 602. In this tray arrangement, the lower carousel 602 can hold six buffer trays 216 and one waste tray 224. The upper carousel 601 can hold six sample trays 214. As a preferred example, each carousel can comprise six sample trays, six buffer trays, one drain tray, and one water tray. This water tray is provided for rinsing the capillary and the first end of the electrode. However, the number of each type of tray provided on the carousel varies depending on the size difference of commercially available microtiter trays.
[0092]
Each of the carousels 601 and 602 in the stacked dual carousel apparatus is provided with a rotor 604 and a motor 608 for selectively rotating the carousels 601 and 602 at selected angular positions. Specifically, the motor 608 selectively rotates the carousels 601 and 602 to place an arbitrary portion 618 in which the sample tray 214, the buffer solution tray 216 or the waste solution tray 224 is stored under the needle array 603. The motor 608 is controlled by the controller 404 to selectively rotate the carousels 601 and 602 toward a predetermined angular position.
[0093]
As a preferred example, this stacked dual carousel device is provided with a motor 608 for each carousel 601, 602. The motor 608 is a stepper motor and is available from Pacific Scientific (Wilmington, Mass.). As another example, this laminated dual carousel device is provided with a DC motor 608 and a clutch mechanism for selectively engaging and rotating the carousels 601 and 602.
[0094]
The angular positions of the two carousels are automatically detected. For this reason, each carousel 601, 602 is further provided with an encoder 612 that is operatively engaged with the rotor 604. The encoder 612 senses the angular position data of the rotor 604 and transmits it to the controller 404. As a preferred example, this encoder 612 (eg, model number AG612XKRR; Stegman Corporation; Bandalia, Ohio) includes an optical sensor and has a 2048 pulse resolution value.
[0095]
This angular position can be detected by arranging holes (holes) 613 linearly adjacent to each part along the periphery of the carousel. For a carousel with eight sites, one lead hole, three coded holes and a tail hole are provided. This lead hole and tail hole merely indicate that a coded hole may exist between them. Each of these three coded holes may be present or absent. This allows coding for 8 = 2x2x2 sites. These holes (holes) are illuminated by LEDs disposed on the upper carousel 601. That is, LED light passes through these holes and is detected by a carousel position detector located below the lower carousel 602. The carousel position detector generates angular position data from a series of holes through which LED visible light is transmitted and communicates this data to the controller 404.
[0096]
The controller 404 uses the angular position data from any of the above examples to determine the rotational position of each carousel 601, 602, and selectively rotates each carousel 601, 602 by the motor 608 to a predetermined angular position. Arrange. More specifically, the controller 404 uses the position data to compensate for the rotational momentum of the carousels 601 and 602 that initially cause the carousels 601 and 602 to exceed a predetermined angular position.
[0097]
The stacked dual carousel arrangement further comprises a DC motor 605, which has a movable member and allows the selected trays 214, 216, 224 to move to the needle array 603 along a common axis as required. Move to move freely. In addition, the controller 404 moves the selected trays 214, 216, and 224 along the common axis via the DC motor 605.
[0098]
The DC motor 605 includes an ammeter and measures the current drawn by the DC motor 605. When a DC motor encounters a load, the current drawn by the motor increases, causing its movable member to move continuously. Accordingly, when the trays 214, 216, and 224 reach the needle array 603 while being raised, or when the trays 214, 216, and 224 are lowered and reach the carousels 601 and 602, the current rapidly increases. When this sudden increase in current is detected, the controller 404 operates to stop the DC motor, assuming that the trays 214, 216, and 224 have reached the correct positions.
[0099]
In the preferred example shown in FIGS. 12a and 12b, the diameter and thickness of each carousel 601 and 602 is 23.5 inches and 3/8 inch, respectively. Further, each carousel 601, 602 has a circular hole 610 having a diameter of 1.375 inches in the center, and can receive the rotor 604. Each carousel 601 and 602 is provided with four holes 611 having a diameter of 5/16 inch at equal intervals along the circumference of a circle concentric with the center of the carousels 601 and 602 having a diameter of 4 inches. The carousels 601 and 602 are bolted to the bearing assembly fixed to the drive shaft through these four holes 611 so that the carousels 601 and 602 are balanced on the bearing assembly so that controlled rotation is possible. It has become.
[0100]
In a preferred example, two rectangular openings 613, 614 form a plurality of portions 618 on each carousel 601, 602. The sizes of the openings on the upper surface 613 of the carousels 601 and 602 are slightly larger than the sizes of the trays 214, 216 and 224. The length and width of the top opening 613 are, for example, 5.95 inches and 4.187 inches, respectively. The sizes of the openings on the bottom surfaces 614 of the carousels 601 and 602 are slightly smaller than the sizes of the trays 214, 216 and 224. As described above, the size of the bottom opening 614 is slightly reduced, so that the peripheral portions of the trays 214, 216, and 224 are placed on the portion 618. The length and width of the bottom opening 614 are, for example, 5.45 inches and 3.687 inches, respectively. A recess 615 is formed in each part 618 on each carousel 601, 602 to match the tabs (ears) of the trays 214, 216, 224 to ensure correct orientation.
[0101]
In a preferred example, the notch 620 is an opening entirely delimited by carousels 601 and 602. In this example, the movable member of the motor 605 moves within the periphery of the carousels 601 and 602 so as to avoid hitting the carousels 601 and 602. This is because the opening is entirely divided by carousels 601 and 602.
[0102]
In another example, the notch 620 is partially separated by carousels 601 and 602 and is not separated along the periphery of the carousels 601 and 602. In this example, the movable member of the motor 605 need not move within the periphery of the carousels 601, 602 to avoid hitting the carousels 601, 602. This is because the opening is not divided along the periphery of the carousels 601 and 602. Therefore, the movable member of the motor 605 may be movable outside the periphery of the carousels 601 and 602 in this example.
[0103]
The sequencer module 600 (FIG. 11) also includes the various members described above that are operated in conjunction with a stacked dual carousel device to perform electrophoresis. These members include a CCD camera 408, a laser 452, a high pressure chamber 422, an array of capillary tubes 454, an enclosure that forms the optical window region 130 and the refrigerant region 300. Hereinafter, the solvent / gel transfer module 800 described with reference to FIGS. 14A to 14C sends the solvent or gel to the sequence module 600 via the solvent / gel inlet 606.
[0104]
FIG. 13 illustrates the operation of the stacked dual carousel apparatus described in FIGS. 11, 12A and 12B. As detailed below in FIGS. 14A-C, after the solvent / gel transfer module 800 has filled the capillary array 454 with gel in step 705, the controller 404 drives the motor 608 and rotor 604, which The carousel 602 rotates to place the notch 620 below the needle array 603 in step 700.1, preventing the trays 216, 224 on the lower carousel 602 from moving vertically in a subsequent step 700.2. ing. Controller 404 further sends commands to motor 608 and rotor 604 to rotate upper carousel 601 and place sample tray 214 under needle array 603 at step 700.1.
[0105]
In step 700.2, the controller 404 drives the motor 605 to move the sample tray 214 of the upper carousel 601 toward the needle array 603 along the common axis. In step 700.3, the controller 404 increases the gas pressure in the high pressure region 422 or a voltage is applied to transfer the sample from the sample tray 214 to the capillary array 454. In step 700.4, the controller 404 drives the motor 605 to move the sample tray 214 away from the needle array 603 along the common axis.
