Abstract:
The invention relates to a sample retrieval apparatus of particular benefit in the field of molecular biology. The apparatus permits the rapid collection of numerous specified fractions of samples: such as DNA, RNA or protein, that are separated by gel electrophoresis; or microorganisms grown on agar plates. The apparatus is capable of multiple sample retrieval from gels without cross-sample contamination from previously excised samples. Specifically, the sample retrieval apparatus is able to engage a cutting tip, cut a desired spot, band or plaque from a gel, deposit the desired band or plaque into a container, such as a multi-well plate for processing, and disengage the used cutting tip. The apparatus is then able to repeat this process many times to facilitate rapid and accurate processing of multiple bands or plaques from a single gel using different cutting tips for each sample retrieval.

Description:
FEDERALLY SPONSORED RESEARCH 
     This research is partially sponsored by: the National Institutes of Health, grant number R01-HG01724; and the Human Genome Institute. The United States Government may have rights in this invention. 
    
    
     CROSS-REFERENCE TO RELATED APPLICATIONS 
     Not Applicable. 
     BACKGROUND OF THE INVENTION 
     In the field of molecular biology, many tools have been developed for analyzing biomolecules. One such tool, electrophoresis, enables one to separate biomolecules, such as DNA, RNA and protein, based upon size, shape and/or charge. A mixture of biomolecules are placed in small wells in a gel, typically, a polyacrylamide gel, and a charge is applied which separates the biomolecules into discrete bands. The ability to separate biomolecules allows for the isolation and purification of many different biomolecule samples or bands in a single gel. Once isolated, a single band can be analyzed to determine a purified biomolecule&#39;s unique properties. In addition, isolation of purified biomolecules facilitates diagnosis of certain diseases and other biological abnormalities. 
     Isolation of the samples has had some shortcomings. One of these shortcomings is cross-sample contamination, which can occur when the same scalpel or razor blade is used to excise different biomolecules from the gel. To isolate samples, the area of the gel containing the desired band is cut with a scalpel or razor blade. Conventional gel cutting tools, such as the tool described in U. S. Pat. No. 5,587,062, do not provide for replacement of the cutting tool for each gel sample excised. Therefore, only one contamination-free sample can be excised. Subsequent samples can not be excised using the same cutting tool without contamination. 
     Agarose gels are used to grow bacteria and other microorganisms. An agarose plate is streaked, for example, with a sample to be tested. Microorganisms typically grow in plaques on the gel. The plaques can be excised from the gel. The contamination problems that exist with electrophoresis gels are also experienced with agarose gels. 
     As a result, there is a need for a gel cutting apparatus capable of multiple sample retrieval without cross-contamination from previously excised samples. 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention provides a sample retrieval apparatus that includes a support platform, a support translation member for moving the support platform to designated coordinates on X and Y axes, a cutter member, a cutter translation assembly for moving the cutter member to designated positions on a Z axis, the Z axis lying in a plane perpendicular to the support platform, and, a controller for designating the X, Y axis coordinates of the support platform and the Z axis positions of the cutter member. The support translation member has an elongate X rail lying on the X axis and an elongate Y rail lying on the Y axis. The X and Y rails each lie on a plane parallel to the other and are operatively connected to each other to permit relative travel of the X and Y rails along the length of the other. The support translation member also includes at least one motor for effecting such travel. 
     The support platform is preferably slidably mounted for travel along the length of the X rail and the X rail is slidably mounted for travel along the length of the Y rail. Alternatively, the support platform is slidably mounted for travel along the length of the Y rail and the Y rail is slidably mounted for travel along the length of the X rail. 
     The cutter member preferably includes a cutter tip having a proximal engagement end and a distal cutting and retrieval end. The cutter tip defines a passage therethrough. A plunger having a proximal engagement portion is preferably provided for slidable movement through the passage of the cutter tip. 
     The cutter translation assembly preferably includes a motor and a shaft operatively connected to the motor for movement along the Z axis. The shaft has an engagement sleeve for releasable engagement of the engagement end of the cutting tip. A coupling member is received within the sleeve for releasable engagement with the engagement portion of the plunger. 
