Abstract:
A system and method for manipulating magnetic particles in a solution to separate nucleic acid molecules from cell components in a cell solution. The system and method employ a device capable of receiving a plurality of tubes, each of which contain respective sample and paramagnetic particles. The device includes heating and cooling devices to facilitate a lysing step to release the nucleic acid molecules from the cells in the cell solution. The device further includes moveable magnets which can be moved proximate to and away from the tube to hold the paramagnetic particles to which the nucleic acid molecules become bound, so that the molecule-bound particles can be separated from the remainder of the solution, and washed as appropriate. The system also employs an electromagnet which is capable of demagnetizing the particles to allow the particles to freely mix with solution, such as elution solutions which are used to unbind the molecules from the particles.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention:  
           [0002]    The present invention relates to a system and method for manipulating magnetic particles in a fluid sample to efficiently and effectively collect DNA or RNA that has been bound to the particles. More particularly, the present invention relates to a system and method employing movable magnets for holding and releasing magnetic particles in a fluid sample so that DNA or RNA bound to the magnetic particles can be separated from the fluid sample.  
           [0003]    1. Description of the Related Art:  
           [0004]    A variety of molecular biology methodologies, such as nucleic acid sequencing, direct detection of particular nucleic acids sequences by nucleic acid hybridization, and nucleic acid sequence amplification techniques, require that the nucleic acids (DNA or RNA) be separated from the remaining cellular components. This process generally includes the steps of collecting the cells in a sample tube and lysing the cells with heat and reagent which causes the cells to burst and release the nucleic acids (DNA or RNA) into the solution in the tube. The tube is then placed in a centrifuge, and the sample is spun down so that the various components of the cells are separated into density layers within the tube. The layer of the nucleic acids can be removed from the sample by a pipette or any suitable instrument. The samples can then be washed and treated with appropriate reagents, such as fluorescein probes, so that the nucleic acids can be detected in an apparatus such as the BDProbeTec®ET system, manufactured by Becton Dickinson and Company and described in U.S. Pat. No. 6,043,880 to Andrews et al., the entire contents of which is incorporated herein by reference. Although the existing techniques for separating nucleic acids from cell samples may be generally suitable, such methods are typically time consuming and complex. Furthermore, although the centrifuging process is generally effective in separating the nucleic acids from the other cell components, certain impurities having the same or similar density as the nucleic acids can also be collected in the nucleic acid layer, and must be removed from the cell sample with the nucleic acids.  
           [0005]    A technique has recently been developed which is capable of more effectively separating nucleic acids from the remaining components of cells. This technique involves the use of paramagnetic particles, and is described in U.S. Pat. No. 5,973,138 to Mathew P. Collis, the entire contents of which is incorporated herein by reference.  
           [0006]    In this technique, paramagnetic particles are placed in an acidic solution along with cell samples. When the cell samples are lysed to release the nucleic acids, the nucleic acids are reversibly bound to the paramagnetic particles. The magnetic particles can then be separated from the remainder of the solution by known techniques such as centrifugation, filtering or magnetic force. The magnetic particle to which the nucleic acids are bound can then be removed from the solution and placed in an appropriate buffer solution, which causes the nucleic acids to become unbound from the magnetic particles. The magnetic particles can then be separated from the nucleic acids by any of the techniques described above.  
           [0007]    Examples of systems and method for manipulating magnetic particles are described in U.S. Pat. Nos. 3,988,240, 4,895,650, 4,936,687, 5,681,478, 5,804,067 and 5,567,326, in European Patent Application No. EP905520A1, and in published PCT Application WO 96/09550, the entire contents of each of said documents being incorporated herein by reference.  
           [0008]    Although the paramagnetic particle technique is very effective in separating and harvesting nucleic acids from cell samples, a need exists for an improved technique for manipulating the paramagnetic particles to provide an even more effective method of separation.  
         SUMMARY OF THE INVENTION  
         [0009]    An object of the present invention is to provide an improved system and method for manipulating paramagnetic particles to which nucleic acid molecules are bound in a solution to effectively separate the nucleic acid molecules from the remaining components of the solution.  
           [0010]    A further object of the present invention is to provide a system and method that is capable of altering the temperature of a cell solution to perform a lysing technique which enables nucleic acid molecules to become bound to paramagnetic particles in the solution, as well as being capable of manipulating the paramagnetic particles to appropriately separate the nucleic acid molecules from the remaining components of the solution.  