[0106]
As shown in FIG. 13, feedback is provided from the stacked dual carousel device to the controller 404 to ensure that the controller 404 issues its command at the correct time. For example, encoder 612 operatively engaged with rotor 604 sends feedback to controller 404 indicating position 1 of carousels 601, 602. However, until the carousel position feedback 1 indicates that the sample tray 214 on the upper carousel 601 is below the needle array 603, the controller 404 moves the sample tray 214 to the capillary inlet 603 in step 700.2. The command is not transmitted to the motor 605.
[0107]
Similarly, the ammeter of the motor 605 sends feedback to the controller 404 indicating the vertical position of the sample tray 2. However, until the tray position feedback 2 indicates that the sample tray 214 has been moved below the needle array 603, the controller 404 does not transmit a command to move the sample to the capillary array 454 in step 700.3. .
[0108]
The pressure transducer in the high pressure region 422 transmits the pressure value in this region (valve state 3) to the controller 404. However, until valve status feedback 3 indicates that the valve in high pressure region 422 is in the correct position, controller 404 moves sample tray 214 away from capillary tube inlet 603 via motor 605 in step 700.4. Does not transmit a command to Finally, controller 404 introduces buffer into capillary array 454 at step 701 until tray position feedback 2 determined by the ammeter of motor 605 indicates that sample tray 214 has returned to upper carousel 601. Does not communicate instructions for
[0109]
Following completion of step 700 as indicated by tray position feedback 2, sequence module 600 introduces buffer into capillary array 454 at step 701. In step 701.5, the controller 404 drives the motor 608 and the rotor 604 to rotate the upper carousel 601 so that the notch 620 is positioned below the needle array 603. In Step 701.5, the controller 404 drives the motor 608 and the rotor 604 to rotate the lower carousel 602 so that the buffer tray 216 is disposed under the needle array 603.
[0110]
In step 701.6, the controller 404 drives the motor 605 to move the buffer solution tray 216 from the lower carousel 602 to the needle array 603 via the notch 620 of the upper carousel 601. In step 701.7, the ultrasonic transducer is activated by the controller 404 to rinse the capillary tube inlet 603.
[0111]
In step 701, feedback is made from the stacked dual carousel device to the controller 404 to ensure that the controller 404 issues its command at the correct time. That is, the encoder 612 sends feedback indicating the position 5 of the lower carousel 602 and the position 5 of the upper carousel 601 to the controller 404. However, until the carousel position feedback 5 indicates that the notch 620 of the upper carousel 601 and the buffer tray 216 of the lower carousel 602 are below the needle array 603, the controller 404 in step 701.6 A command for moving 216 to the capillary tube inlet 603 is not transmitted to the motor 605.
[0112]
Similarly, the ammeter of motor 605 sends feedback to controller 404 indicating the vertical position of buffer tray 216. However, until the tray position feedback 6 indicates that the buffer tray 216 has been moved below the needle array 603, the controller 404 does not transmit a command to operate the ultrasonic transducer in step 701.7. Finally, the ultrasonic transducer sends feedback indicating the condition to the controller 404. The controller 404 does not initiate electrophoresis at step 702 until the transducer state 7 indicates that the capillary inlet 603 has been rinsed.
[0113]
The stacked dual carousel device regenerates the capillary array 454 at step 704 as described below in the description of the solvent / gel transfer module of FIGS. In step 704.15, the controller 404 drives the motor 608 and the rotor 604 to rotate the upper carousel 601 so that the notch 620 is positioned below the needle array 603. In step 704.15, the controller 404 drives the motor 608 and the rotor 604 to rotate the lower carousel 602 so that the waste liquid tray 224 is disposed under the needle array 603. In Step 704.16, the controller 404 drives the motor 605 to move the waste liquid tray 224 from the lower carousel 602 to the needle array 603 via the notch 620 of the upper carousel 601.
[0114]
In a preferred example, as previously described in the description of the stacked dual carousel device, the controller 404 issues commands to members of the electrophoresis device to operate electrophoresis and DNA analysis. Therefore, the controller 404 operates the operations listed in the left column of FIG. Data processing computer 406 is exclusively used to process data obtained from the execution of DNA analysis (step 703). This is because DNA data processing is of typical computer intensive nature. Accordingly, the data processing computer 406 operates the operations listed in the right column of FIG. Controller 404 and data processing computer 406 are connected to a local area network and are connected via a data processing user equipment interface 706.
[0115]
14A-C illustrate a solvent / gel transfer module 800 that is used to regenerate the capillary array 454 and refill the capillary array 454 with gel after DNA analysis. 14A is a front view of the solvent / gel transfer module 800, FIG. 14B is a side view thereof, and FIG. 14C is a rear view thereof. During DNA analysis, the sample is moved from right to left through capillary array 454 from capillary inlet 603 in FIG.
[0116]
During the regeneration of the capillary, the solvent is moved from the left to the right from the solvent containers 801 to 803 shown in FIG. 14A through the solvent / gel inlet 606 of FIG. 11 and further through the capillary array 454 of the sequence module 600 of FIG. The Similarly, while the capillary array 454 is refilled with gel, the gel passes from the gel syringe 804 shown in FIG. 14B through the solvent / gel inlet 606 of FIG. 11 and further through the capillary array 454 of the sequence module 600 of FIG. Move from left to right.
[0117]
The solvent containers 801, 802, and 803 contain methanol, water, and soap water, respectively. A feeder tube 806 in each solvent container 801-803 transfers the solvent toward the HPLC pump and washing solvent system 807. Similar to the previous example, the cleaning solvent system 807 includes a high pressure cell (HP cell) that produces a pressure increase for more rapid regeneration of the capillary array 454.
[0118]
Support rail 808 provides the necessary structure to hold the components of solvent / gel transfer module 800. These members include solvent containers 801-803, gel syringe 804, HPLC pump, wash solvent system 807 and controller 404. The controller 404 regenerates the capillary array 454 via other members of the solvent / gel transfer module 800 and refills the capillary array 454 with the gel material.
[0119]
15A to 15C explain the gel syringe 804 shown in FIG. 14B in more detail. In contrast to the solvent in solvent containers 801-803, the gel is too viscous to be pumped. Therefore, the gel syringe 804 is used for transferring the gel in the electrophoresis apparatus. The gel syringe 804 includes a gel tube carriage 891 and holds a gel cartridge that stores the gel substance 892 therein. Since this gel cartridge is of a disposable type, the capillary array 454 can be removed from the gel tube carriage 891 after refilling with gel material and used in subsequent electrophoresis and DNA analysis runs. Replaced with a new gel cartridge.
[0120]
The stepper motor linear actuator 893 has a movable actuator shaft 897 with a pressing member 898. The pressing member 898 has a front surface in contact with a Teflon plunger 894 disposed at one end of the gel syringe 804, and the gel substance is removed via a syringe cap 899 and a high-pressure attachment member 895 provided at the other end of the gel syringe 804. It is designed to be distributed. The controller 404 shown in FIG. 14C selectively drives the stepper motor linear actuator 893 to control gel transfer. A cylindrical tube 896 forms the outer structure of the gel syringe 804. The O-ring prevents gel material 892 from leaking around Teflon plunger 894 as it moves toward high pressure mounting member 895.