     In an alternative embodiment, the cutter tip does not include the plunger and the plunger forms part of the cutter translation assembly. 
     In the preferred embodiment, the distal cutting end of the cutter tip is configured for cutting into a section of gelatinous material for retrieving and holding the section of material. There are preferably a plurality of disposable cutter tips with associated disposable plungers. Alternatively, the cutter tips and associated plungers may be reusable following appropriate cleaning or sterilization. 
     The support platform is configured to hold at least one gelatinous sample container, at least one array of cutter members and at least one transfer container. The configuration may take the form of clasps to hold the various elements in place, precisely configured depressions or raised boundaries for receiving the various elements. 
     An imager is provided for providing images of samples positioned on the support platform in the sample containers. A display unit is preferably provided for receiving and displaying images of the samples from the imager. The controller, which is preferably a computer in communication with the support translation member and the cutter translation assembly, may have an input device for selecting sample sections represented by the sample images displayed on the display unit that are to be retrieved and a memory and execution tool for determining the X, Y coordinates of the selected sample section on the support platform, determining the distance between the sample X, Y coordinates and moving the X, Y coordinates of the point of intersection between the Z axis and the support platform, and moving the support platform that distance such that the selected sample is aligned with the Z axis and moving the cutter member along the Z axis into position for cutting and retrieving the selected section of the sample. 
     The present invention also provides a method for retrieving one or more selected samples from a gel. The method may include positioning at least one gel containing samples of interest, a plurality of cutter tips and at least one receiving container on a support platform; taking an image of the gel and displaying the image; and selecting a sample from the displayed image of the gel for retrieval. Thereafter, the support platform is moved to first coordinates along an X axis and a Y axis to place the one of the plurality of cutter tips into alignment with a cutter member. The cutter member is moved into contact with the cutter tip to attach it to the cutter member. The cutter member and the now attached cutter tip are moved out of contact with the plurality of cutter tips. The method continues by moving the support platform to second coordinates along the X and Y axes to place the selected sample into alignment with the cutter member, moving the cutter member along a Z axis into contact with the selected sample, piercing the portion of the gel containing the selected sample with the cutter tip, and withdrawing the cutter member to retrieve the selected sample. Then, the support platform is moved to third coordinates along the X and Y axes to place a receiving container in alignment with the cutter member. The selected sample is released to the receiving container. Where retrieval of additional samples is desired, the method further includes releasing the cutter tip following release of the selected sample and repeating the foregoing steps for each of a desired number of different samples using a different one of the plurality of cutter tips for each sample retrieval. 
     The invention is particularly useful in the field of molecular biology for rapidly collecting numerous specified fractions of samples of biomolecules, such as nucleic acid fragments, DNA, RNA or protein, that are separated by gel electrophoresis; or microorganisms, such as bacteria or yeast that is grown on agarose plates. The apparatus of the present invention is capable of multiple sample retrieval from such gels without cross-contamination from previously excised samples. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a perspective cut away view of the sample retrieval apparatus of the present invention. 
     FIG. 2 is a side view of the gel cutting assembly used in the sample retrieval apparatus of FIG.  1 . 
     FIGS. 3 (a)-(e) illustrates the sequence of operations involved in excising a selected sample from a gel. 
     FIG. 4 is a schematic of the gel imaging and cutting routine where one cutting tip is used to make one cut per sample. 
     FIG. 5 is a schematic of the gel imaging and cutting routine where one cutting tip is used to make multiple cuts per sample. 
     FIG. 6 is a schematic of the gel imaging and cutting routine where different sized cutting tips are used to make one cut per sample. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     An embodiment of the sample retrieval apparatus  10  is shown in FIG.  1 . The apparatus shown includes two light-tight housing structures  12  and  14  which house, respectively, a support platform  24  and a support translation member  16  in the base housing  12  and a gel imaging system  20  and a cutter assembly  18  in the upper housing  14 . 
     The housing structures  12 ,  14  may be constructed of any suitable material for light-tight imaging, such as a metal, black acrylic, or other plastics. While shown in upper and base housing structures, those skilled in the art will recognize that any housing will suffice. The housing structures may be modular or may form a unitary structure. A single housing, not separated into apparent sections may also be used. 