           [0011]    A further object of the present invention is to provide a system and method for use in a nucleic acid assay preparation system, that is capable of heating and cooling sample solutions as appropriate to perform a lysing technique, and which is further capable of manipulating paramagnetic particles to which nucleic acid molecules of the lysed cell samples become bound, so that the assay preparation system can properly wash the nucleic acid molecules and place the nucleic acid molecules in a sample assay.  
           [0012]    These and other objects are substantially achieved by providing a system and method for manipulating nucleic acid molecule-bound paramagnetic particles in a sample solution to separate the molecules from the remaining components in the solution. The system and method includes a tube receiver for receiving at least one sample tube containing a cell solution, paramagnetic particles such as iron oxide particles, and an acidic solution. The tube receiver is adapted for use with a system for preparing nucleic acid assays. The tube receiver includes a heating and cooling unit, such as a thermoelectric element, which is capable of heating the cell solution to lyse the cell and enable the nucleic acid molecules to become bound to the paramagnetic particles. The thermoelectric elements can also be used to quickly cool the solution as necessary. The tube receiver further includes movable magnets which can be moved proximate to the outer wall of the tubes to attract the molecule-bound paramagnetic particle to the sides of the tubes, while the assay preparation system removes the remainder of the cell solution and washes the particles. The movable magnets can then be moved away from the tubes so that the molecule-bound paramagnetic particles are released from the walls of the tubes, so that the assay preparation system can eject an elution reagent, such as a suitable buffer solution, which causes the nucleic acid molecules to become unbound from the paramagnetic particles. The tube receiver further includes electromagnets which are activated to provide a magnetic field to the tubes to degauss the paramagnetic particles to allow the paramagnetic particles to mix with the elution reagent. The movable magnets can then be moved proximate to the sample tubes to adhere the paramagnetic particles to the walls of the sample tubes while the assay preparation system aspirates the nucleic acid molecules from the sample tubes. The assay preparation system can then place the nucleic acid molecules in the appropriate microtiter trays for reading by an assay reading system. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0013]    These and other objects, advantages and novel features of the invention will be more readily appreciated from the following detailed description when read in conjunction with the accompanying drawings, in which:  
         [0014]    [0014]FIG. 1 is a diagram of an example of a nucleic acid assay preparation system employing a nucleic acid molecule extractor according to an embodiment of the present invention;  
         [0015]    [0015]FIG. 2 is a perspective view of the nucleic acid molecule extractor shown in FIG. 1;  
         [0016]    [0016]FIG. 3 is a top view of the nucleic acid molecule extractor shown in FIG. 2;  
         [0017]    [0017]FIG. 4 is a exploded perspective view of an example of a tube rack used with the nucleic acid molecule extractor shown in FIGS.  1 - 3 ;  
         [0018]    [0018]FIG. 5 is a detailed view of an example of the shape of one of the openings in the tube rack shown in FIG. 4;  
         [0019]    [0019]FIG. 6 is a cross-sectional view of the nucleic acid molecule extractor taken along lines  6 - 6  in FIG. 3;  
         [0020]    [0020]FIG. 7 is a detailed view of the portion of the nucleic acid molecule extractor designated in FIG. 6;  
         [0021]    [0021]FIG. 8 is a exploded perspective view showing an example of the relationship between the tube blocks, electromagnets and thermoelectric devices included in the nucleic acid molecule extractor shown in FIGS.  1 - 3 ,  6  and  7 ;  
         [0022]    [0022]FIG. 9 is a side view of the electromagnet printed circuit board shown in FIG. 8;  
         [0023]    [0023]FIG. 10 is diagrammatic view illustrating the relationship of the fixed side and sliding cam of the nucleic acid molecule extractor shown in FIGS.  1 - 3 ,  6  and  7  when the movable magnets are positioned as shown in FIGS. 6 and 7;  
         [0024]    [0024]FIG. 11 is a diagrammatic view illustrating the relationship between the fixed side and sliding cam of the nucleic acid molecule extractor shown in FIGS.  1 - 3 ,  6  and  7  when the magnets are being moved in a downward direction away from the tubes; and  
         [0025]    [0025]FIG. 12 is a diagrammatic view illustrating the relationship between the fixed side and sliding cam of the nucleic acid module extractor shown in FIGS.  1 - 3 ,  6  and  7  when the movable magnets are positioned at the downward most position away from the tubes. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0026]    [0026]FIG. 1 illustrates a sample assay preparation system  100  for which a nucleic acid molecule extractor  102  is adapted for use. The system  100  includes a robot  104 , such as a robot manufactured by Adept Corp. of San Jose, Calif., or any other suitable robot. The robot includes a pipette holding mechanism  106 , which can releasably couple to a plurality of pipette tips (not shown) stored in pipette tip racks  108 . The robot  104  further includes a suction mechanism (not shown) that can be activated to create a vacuum in tubing  110  to draw fluid into the pipette tips, or to create pressure in tubing  110  to eject fluid from the pipette tips for reasons discussed in more detail below.  