[0121]
In a preferred example, a stepper motor linear actuator (available from AMSI Corporation, Smithtown, NY) can output 140 pounds of linear force and can discharge 10 mL of gel with 6,000 pulses. The cylindrical tube 896 is made of standard acrylic resin, and the syringe cap 899 is made of stainless steel. This high pressure mounting member 895 is available from Swagelock.
[0122]
FIG. 16 shows the gel syringe 804 and HPLC wash solvent system 807 combined with the solvent / gel transfer module 800. The solvent manifold 850 has three inlets from the feeder tubes 806 of the solvent containers 801 to 803 connected to the outlets. A feeder tube 806 from the solvent containers 801 to 803 is connected to the inlet of the solvent manifold 850 by a tube 860. The controller 404 shown in FIG. 14C controls the solvent manifold 850 to select one solvent from the three solvent containers 801-803. The inlet of the HPLC pump 807 is connected to the outlet of the solvent manifold 850 via the tube 861, and the outlet of the HPLC pump 807 is connected to the inlet of the valve manifold 851 via the tube 862.
[0123]
The valve manifold 851 connects two inlets and outlets. One inlet of the valve manifold 851 is connected to the gel syringe 804 via the tube 863, and the other inlet of the valve manifold 851 is connected to the outlet of the HPLC pump 807. The outlet of the valve manifold 851 is connected to the solvent / gel inlet 606 via a tube 864. The controller 404 shown in FIG. 14C is configured to select either the inlet connected to the gel syringe 804 or the inlet connected to the HPLC pump 807 via the valve manifold 851.
[0124]
In a preferred example, the tube 860 connecting the feeder tubes 806 of the solvent containers 801-803 to the inlet of the solvent manifold 850 is a standard 1/8 inch diameter Teflon tube. A tube 861 connecting the outlet of the solvent manifold 850 to the inlet of the HPLC pump 807 is a PEEK tube having a diameter of 1/16 inch. Further, a tube 861 connecting the outlet of the solvent manifold 850 to the inlet of the HPLC pump 807, a tube 862 connecting the outlet of the HPLC pump 807 to the inlet of the valve manifold 851, and the gel syringe 804 are connected to the inlet of the valve manifold 851. The connected tube 863 and the tube 864 connecting the outlet of the valve manifold 851 to the solvent / gel inlet are PEEK tubes having a diameter of 1/16 inch.
[0125]
In a preferred example, this HPLC pump 807, available from Alltech Corporation (Model No. 301300), has a non-metallic pump head. The valve manifold 851 available from All-Tech Corporation (Model No. 97500) is a non-metallic valve.
[0126]
FIG. 13 illustrates the operation of the solvent / gel transfer module 800 shown in FIGS. 14A and 16. Following the DNA analysis in step 703, the precise rotational position of the carousels 601, 602 in step 704.15, and the precise movement of the waste tray in step 704.16, the controller 404 in step 704.17 causes the solvent / gel transfer module 800 to be used. Is driven, and the used gel is discharged from the capillary array 454 through the HP cell.
[0127]
To drain the spent gel, the controller 404 selects the inlet from the water container 802 via the solvent manifold 850 and the inlet from the HPLC pump 807 via the valve manifold 851, as shown in FIG. Is selected. The HPLC pump 807 transfers water from the solvent manifold 850 through the valve manifold 851 to the HP cell, creating a pressure increase for more rapid regeneration of the capillary array 454 as described above. Waste tray 852 that has been correctly moved in step 704.16 collects spent gel from capillary array 454.
[0128]
After the gel is removed from the capillary array 454, the solvent / gel transfer module 800 is driven by the controller 404 in step 704.18, and the rinsing liquid is passed through the capillary array 454 through the HP cell. To perform this rinse, the controller 404 selects the inlet from the methanol container 801 via the solvent manifold 850 and the inlet from the HPLC pump 807 via the valve manifold 851 as shown in FIG. The The HPLC pump 807 transfers methanol from the solvent manifold 850 through the valve manifold 851 to the HP cell, creating a pressure increase for faster rinsing of the capillary array 454 as described above. Waste tray 852 that has been correctly moved in step 704.16 collects spent solvent from capillary array 454.
[0129]
After the capillary array 454 is rinsed with methanol, the controller 404 drives the solvent / gel transfer module 800 and rinses the capillary array 454 with the soap solution from the container 803. To perform this rinse, the controller 404 selects the inlet from the soap container 803 via the solvent manifold 850 and the inlet from the HPLC pump 807 via the valve manifold 851, as shown in FIG. The The HPLC pump 807 transfers soap solution from the solvent manifold 850 through the valve manifold 851 to the HP cell, creating a pressure increase for faster rinsing of the capillary array 454 as described above. Waste tray 852 that has been correctly moved in step 704.16 collects spent solvent from capillary array 454. Further, the solvent / gel transfer module 800 is driven by the controller 404 and the regeneration process using the solvent from the three solvent containers 801 to 803 is continued until the capillary array 454 is cleaned.
[0130]
As shown in FIG. 13, feedback is provided from the solvent / gel transfer module to the controller 404 to ensure that the controller 404 issues its command at the correct time. For example, the controller 404 does not drive the solvent / gel transfer module 800 to pass the rinsing liquid through the capillary array 454 until the HP pump status feedback 17 indicates that the gel has been ejected from the capillary array 454. . Similarly, controller 404 issues a command to refill capillary array 454 with gel material in step 705 until HP pump status feedback 18 indicates that capillary array 454 has been rinsed with methanol and soapy water. There is no.
[0131]
In the preferred embodiment, controller 404 determines HP pump status feedback 17. The controller 404 calculates the amount of solution passing through the capillary array 454 from the HP pump flow rate specified by the manufacturer, and calculates the elapsed time since the pump was started, thereby providing HP pump status feedback. 17 is determined.
[0132]
Following capillary regeneration in step 704, the controller 404 drives the solvent / gel transfer module 800 in step 705.19 to refill the capillary array 454 with a new gel via the HP cell. To refill the capillary array 454 with a new gel, the controller 404 selects the inlet from the gel syringe 804 via the valve manifold 851, as shown in FIG. The gel syringe 804 transfers the gel to the HP cell via the valve manifold 851, creating a pressure increase for faster refilling of the capillary array 454 as described above. Waste tray 852 that has been correctly moved in step 704.16 collects any solvent or gel that exits capillary array 454 in step 705.19.
[0133]
After the HP cell and capillary array 454 are filled with gel in step 704.18, the process can be continued in one or two ways. As an example, the gel in the HP cell can be used as a buffer in electrophoresis after step 702. In this case, the process continues with step 705.20. As another example, prior to step 705.20, controller 404 drives solvent / gel transfer module 800 to transfer gel from the HP cell and fill the HP cell with buffer using the same process as step 704. Let
[0134]
The laminated dual carousel is allowed to participate in the remaining operations of step 705. In step 705.20, the controller 404 rotates the upper carousel 602 via the rotor 604, and moves the notch 620 below the needle array 603. Further, in Step 705.20, the controller 404 rotates the lower carousel 604 through the rotor 604, and moves the buffer tray 216 below the needle array 603. In Step 705.21, the controller 404 moves the buffer tray 216 from the lower carousel 602 to the needle array 603 through the large opening of the upper carousel 601 via the motor 605.