     With reference again to FIG. 1, the base housing structure  12  has an access door  40  for inserting and removing the support platform  24  and its contents. The support platform  24  may be constructed of any suitable material. In a preferred embodiment, the support platform  24  is constructed of black acrylic. The support platform  24  is preferably configured to hold one or more containers  34  of sample containing gelatinous materials, at least one cutting tip rack  30 , and at least one receiving container  32 . In a preferred embodiment, the support platform  24  is compartmentalized so as to have either preformed depressions or raised walls outlining where the gel  34 , cutting tip rack  36  and receiving container  32  are positioned. Any suitable receiving container will suffice, such as a multi-well processing plate, a rack of one or more test tubes (not shown), or another gel container, depending on the nature of the sample and the desired further use thereof following retrieval. In use, the gel  34 , the cutting tip rack  30  and receiving container  32  are positioned on the support platform  24 . The gel  34 , rack  30  and container  32  are preferably removable and are positioned on the support platform  24  when the apparatus  10  is being prepared for use. In an alternative embodiment, the rack  30  may be fixed to the support platform  24 . 
     The support platform  24  is able to move in two dimensions along an X axis and a Y axis through its connection to the support translation member  16 . The translation member  16  includes an X axis rail  26  and a Y axis rail  28 . In a preferred embodiment, the Y axis rail  28  rests on top of, and is slidably attached to, the X axis rail  26  while the support platform  24  rests on top of, and is slidably attached to, the Y axis rail  28 . The Y axis rail  28  moves along the X axis rail  26  in the direction of the X axis to designated X axis coordinates. The support platform  24  moves along the Y axis rail  28  in the direction of the Y axis to designated Y axis coordinates. Alternatively, the positions of the X and Y axis rails  26 ,  28  may be reversed. The movement is effected by motors  21  and  22  which are in communication with and controlled by a control device, such as computer  58 . Each motor is able to move the support translation member  16  in one of the X axis or Y axis directions. Alternatively, a single motor capable of moving the support translation member  16  in both the X axis and Y axis directions may be used. In one embodiment, the support translation member  16  is connected via the motor  22  to a port in a workstation or other compatible system, such as a Silicon Graphic Inc. workstation, by means of serial communication, for example through an RS232 connector. In use, as the support platform  24  moves in either the X axis and/or Y axis directions, gel  34 , cutting tip rack  30  and receiving container  32  all move together as a unit. 
     The upper housing structure  14  contains a cutter assembly  18  and a gel imaging system  20 . The gel imaging system  20  includes a camera, filters and light source. The system  20  images the gel  34  so the user can determine which spots, bands or plaques should be excised. The gel imaging system  20  can use many different kinds of light and in multiple wavelengths, including ultraviolet, visible and far red, depending on the stain used in the gel. By imaging the gel  34  in multiple wavelengths, the gel&#39;s image may be projected to a display device  50 , such as a computer monitor. 
     In a preferred embodiment, the gel imaging system  20  includes a CCD camera and filter wheel. Two fiber optic cables connected to two 100W quartz-tungsten-halogen lamps are fitted with the motorized filter wheels. In addition, a video camera may be connected to a fiber optic scope aimed at the cutter assembly  18 , and in particular, at the cutter member  38  to allow the user to monitor the process without opening the housing and exposing the interior to outside light. In the preferred embodiment, the CCD camera is connected to a computer  58  via the SCSI port. The filter wheel controllers are connected to computer  58  via a parallel port. Those familiar with computer design and architecture will understand that other means of communication between the elements of the imaging system and the computer or other control or display device may be substituted. 
     The upper housing structure  14  also contains the cutter assembly  18 , as shown in FIG.  2 . The cutter assembly  18  includes a cutter member  38  and a cutter translation assembly  42 . The cutter member  38  includes cutting tips  36  and, in the preferred embodiment, an associated plunger  70 . The cutting tip  36  has a proximal engagement end  46  and a distal cutting and retrieval end  48  and defines a passage  44  therethrough. The plunger  70  is slidably mounted in the passage  44  and has a proximal engagement portion  49 . 