         [0027]    As further shown in FIG. 1, a plurality of sample input tubes  112  in a sample tube holder are positioned at a predetermined location with respect to the area of movement of the robot  104 . In addition, bulk reagent containers  114 , which include different reagents as discussed in more detail below, and a plurality of microtiter trays  116  are located at predetermined position with respect to the robot  104 .  
         [0028]    Further details of the extractor  102  are shown in FIGS.  2 - 9  as will now be discussed. The extractor  102  includes a removable rack  118  into which can be placed a plurality of tubes  120  containing paramagnetic particles such as those described in U.S. Pat. No. 5,973,138 referenced above. The extractor  102  further includes fixed sides  122  and cam plates  124  which extend parallel or substantially parallel to fixed sides  122  as shown. The extractor further includes a stepper motor  126  connected to a lead screw  128  which is controlled by a controller (not shown) of the system  100  to slide the cam plates  124  with respect to the fixed sides  122  for reasons discussed in more detail below. As shown, in particular, in FIG. 3, the extractor  102  includes a home sensor  130  that is connected to the controller (not shown). The home sensor detects the position of a home flag  132  to indicate to the controller the position of the cam plates  124  with respect to the fixed sides  122  for reasons discussed below.  
         [0029]    As discussed above, the extractor  102  includes and is adaptable for use with a rack  118 , the details of which are shown with more specificity in FIGS. 4 and 5. In particular, the rack  118  includes a bottom  134  and a top  136 . The bottom  134  includes a plurality of legs  138 , a handle  140  and a plurality of openings  142  therein. As shown in FIG. 5, the openings  142  include edges  144  which are configured to engage with projections  146  on the exterior of the tubes  120  to prevent the tubes  120  from rotating within the openings  142  when, for example, a cap (not shown) is being screwed onto a top of the tube  120 .  
         [0030]    As further shown in FIG. 4, the bottom  134  of rack  118  includes two openings, each having a press-in nut  148  inserted therein. Each nut receives the threaded portion of a captive thumb screw  150  which secures the top  136  of the rack  118  to the bottom  134  after the tubes  130  have been inserted into the opening  142 . The top  136  abuts against a shoulder  152  which is positioned proximate to the tops of the tubes  120 , and thus prevents the tubes  120  from falling out of the rack  118 , or being inadvertently lifted out of the rack by the pipette tips discussed above, when the robot  104  is adding or removing solution to and from the tubes  120 .  
         [0031]    Further details of the extractor  102  are shown in FIGS.  6 - 9  as will now be described. As illustrated, the extractor  102  includes a plurality of heat sink blocks  154  disposed between the fixed sides  122  and thus, in the interior of the extractor  102 . In this example, the extractor includes six heat sink blocks  154 . The heat sink blocks are supported by a base plate  156  of the extractor  102  as shown, in particular, in FIG. 6. Each fixed side  122  includes a cam slot  158  which extends in a vertical or substantially vertical direction. The cam slots receive shoulder screws  160  (see FIGS. 2 and 3) which pass through cam slots  162  (see FIG. 2) and to respective cam slots  158 . As described in more detail below, each pair of shoulder screws  160  (two aligned shoulder screws on opposite sides of the extraction  102 ) are coupled to a respective magnet carrier  164  to which is mounted a permanent magnet  166 . In this example, the extractor  102  includes seven pairs of shoulder screws  160  and seven corresponding magnet carriers  164  and magnets  166 . As discussed in more detail below, when the stepper motor  126  which is connected to the motor mount  125  and the cam plates  124 , moves the cam plates  124  in a horizontal or substantially horizontal direction with respect to the fixed sides  122 , the cam slots  162  force the shoulder screws  160  to move in a vertical direction along the fixed cam slots  158  and therefore raise or lower the magnet carriers  164  and their respective magnets  166  for reasons discussed below.  