[0135]
Step 705 is completed with the step of equilibrating the capillary array 454 and circulating the buffer solution through the capillary array 454 (step 705.22). Due to the pulling difference through the capillary array 454, bubble and pressure differences are generated in the gel in the capillary array 454 during gel transfer in step 705.19. Step 705.22 removes this pressure difference and bubbles, which is accomplished by equilibrating the capillary array 454 and circulating a buffer through the capillary array 454. This technique is similar to the electrophoresis in step 702, except that DNA is clearly present in the electrophoresis in step 702, whereas in step 705.22, there is no DNA in the capillary array 454. . Specifically, a voltage is applied across the capillary array 454 to induce gel relaxation and buffer movement through the capillary array 454. After circulating the buffer through the capillary array 454 at step 705.22, the process for the next DNA analysis beginning with introducing the sample at step 700 is repeated.
[0136]
As shown in FIG. 13, as in step 704, feedback from the solvent / gel transfer module to the controller 404 is continued in step 705. The controller 404 processes this feedback to ensure that the command is issued at the correct time. For example, the controller 404 does not issue any command beyond step 705.19 until the gel syringe feedback 19 indicates that the capillary array 454 is filled with a new gel. Similarly, controller 404 will not issue a command to rotate upper carousel 601 and lower carousel 602 in step 705.20 until HP pump status feedback 19 indicates that the gel has been fed into capillary array 454. .
[0137]
In a preferred embodiment, controller 404 determines gel syringe feedback 19. That is, the gel syringe feedback 19 is determined by calculating the amount of the solution passing through the capillary tube array 454 from the replacement speed of the gel syringe and calculating the elapsed time since the gel syringe is started.
[0138]
Similarly, at step 705, the encoder that is operatively engaged with the rotor 604 sends feedback to the controller 404 indicating the position of the carousel 20. However, until the carousel position feedback 20 indicates that the large opening in the upper carousel 601 and the buffer tray 216 in the lower carousel 602 are located below the needle array 603, the controller 404 buffers in step 705.21. A command for moving the liquid tray 216 is not issued.
[0139]
The ammeter of the motor 605 also sends feedback indicating the vertical position of the buffer tray 216 to the controller 404. However, until the tray position feedback 21 indicates that the buffer tray 216 has been moved to the needle array 603, the controller 404 does not transmit a command to circulate the buffer to the capillary array 454 at step 705.22. . Finally, the controller 404 accepts feedback on the high voltage state of the capillary tube current 22. Note that the controller 404 does not proceed to step 700 for introducing the next DNA sample until this high voltage state 22 indicates that the buffer is circulating through the capillary array 454.
[0140]
FIG. 17 shows an off-line capillary regenerator 900 that is used to periodically thoroughly clean the capillary cartridge 909. To completely clean the capillary cartridge 909, the operator removes it from the automated electrophoresis system and attaches it to the off-line capillary regenerator 900. Therefore, the operator can attach another clean capillary cartridge 909 to the automatic electrophoresis system and perform DNA analysis using this capillary cartridge, while the previously used capillary cartridge 909 is replaced with an offline capillary regenerator. Thoroughly clean at 900. The complete cleaning of the capillary cartridge takes 20 to 30 minutes, but since there is no need to wait for the complete cleaning of the capillary cartridge 909 during the continuous execution of the DNA analysis, the offline capillary regenerator is an automated electrophoresis This will improve system throughput.
[0141]
This offline capillary regenerator 900 is a low-cost and simplified version of the solvent / gel transfer module 600 described above with reference to FIG. This is because it does not include all items included in the solvent / gel transfer module 600. For example, this off-line capillary regenerator 900 does not have a camera, laser or gel syringe. Further, the off-line capillary regenerator 900 does not include a gel syringe for gel transfer. This is because if the gel is sent off-line before the capillary cartridge is moved off, undesired hardening of the gel may occur at the end of the capillary array 454 while moving the capillary cartridge from the off-line capillary regenerator 900 to the automatic electrophoresis system. It is because it may occur. The simplicity of this off-line capillary regenerator 900 provides the advantage of increasing system throughput at low cost.
[0142]
The solvent containers 901, 902, and 903 contain methanol, water, and soapy water, respectively. A feeder tube 906 in each of the solvent containers 901 to 903 transfers the solvent toward the solvent manifold 905. This solvent manifold 905 connects three inlets to one outlet. The three inlets of the solvent manifold are connected to the feeder tubes 906 of the solvent containers 901-903, establishing a one-to-one correspondence between the set of inlets and the set of feeder tubes 906.
[0143]
The HPLC pump 906 has one inlet that is connected to the outlet of the solvent manifold, and the HPLC pump 906 has one outlet that is connected to the solvent inlet 907 at one end of the capillary cartridge 909. ing. The off-line capillary regenerator 900 also has an HP cell, similar to the solvent / gel transfer module 600 described in FIG. 14A, causing a pressure increase for more rapid regeneration of the capillary cartridge 909. The controller also controls the operation of the solvent manifold 905 and the HPLC pump 906. A waste container 904 collects waste liquid during capillary regeneration at the other end of the capillary cartridge 909.
[0144]
In the preferred example, the controller is a simple low-cost digital signal processing system that accepts status feedback and issues commands to the HPLC pump 906 and solvent manifold 905 in a predetermined order as described below. Alternatively, the off-line capillary regenerator may be managed by executing a simple control program using a general-purpose computer such as a personal computer.
[0145]
After the operator installs the capillary cartridge 909 in the off-line capillary regenerator 900, the internal controller selects the inlet from the water container 901 via the solvent manifold, and the HPLC pump 906 is activated. The HPLC pump 807 transfers water from the solvent manifold 850 to the HP cell, producing a pressure increase for faster regeneration of the capillary cartridge 909 as before. A waste container 904 that has been correctly moved in advance collects the used gel from the capillary cartridge 909.
[0146]
After the gel is removed from the capillary cartridge 909, the capillary cartridge 909 is rinsed using methanol by the offline capillary regenerator 900 via the HP cell. First, the controller selects the inlet from the methanol container 902 via the solvent manifold 905. The HPLC pump 906 transfers methanol from the solvent manifold 905 to the HP cell, creating a pressure increase for more rapid regeneration of the capillary cartridge 909. The waste liquid container 904 that has been correctly moved in advance collects the used solvent from the capillary cartridge 909.
[0147]
Next, the capillary cartridge 909 is rinsed with soap water from the container 903 through the HP cell by the off-line capillary regenerator 900. First, the controller selects the inlet from the soap container 903 via the solvent manifold 905. The HPLC pump 906 transfers soapy water from the solvent manifold 905 to the HP cell, creating a pressure increase for faster rinsing of the capillary cartridge 909. The waste liquid container 904 that has been correctly moved in advance collects the used solvent from the capillary cartridge 909. The controller 404 drives the off-line capillary regenerator 900, and the regeneration process using the solvent from the three solvent containers 801 to 803 is repeated until the cleaning of the capillary array 454 is completed.
[0148]
FIG. 18 illustrates another preferred example of the capillary cartridge 1180. In this example, a capillary tube extends from a first end 1188 disposed within the electrode / capillary array 1181. These capillary tubes then pass inside the multi-lumen tube 1183. This multi-lumen tube is described in detail in US patent application Ser. No. 08 / 866,308, which is hereby incorporated by reference. The multiple lumen tube 1183 is held at a predetermined position by a tube holder 1185. In addition, these capillary tubes pass through the optical detection region 1187 without protection by a multi-lumen tube. Beyond the optical sensing region 1187, the capillary tubes have a common end, are bundled together, and are secured in a high pressure T-shaped mounting member 1182 made of a conductive material. The attachment member 1182 is grounded during electrophoresis.
[0149]
The tube holder 1185 and the T-shaped attachment member 1182 are fixed to the cartridge base 1186. The cartridge base 1186 is made of polycarbonate for electrical insulation. The base 1186 is detachably attached to the shuttle 1179. The shuttle 1179 includes a set of rail couplings 1184 protruding from the bottom thereof. These rail couplings 1184 are arranged to fit into the sequencer module 400 in FIG. 10 or the rail system (not shown in FIG. 18) of the sequencer module 600 in FIG. This rail system allows the shuttle 1179 to move in and out of a predetermined position. The base 1186 can also be removed from the shuttle 1179 and the cartridge 1180 can be discarded (or washed) or a new (or cleaned) capillary cartridge can be attached when the shuttle 1179 is removed from place. I am doing so. With this rail system and shuttle 1179 combination, the newly installed capillary cartridge was discarded in the context of the camera and laser (not shown in FIG. 18) when the shuttle 1179 was in place. It can be repeatedly arranged at the same position as the capillary cartridge.
[0150]
In a preferred example, the shuttle 1179 extends the length of the base 1186 with an opening that houses the electrode / capillary array 1181. That is, the shuttle 1179 is attached to the base 1186 by a plurality of detachable fasteners 1178.
[0151]
The electrode / capillary array 1181 is held at a predetermined position by a current supply / monitor board 1190 shown in FIG. The board 1190 is preferably a printed circuit board for supplying high voltage.
[0152]
The board 1190 preferably includes a plurality of large holes 1193 so that the board 1190 can be attached to the base 1186 using a set of fasteners. However, the board 1190 can be attached to the base 1186 using any other means, such as bonding.
[0153]
The board 1190 further includes a plurality of tube holes 1194 arranged to align with the holes provided in the base 1186. Thus, when the board 1190 is attached to the base 1186, the first end of the capillary tube can protrude through the tube hole 1194. A plurality of pins 1195 (preferably gold plated) are further disposed on the board 1190. At least one pin is placed close to each tube hole to form a pin-hole pair 1192. Each pin and hole pair 1192 is immersed in the sample well of the sample microtiter tray.
[0154]
The board 1190 is further provided with a high voltage lead 1198. The lead wire 1198 connects each pin 1195 to a corresponding connector terminal 1196 formed around the board 1190. Each connector terminal 1196 is preferably configured to accept a high voltage connector having about 50 electrical connections. This high voltage connector connects lead 1198 to a power supply line from a high voltage power supply (preferably Bertan) (not shown in FIG. 19). This forms a closed electrical circuit from the pin 1195 to the second electrode connected to the high voltage T-shaped mounting member 1182 when the capillary tube is filled with gel. This second electrode is preferably connected to the ground of the system.
[0155]
In addition, the high voltage connector is also connected to a current monitor that monitors the current. In current monitoring, the current flowing through each power supply line is preferably monitored simultaneously or sequentially during multiple power supply lines. This makes it possible to perform integrated current measurement by this current monitor.
[0156]
FIG. 20 shows a multiwavelength beam generator. This wavelength beam generator comprises a laser head 1200, preferably an argon ion laser capable of generating a multi-wavelength laser beam at wavelengths of 457 nm, 476 nm, 488 nm, 496 nm, 502 nm and 514 nm. The beam generator further includes a laser emitter tube 1207. This laser emitter tube 1207 is connected to an argon ion laser 1200 by an optical coupling assembly 1202.
[0157]
The optical coupling assembly 1202 includes a fiber coupler 1201 connected to the laser head 1200 and an optical fiber cable 1203 connecting the fiber coupler 1201 to the laser emitter tube 1207. The fiber coupler 1201 aligns the laser head 1200 with a colorless optical fiber cable 1203. The optical coupling assembly 1202 enables the laser emitter tube 1207 to be positioned far from the laser head 1200 and generates a laser beam, ie, coherent light, in the laser emitter tube 1207. Further, this makes it possible to position the laser head 1200 that generates heat in a region where the sensitivity of the sequencer is small.
[0158]
The laser emitter tube 1207 includes a one-dimensional concentrator 1208. This one-dimensional concentrator 1208 is preferably a positive cylindrical optical lens having a focal length of 10 cm and is arranged at the output end 1204 of the laser emitter tube 1207. The laser emitter tube 1207 further includes a fiber emitter tube 1205 for receiving the optical fiber cable 1203, and a negative cylindrical optical lens preferably having a focal length of 1.9 cm. The fiber emitter tube 1205, the one-dimensional concentrator 1208, and the like. And a beam expander 1209 disposed between the two. The laser emitter tube 1207 is preferably housed in a 1 inch inner diameter and 6 inch long tube.
[0159]
FIG. 22 schematically shows an optical process performed by the laser emitter tube 1207. Laser light generated by the fiber emitter tube 1230 is expanded by a beam expander 1231. This laser beam is then focused only in one direction by a one-dimensional concentrator 1233. The resulting beam is directed toward the capillary array 1235. The resulting laser beam is narrowed in one direction and long in the other direction. 22A to 22C show the footprint of the laser beam in each process step.
[0160]
FIG. 21 shows another example of the multiwavelength beam generator, in which two laser heads 1211 and 1215 are provided. The first laser head 1211 is similar to the laser head 1200, and the second laser head 1217 generates laser beams having different wavelengths. The second laser head 1217 is preferably a solid state laser that generates a laser beam having a wavelength exceeding 532 nm. As another example, the second laser head 1217 generates a multi-wavelength laser beam having a wavelength different from that of the beam generated by the first laser head 1211.
[0161]
In this example, two optical coupling assemblies having fiber couplers 1213 and 1217 and optical fiber cables 1225 and 1221 are provided. These two optical coupling assemblies function similarly to the optical coupling assembly 1202 of FIG. Laser beams generated by the two laser heads 1211, 1215 and transported by this optical coupling assembly are combined in a laser emitter tube 1224 designed to accept laser beams from two laser sources.
[0162]
The laser emitter tube 1224 has two input terminals 1210 and 1212 and one output terminal. That is, the first fiber emitter tube 1227 that receives the laser beam from the first laser head 1211 is positioned at the first input terminal 1210; the second fiber that receives the laser beam from the second laser head 1215. The emitter tube 1223 is located at the site of the second input terminal 1212; a one-dimensional concentrator 1226 (preferably a positive cylindrical optical lens with a focal length of 10 cm, which outputs a combined laser beam) is an output terminal 1214. It is located in the part of.
[0163]
Laser beams received by the first and second fiber emitter tubes 1227 and 1223 are combined by a dichroic filter 1229. The dichroic filter 1229 transmits the laser beam from the first fiber emitter tube 1227 and reflects the laser beam from the second fiber emitter tube 1223, whereby the first and second fiber emitter tubes 1227, The laser beam from 1223 is combined.
[0164]
A beam expander 1222 which is preferably a negative cylindrical optical lens having a focal length of 1.5 cm is disposed between the dichroic filter 1229 and the one-dimensional concentrator 1226. This combination of beam expander 1222 and concentrator 1226 functions optically in substantially the same manner as the optical process described in FIGS. 22 and 22A-C.