         [0032]    As further illustrated in FIGS. 6 and 7, a thermoelectric device  168  is mounted to the top of each of the respective heat sink blocks  154 . A respective tube block  170  is positioned on the top of each of the thermoelectric devices  168  as illustrated.  
         [0033]    As further shown in FIGS. 8 and 9, each respective tube block  170  includes a plurality of openings  172 , which are each adapted to receive a respective tube  120 . Also, in this example, three thermoelectric devices  168  are associated with each tube block  170  and therefore, three thermoelectric devices are mounted on the top of each respective heat sink block  154 . The thermoelectric devices  168  can be controlled to apply heat to tube block  170  or to extract heat from tube  170 , as can be appreciated by one skilled in the art, under the control of the controller (not shown). Each tube block  170  also has a resistive temperature device (RTD) sensor  174  for sensing the temperature of the tube block and providing a signal to the controller so that the controller can appropriately control the thermoelectric devices  168 .  
         [0034]    As further illustrated, each tube block  170  has a slotted opening  176  into which is received an electromagnet circuit board  178  having a plurality of electromagnets  180  mounted thereon. The electromagnets  180  each include a preform coil  182  surrounding an electromagnetic core  184 , and are coupled in series to PCB traces  186 , which are coupled via connection pads  188  to the controller (not shown). As discussed in more detail below, the controller applies a current to electromagnets  180  which causes the electromagnets to generate an alternating current (AC) magnetic field.  
         [0035]    As further shown in FIGS. 6 and 7, the adjacent tube blocks  170  are spaced at a sufficient distance to allow magnet carriers  164  and permanent magnets  166  to slide proximate to the tube openings  172  and therefore proximate to the tubes  120  for purposes discussed in more detail below. In this example, each tube block  170  includes tube rows, each having eight openings  172 . The extractor  102  includes six tube blocks  170 . Thus, the extractor  102  includes  96  openings  172 .  
         [0036]    The operation of the extractor  102  with respect to the system  100  will now be described with reference to FIGS.  1 - 3 ,  6 ,  7  and  10 - 12 . Initially, samples containing cells are provided in sample input tubes  112 . These samples may be of any type, including biological fluids such as blood, urine and cerebrospinal fluid, tissue homogenates and environmental samples, that are to be assayed for nucleic acids (DNA or RNA) of interest. The robot  104  is first controlled to move to the pipette tip racks  108  to pick up a plurality of pipette tips, for example, four pipette tips (not shown). The robot  104  is then controlled to position the pipette tips over a respective number of sample tubes  112  and draw the samples into the respective pipette tips. The robot then moves the pipette tips over to the extractor  102 , and releases the samples into respective sample tubes  120  that have been loaded in advance into the rack  118  positioned on the extractor  102 .  
         [0037]    Each sample tube  120  has been previously supplied with paramagnetic particles. Although any type of paramagnetic particle may be used, including particles having polymeric coatings, the particles disclosed in U.S. Pat. No. 5,973,138 referenced above are preferred. Each of the sample tubes  112  also has lyse solution which lyses the cell samples.  
         [0038]    The above process continues until all of the samples from the sample input tubes  112  have been inserted into the corresponding tubes  120  in the extractor  102 . It is noted that the number of samples drawn at each time (i.e., four samples in this example) can vary as desired. It is also noted that each time the robot draws its samples from sample tubes  112  into pipette tips and then dispenses those samples into corresponding tubes  120 , the robot moves to a discard position to discard the pipette tips. The robot  104  then selects four new pipette tips to transfer four new samples from the input tubes  112  to the tubes  120 .  
         [0039]    Once all of the samples have been loaded into the respective sample tubes  120 , the controller controls the thermoelectric devices  168  to apply heat to the solutions in the tube  120  to lyse the samples. Once the lysing has been completed, the controller controls the thermoelectric device  168  to extract heat from the tube blocks  170 , the sampling tubes  120  and the solutions contained therein, to cool the solutions to substantially room temperature.  
         [0040]    Once the lysing and cooling processes are completed, the robot  104  is controlled to transfer a suitable acidic solution, such as that described in U.S. Pat. No. 5,973,138, into the sample tubes  120 . To do this, the robot  104  moves back and forth between the pipette tip racks  108 , the bulk reagent containers  114 , extractor  102 , and the pipette disposal section (not shown) to transfer the acidic solution to, for example, four tubes  120  at a time. The robot  104  transfers acidic solution to four corresponding tubes  120  and mixes the solution in the tubes  120  by drawing the solution into the pipette tips and discharging the solution back into the tubes  120  in a controlled manner, while raising and lowering the pipette tips into and out of the tubes  120  in a controlled manner to maintain minimum tip submersion.  