[0165]
In the preferred example, the polarization in the laser heads 1200, 1211, 1215 is removed to maximize the laser output. In the example described in FIG. 21, the combination of multi-line and non-polarized emission increases the laser output by more than 4 times compared to a polarized single line laser.
[0166]
Although FIG. 22 shows a laser beam applied to the capillary array 1235 without an angle, the small angle incidence excitation configuration as shown in FIG. 23 has an excitation laser beam coupling efficiency in the detection window. To improve. This further reduces the laser power required to excite a 96 (or more) capillary array, allowing the use of a portable air-cooled argon ion laser in a DNA sequencer instrument. In other words, the multiwavelength laser emitter tube described in FIG. 20 or 21 irradiates a large number of capillary arrays (for example, 1000 capillary tubes) while maintaining the focusability of the laser beam over a wide range of capillary arrays. Is consistent.
[0167]
24 and 25 show a CCD camera 1257 combined with a laser emitter tube 1243. As a preferred example, the laser emitter tube 1243 is arranged perpendicularly to the surface of FIG. 25 with a small inclination angle, so that the laser beam from the laser emitter tube 1243 hits the capillary array 1200 as shown in FIG. .
[0168]
The laser emitter tube 1207 is arranged at the same level as the capillary detection window, and the beam emission end portion of the laser emitter tube 1207 faces in the opposite direction to the operator. As a preferred example, the laser emitter tube 1207 is fixed to the lower end of a tube control arm 1267 that is rotatably connected to an arm mount 1265. The arm mount 1265 is attached to the lower end of the flexible rotor 1261. The upper end of the rotor 1261 is connected to a dial accessible from the outside of the sequencer. The arm mount 1265 is further mounted on the laser emitter positioning rail 1255 so that it can be moved by the arm mounting position controller 1253. By using such a dial and controller 1253, the laser emitter tube 1207 can be optimally arranged to irradiate the capillary array with the output laser beam.
[0169]
The CCD camera 1257 is mounted on a camera mount (not shown in FIG. 25), and this camera mount is mounted on a laser emitter positioning rail 1255. A camera rotation cable (not shown in FIG. 25) moves the CCD camera plate on the rail 1255 in the horizontal direction. A camera focus gear 1271 is connected to the gear control cable 1254 to control the movement of the CCD camera lens assembly 1269. The preceding camera controller focuses the CCD camera 1257 on the portion of the capillary array irradiated by the laser beam from the laser emitter tube 1243.
[0170]
Another preferred example of a high viscosity liquid transfer system is shown in FIG. This transfer system comprises a high pressure chamber 1401 holding a low viscosity liquid 1413 such as water and a compressible disposable bag 1411 containing a high viscosity liquid such as a gel.
[0171]
The high pressure chamber 1401 includes a cylinder 1402 made of a metal such as aluminum or stainless steel so as to withstand an internal pressure of preferably up to 2000 psi. That is, it has a substantially hollow body whose bottom is closed and whose top is open. A cap 1404 is detachably attached to the top of the cylinder 1402. The cylinder 1402 and the cap 1404 form a high-pressure chamber 1401 that stores a liquid 1413 and a disposable bag 1411.
[0172]
This gel container 1411 is detachably attached to the outlet assembly 1410 (preferably by Swagelock 1409). When the pressure in the chamber increases, viscous liquid is forced through the outlet assembly 1410. The outlet assembly 1410 is fitted to the cap 1404 using a liquid tight mounting member (available from Swagelock). The outlet assembly 1410 further includes a gel release tube 1403. Since the pressure around the compressible gel bag 1411 is uniformly applied by the liquid 1413, the pressure resistance requirement of the gel container can be minimized. Thus, the gel bag 1411 can be made economically from a disposable bag having sufficient capacity for multiple runs of gel.
[0173]
The pressure in the high pressure chamber 1401 can be increased or decreased by a pressure control assembly 1406. As more liquid is supplied into the chamber 1401 by the pressure control assembly 1406, the pressure in the chamber 1401 increases. When this internal pressure is increased by an excess amount of liquid, or when the cap 1404 is opened and the bag 1411 is replaced, the liquid in the chamber 1401 is released by the pressure control assembly 1406 to reduce its internal pressure.
[0174]
The pressure control assembly 1406 includes a high pressure pump 1423 controlled by a controller 1404. As a preferred example, the high-pressure pump 1423 is composed of another HPLC pump. The pressure control assembly 1406 further includes an inlet tube 1421 connected to a pump 1423, which is fitted to the bottom of the cylinder by a liquid tight mounting member 1419.
[0175]
An outlet tube 1427 is further provided in the pressure control assembly 1406. The outlet tube 1427 is preferably fitted to the bottom of the cylinder 1402 by a second liquid tight mounting member 1417. The outlet tube 1427 is provided with a release valve 1425 and a pressure transducer 1429 for generating a feedback signal (preferably less than 6 volts). The pressure transducer 1429 is in communication with the controller 1404. The release valve 1425 is controlled by the controller 1404.
[0176]
The controller 1404 may be preferably configured as a part of the central computer controller 404 shown in FIG. 10 to save space. However, as another example, central computer controller 404 may be replaced with another type of controller, such as another computer, a microprocessor or other electronic device that can control the water pump and valves.
[0177]
By monitoring a feedback signal indicating the pressure in the chamber 1401, the controller 1404 functions as follows: (1) When the pressure in the chamber 1401 needs to be increased to some extent, the controller 1404 1423 is driven and liquid is introduced into the chamber 1401 via the inlet tube 1421; or (2) When the pressure in the chamber 1401 needs to be reduced to some extent, the controller 1404 opens the release valve 1425 and opens the chamber The liquid is released from inside 1401. Once a sufficient amount of gel has been pushed out, the pump stops and the pressure in the chamber decreases and returns to its original equilibrium state. A preferred gel filling pressure is 500 psi.
[0178]
FIG. 27 shows a preferred example of a solvent / gel transfer module 1800 used to regenerate the capillary tube array after DNA analysis and refill the capillary array with the gel. FIG. 28 is a rear view of the solvent / gel transfer module 1800. This solvent / gel transfer module 1800 is preferably located adjacent to the sequencer. The purpose of this solvent / gel transfer module 1800 is to provide continuous and automatic gel transfer and capillary tube regeneration.
[0179]
FIG. 29 shows a high pressure gel transfer system 1805 (gel transfer syringe 804 of FIG. 16 or high pressure chamber 1401 of FIG. 26) combined with the solvent / gel transfer module 1800 of FIG. In this preferred example, solvent manifold 1850 connects four inlets from feeder tubes 1806 of solvent containers 1801-1804 to one outlet. Feeder tube 1806 from solvent containers 1801-1804, two bottles filled with methanol, one bottle filled with polyvinylpyrrolidone (PVP) (preferably 2% PVP), and one filled with buffer The bottle is connected to the inlet of the solvent manifold 1850. The inlet of the HPLC pump 1807 is connected to the outlet of the solvent manifold 1850 by a pump connection tube 1861. The outlet of the HPLC pump 1807 is connected to the inlet of the solvent manifold 1850 by a pump outlet tube 1862. The outlet from the HPLC pump 1807 (not shown in FIG. 29) is connected to the high pressure chamber 1805 as described above.
[0180]
A valve manifold 1851 connects two inlets to one outlet. One inlet of this valve manifold 1851 is connected to a gel transfer system 1805 by a gel outlet tube 1863. The other inlet of the valve manifold is connected to the outlet of the HPLC pump 1807. The outlet of the valve manifold 1851 is connected via a manifold outlet tube 1864 to a liquid transfer chamber 1810 (preferably the high pressure T-shaped mounting member 1182 of FIG. 18). The liquid transfer chamber 1810 includes a purge valve 1867 for discharging the waste liquid therein to the waste liquid container 1865.
[0181]
The controller 404 shown in FIG. 10 has connections to a solvent manifold 1850, an HPLC pump 1807, a pressure control assembly for the gel transfer system 1805, a valve manifold 1851, and a drain valve 1867 for controlling the connected components. Have each.
[0182]
For example, the controller 404 controls the solvent manifold 1850 to select a solvent from the four solvent containers 1801-1804, or to drive the valve manifold 1851 to receive an inlet or solvent connected to the chamber 1804 to receive the gel. Select the inlet connected to the HPLC pump 1807 for.
[0183]
As a preferred example, the length of the pump connection tube 1861, pump outlet tube 1862, and manifold outlet tube 1864 is minimized to reduce gel and solvent waste. In particular, the pump connection tube 1861 and the pump outlet tube 1862 retain old solvent when new solvent needs to be supplied to the valve manifold 1851, thereby wasting this old solvent. Similar waste occurs between the solvent in the manifold outlet tube 1864 and the gel. Accordingly, the length of the manifold outlet tube 1864 is less than 50 cm, preferably less than 25 cm, more preferably less than 10 cm.
[0184]
As shown in FIG. 28, the solvent / gel transfer module 1800 has a blower 1801 for the laser head located in the sequencer. This laser head is housed on the bottom surface of the sequencer module. The cooling blower is designed to draw air from the sequencer module and blow it out to the exhaust of the washer module 1800. As a result, the cold air moves across the laser and there is no risk of causing significant disruption to the sequencer module. A 5-inch diameter flexible hose is connected from the rear of the laser toward the blower intake. Hot exhaust gas is carried away through another 5 inch diameter hose (connected to the ceiling duct) and released to the outside (not shown in FIG. 28).
[0185]
Similar to Steps 700 to 703 in FIG. 13, Steps 1901, 1903, and 1905 in FIG. 31 describe the operation of the stacked dual carousel device described in FIGS. 11 and 12A. Since these steps are substantially the same as each other and the difference between them is self-evident, detailed description of steps 1901, 1903, and 1905 is omitted.
[0186]
Further, steps 1907, 1909, 1911, 1912 of FIG. 31 illustrate the operation of the solvent / gel transfer module 1800 described in FIGS. Since the operation of the solvent / gel transfer module 1800 is also substantially the same as that of the solvent / gel transfer module 800, redundant description is omitted. However, the following differences will be described.
[0187]
In step 1907.17, the rinsing step is performed by rinsing the capillary tube with methanol for 24 minutes and then with PVP for 8 minutes. In step 1911.23, the current monitor attached between the current / monitor board and the power supply is driven. In steps 1912.25 and 1912.26, a rinsing tray is utilized for rinsing pins protruding from the first end of the capillary tube and the current supply / monitor board.
[0188]
While the invention has been described with reference to certain preferred examples, it should be understood that the scope of the invention is not limited by these examples. That is, it will be easy for those skilled in the art to make various modifications, but these are also included in the scope of the present invention, and the scope is regulated by the following claims.
[Brief description of the drawings]
FIG. 1 is a side view showing an arrangement of an apparatus of the present invention.
FIG. 2A is a plan view showing one example of the cartridge of the present invention.
FIG. 2B is a side view showing one example of the cartridge of the present invention.
FIG. 3A is a side view showing a tube assembly for the cartridge of the present invention.
FIG. 3B is a side view showing the mounting arrangement of the tube assembly.
FIG. 4 is a plan view showing a monitor plate used together with a needle row.
FIG. 5 is a plan view showing the arrangement of devices used with the cartridge shown in FIGS. 2A and 2B.
FIG. 6A is a side view showing a second example of the cartridge of the present invention.
FIG. 6B is a plan view showing a second example of the cartridge of the present invention.
7 is a side view showing a valve arrangement for a pressure cell similar to that shown in FIGS. 6A and 6B. FIG.
FIG. 8 is an exploded longitudinal sectional view of a pressure cell.
FIG. 9 is a plan view of a second mounting plate of a pressure cell having an alternating arrangement of plate holes.
FIG. 10 is a side view showing an electrophoresis apparatus of the present invention.
FIG. 11 is a side view showing a sequencer module including a stacked dual carousel device.
FIG. 12 is a diagram showing in detail a carousel included in a stacked double array.
FIG. 13 is a flowchart for explaining the operation of the present invention.
14A is a front view of a solvent / gel transfer module in the system. FIG.
FIG. 14B is a side view of the solvent / gel transfer module in the system.
FIG. 14C is a back view of the solvent / gel transfer module in the system.
FIG. 15A is a detailed view of a gel syringe included in the gel transfer module.
FIG. 15B is a detailed view of the gel syringe included in the gel transfer module.
FIG. 15C is a detailed view of a gel syringe included in the gel transfer module.
FIG. 16 shows the gel and solvent flow through the solvent / gel transfer module to the sequencer module.
FIG. 17 shows an off-line capillary regenerator.
FIG. 18 is a side view of the capillary cartridge of the present invention.
FIG. 19 is a plan view showing a current supply / monitor board.
FIG. 20 is a diagram showing a multiwavelength beam generator using one laser head.
FIG. 21 is a diagram showing a multi-wavelength beam generator using two laser heads.
FIG. 22 is a side view showing an optical processing function of a laser emitter tube.
FIG. 22A shows a footprint of a light beam at the output of a laser emitter.
FIG. 22B is a diagram showing a footprint of a light beam after passing through a beam expander.
FIG. 22C shows a footprint of a light beam after passing through a one-dimensional focus lens.
FIG. 23 is a diagram showing a direction in which a light beam from a laser emitter collides with a capillary row.
FIG. 24
FIG. 25 is a side view showing a structure around a detection region.
FIG. 26 is a side view showing a high-pressure chamber for supplying a high-viscosity gel.
FIG. 27 shows a solvent / gel transfer module in a preferred embodiment.
FIG. 28 shows the back of the solvent / gel transfer module.
FIG. 29 illustrates gel and solvent flow through a solvent / gel transfer module in a preferred embodiment.
FIG. 30 is a flowchart illustrating an operation in a preferred embodiment.