         [0041]    Also at this time, the controller controls the electromagnets  178  to generate an AC magnetic field, which demagnetizes the particles  190  so that the particles can freely mix with the acidic solution. Once the robot  104  has transferred acidic solution to four corresponding tubes  120  and has performed the mixing operations, the controller turns off the electromagnets to remove the AC magnetic field. The acidic solution that has been added to the cell sampling tube  120  causes the nucleic acid molecules to become bound to the paramagnetic particles  190 . Once the acidic solutions have been added to the samples in the sample tubes  120 , the controller controls the stepper motor  126  to move the cam plates  124  in a direction indicated by arrow A in FIG. 10. This drives the shoulder screw  160  in an upward direction along fixed cam slots  158  so that the magnets  164  are positioned proximate to the tubes  120 . Therefore, the molecule-bound particles  190  become adherent to the sides of the tubes  120  as shown, for example, in FIG. 7.  
         [0042]    The robot  104  is then controlled to use the pipette tips to remove the solution from the tubes  120  and discard the solution in a waste container (not shown). As in the operations discussed above, each time the robot  104  uses pipette tips to remove solution from respective tubes  120 , the robot  104  discards the pipette tips and uses new pipette tips before repeating the process on the remaining tubes  120 .  
         [0043]    The robot  104  is then controlled to add a washing solution to each of the tubes  120 . When the wash solution is being added to the tubes  120 , the controller controls the cam plates  124  to move in the direction indicated by arrow B in FIGS. 11 and 12, which causes the shoulder screws  160  to drive the magnet carriers  164  and, hence the permanent magnets  166 , in a downward direction in their respective fixed cam slots  158 . When the magnets  166  are moved away from the tubes  120 , the particles  190  are allowed to fall back into the bottoms of the tubes  120 . At this time, the controller controls the electromagnets  178  to generate an AC magnetic field, which demagnetizes the particles  190  so that the particles can freely mix with the wash solution being added to the tubes  120 . A rapid sequence of 5 aspirate and dispense cycles is used to perform the mix the particles with the wash solution. Once the robot  104  has completed mixing the wash solution, the controller turns off the electromagnets to remove the AC magnetic field.  
         [0044]    After the wash solution has been added and mixed with the particles, the controller controls the stepper motor  126  to move the cam plates  124  in the direction along arrow A shown in FIG. 10, to drive the magnets  166  in the upward direction to be proximate to the tubes  120 . The magnets  166  thus secure the molecule-bound particles  190  to the sides of the tube again as shown in FIG. 7. The robot  104  is then controlled to use the pipette tips (not shown) to remove the wash solution from the tubes  120 . This wash step may be repeated as many times as necessary to wash the particles, e.g., two times.  
         [0045]    The robot  104  is then controlled to add an elution reagent, such as those described in U.S. Pat. No. 5,973,138 referenced above, to the tubes  120 . The elution solution causes the molecules to become unbound from the particles  190 . In a manner similar to that described above, the robot  104  uses new pipette tips for each group of tubes  120  to which the elution solution is being added from the bulk reagent tank  114 .  
         [0046]    After the elution solution has been added to and mixed within all of the tubes  120 , the stepper motor  126  is controlled to move the cam plates  124  along direction A, as shown in FIG. 10, to move the magnets  166  proximate to the tubes  120 . The robot  104  is then controlled to use the pipette tips to transfer the elution solution containing the nucleic acid molecules that have been released from the particles  190  into the microtiter trays  116 . As with the operations described, the robot  104  uses fresh groups of pipette tips to transfer each group of sample to the respective wells and the microtiter trays  116 . Once all the samples have been transferred, the microtiter trays  116  can be placed in a suitable reading device, such as the BDProbeTec®ET system described above. In an alternative embodiment, microtiter trays  116  can be configured on a conveyer and conveyed automatically into the BDProbeTec®ET system.  
         [0047]    Although only one embodiment of this invention has been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiment without materially departing from the novel teachings and advantages of this invention. All such modifications are intended to be included within the scope of this invention as defined in the following claims.