Claims (17)

A capillary electrophoresis system for performing capillary electrophoresis on a plurality of samples, the system comprising:
A plurality of capillary tubes, each capillary tube having a first end and a second end, wherein the first ends are arranged in a two-dimensional array, and the distance between them is the microtiter tray Corresponding to the spacing of the array of wells, the second end having a common termination;
A positioning device comprising a dual carousel comprising a stack of upper and lower carousels each carrying a plurality of microtiter trays, wherein the positioning device corresponds to a two-dimensional array of capillary ends corresponding to the microtiter tray Arranged to position one of the microtiter trays for insertion into the well;
An apparatus arranged to selectively transfer a selected one of a gel and a plurality of liquids to a second end of the capillary tube;
A light source arranged to illuminate the sample;
Camera means arranged to detect light emitted by the sample;
A capillary electrophoresis system comprising: 2. The capillary electrophoresis system according to claim 1, wherein the light source is: a laser head arranged to generate a light beam;
A laser emitter tube disposed away from the laser head;
A first optical coupling assembly for connecting the laser head to the laser emitter tube, thereby guiding a light beam from the laser head to the laser emitter tube;
A capillary electrophoresis system comprising: In capillary electrophoresis system of claim 2, further capillary electrophoresis system comprising comprising: means for adjusting the position of the emitter tube. The capillary electrophoresis system according to claim 1, wherein
The upper and lower carousels are aligned and spaced apart from each other along a common axis, the upper carousel has a notch, and the shape and dimensions of the notch are such that the microtiter tray is placed on the lower carousel. The microtiter tray can be passed through ,
The positioning device comprises:
A first motor operably coupled to the carousel, wherein the first motor is arranged to selectively rotate each of the carousels to a predetermined angular position;
A second motor coupled to act on the lift member, wherein the lift member is the microtiter tray, the microtiter tray is placed on the lower carousel, and the micro position When the titer tray is raised above the surface of the upper carousel and the capillary end array is placed on the microtiter tray, the capillary end array is inserted into the corresponding well of the microtiter tray. Arranged to move along said common axis between a second position of
A controller arranged to control the first and second motors;
A capillary electrophoresis system comprising: In capillary electrophoresis system of claim 1, further capillary electrophoresis system formed by including the placed sensors to determine the respective angular position of the carousel. The capillary electrophoresis system according to claim 1, wherein
The light source is a light source for irradiating a sample moving through a plurality of capillaries ;
A first laser head for generating a first light beam at a first wavelength;
A laser emitter tube having a first input terminal and an output terminal, wherein the laser emitter tube receives the first light beam at the first input terminal and is focused at the output terminal. Arranged to output one light beam and further away from the first laser head;
A first optical coupling assembly connected to the first laser head at a first end and connected to a first input terminal of the laser emitter tube at a second end, the first optical assembly comprising: Guiding a light beam from a laser head of the first to a first input terminal of the laser emitter tube;
Comprising:
A capillary electrophoresis system in which an irradiation beam from a first input terminal of the laser emitter tube is directed to the sample. The capillary electrophoresis system according to claim 6 , wherein the laser emitter tube of the light source is;
A fiber emitter tube disposed to receive a second end of the first optical coupling assembly at the first input terminal;
A one-dimensional concentrator disposed in the vicinity of the output terminal;
A beam expander disposed between the fiber emitter tube and the one-dimensional concentrator;
A capillary electrophoresis system comprising: The capillary electrophoresis system according to claim 6 , wherein the light source is
A second laser head for generating a second light beam of a second wavelength;
A second input terminal formed on the laser emitter tube, wherein the laser emitter tube receives the second light beam at the second input terminal and is spaced apart from the second laser head; And what was done;
A second optical coupling assembly connected to the second laser head at a first end and connected to a second input terminal of the laser emitter tube at a second end;
Further comprising
Accordingly, the capillary electrophoresis system in which the first and second light beams are guided to the laser emitter tube via the first and second optical coupling assemblies. 9. The capillary electrophoresis system of claim 8 , wherein the laser emitter tube further comprises:
A first fiber emitter tube disposed at the first input terminal for receiving a second end of the first optical coupling assembly;
A second fiber emitter tube disposed at the second input terminal for receiving a second end of the second optical coupling assembly;
A dichroic filter disposed within the laser emitter tube that optically interfaces with the light beams from the first and second fiber emitter tubes;
A one-dimensional concentrator disposed in the vicinity of the output terminal;
A beam expander disposed between the fiber emitter tube and a one-dimensional concentrator;
A capillary electrophoresis system comprising: 2. The capillary electrophoresis system according to claim 1, further comprising a position adjusting device for adjusting a position of an emitter tube arranged to output a light beam toward a plurality of samples moving through the corresponding plurality of capillaries. With
The position adjusting device is:
An arm that holds the emitter tube so that the output of the emitter tube is directed toward the sample;
An arm mount for holding the arm;
Rail means for moving the arm mount along a first direction;
A first flexible rotator connected to the arm mount and moving the arm mount along the rail means;
A second flexible rotator connected to the arm mount and moving the arm mount in a direction intersecting the first direction;
A capillary electrophoresis system comprising: The capillary electrophoresis system according to claim 1,
The equipment for selectively transferring the selected one of the gel and a plurality of liquid into a plurality of capillaries:
A first manifold arranged to selectively connect one of the plurality of liquid holding containers to the first supply line;
One of the gel supply lines and a second manifold arranged to selectively connect the first supply line to a second supply line;
A first end connected to the second supply line; a second end connected to the second end of the capillary; and a third end connected to a waste container. A three-way connector, wherein the third end comprises a valve means;
A capillary electrophoresis system, wherein the length of the second supply line is less than 50 cm. In capillary electrophoresis system of claim 11, wherein the capillary electrophoresis system length of the second supply line is less than 25 cm. In capillary electrophoresis system of claim 12, wherein the capillary electrophoresis system length of the second supply line is less than 10 cm. The capillary electrophoresis system according to claim 1, wherein
With the device gel transfer system for selectively transferring the selected one of the gel and a plurality of liquid into a plurality of capillaries, the gel transfer system is;
A tube having a longitudinal axis, a first end with an outlet having a high-pressure attachment, and a second end with a surface movable toward the first end along the longitudinal axis A gel-filled container;
A plunger provided in contact with the movable surface;
A stepper motor operatively engaged with the plunger to apply a force to the plunger to supply gel from a first end of the container;
The plunger is selectively moved via the motor, and a predetermined amount of gel is discharged from the outlet and sent to a passage connected to the capillary tube, whereby the capillary tube is filled with the gel simultaneously. With a controller;
A capillary electrophoresis system comprising: The capillary electrophoresis system according to claim 1, wherein
With the device gel transfer system for selectively transferring the selected one of the gel and a plurality of liquid into a plurality of capillaries, the gel transfer system is;
A substantially rigid chamber having a first end having an outlet and a second end having an inlet, substantially filled with media;
A friable gel-filled container disposed within the chamber and substantially surrounded by the medium, having an opening communicating with the outlet;
Pump means connected to the chamber inlet;
Connected to the pump means, and through the pump means, additional medium is fed into the chamber, thereby at least partially collapsing the gel-filled container and extruding a predetermined amount of gel from the outlet and connected to a capillary tube A controller that feeds into the passageway, thereby filling the capillary tube with gel simultaneously;
A capillary electrophoresis system comprising: The capillary electrophoresis system according to claim 15,
The gel transfer system,
A pressure transducer arranged to sense the pressure of the medium and supplying the read pressure value to the controller;
A release valve connected to the chamber;
A capillary electrophoresis system, wherein the controller is arranged to operate a release valve in response to the pressure value. The capillary electrophoresis system according to claim 1, wherein
Furthermore, for cleaning a plurality of capillary tubes having first and second ends
Comprising a cleaning device, the cleaning device;
A plurality of containers each holding a solvent;
A manifold that selectively supplies one of the solvents to the second end of the capillary;
Pump means for pumping the selected solvent to the second end of the capillary;
A controller connected to the pump and manifold and configured to control the transfer of the solvent to the capillary tube in a predetermined sequence and at a predetermined time;
A waste container for receiving waste liquid exiting from the first capillary end;
A capillary electrophoresis system comprising:

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