     The cutter translation assembly  42  is mounted to the upper housing structure  14  in such a manner that it is capable of moving in a third dimension, along a Z axis. The cutter translation assembly  42  includes a motor mount  62  fixedly attached to the upper housing structure  14 , a motor mount sleeve  64  attached to the motor mount  62 , and a linear stepping motor  60  which is fixedly attached to the motor mount sleeve  64 . The motor mount sleeve  64  is moveably attached to the motor mount  62  such that the sleeve  64  passively travels or slips a desired distance along the Z axis. 
     In the preferred embodiment, the distance of travel or slippage of the sleeve  64  in the mount  62  is approximately ¼ inches. Greater or lesser distances may be used to accommodate the dimensions of the overall apparatus design. Attached to the bottom surface of the linear stepping motor  60  is a hollow tip holding sleeve  66 . The linear stepping motor  60  and its attached tip holding sleeve  66  move along the Z axis, which movement is controlled, in one embodiment, by means of an operative connected to a computer  58  via a serial connection, such as an RS232 connector. Other suitable known means of control will suffice. 
     Inside the hollow tip holding sleeve  66 , the linear stepping motor  60  drives a drive shaft  72  which pushes a spring-loaded coupling shaft  74  in the Z′ axis direction. The drive shaft  72  and the spring-loaded coupling shaft  74  can be one or two pieces, so they may or may not be threaded together. The tip holding sleeve  66  engages the proximal engaging end  46  of cutting tips  36 , which fit snugly onto the end of the tip holding sleeve  66 . The coupling shaft  74  has a knurled end  76  which is inserted into a slot  68  in the proximal end  49  of a plunger  70 . 
     The spring-loaded coupling shaft  74  acts as a piston to move the plunger  70  in the Z′ axis direction. The plunger  70  may be fixedly attached to coupling shaft  74  or form a contiguous structure with shaft  74  provided the plunger  70  does not contact the samples in use. However, in the preferred embodiment, the plunger  70  and a cutting tip  36  form the cutter member  38 . The plunger  70  and the cutting tip  36  are preferably disposable, but may be reusable after cleaning. The plunger  70  is proportioned to fit and slide freely within an axial passage  44  through the cutting tip  36  while still maintaining a fit that is tight enough to create suction. The spring-loaded coupling shaft  74  moves the plunger  70  upwards and downwards, such that the plunger  70  travels freely inside the cutting tip  36 . In one embodiment, the motor controller for the coupling shaft  74  is operatively connected to a computer  58  through a second port via a serial connection, such as an RS232 connector. Any suitable known device for controlling the movement of the coupling shaft  74  and plunger  70  will suffice. 
     The apparatus  10  is also comprised of a display device  50 , an input device  52 ,  54 ,  56  and a control device  58  as shown in FIG.  1 . The display device  50  is used for displaying an image of the sample, such as spot, band or plaque patterns on the gel  34 . The input devices  52 ,  54  or  56  allow the user to choose specific spots, bands or plaques of interest for excision. For example, the computer mouse  54  may be used to highlight and click on the selected sample. Alternatively, an electronic pen  56  may be used to circle the selected sample. The computer keyboard  52  may be used to key in coordinates matching the location of the selected sample on a grid overlay or cursor keys may be used to identify the selected sample. In yet another embodiment, the selection of the sample from the image may be automatic by means of software programmed to recognize and select specified spots, bands or plaques on a gel. 
     The control device  58 , controls both the support platform  24  and the cutter translation assembly  42  so as to cause the cutter translation assembly  42  to move into the correct position to excise the desired spots, bands or plaques from the gel  34 . The input device, such as a computer mouse  54 , allows the user to select a desired spot, band or plaque by “clicking” on the spot&#39;s, band&#39;s or plaque&#39;s image while displayed on the display device  50 . The control device  58 , a computer workstation or other suitable system, employs software to interpret the input data received from the input device and controls both the support platform  24  and the cutter member  38  so as to cause the cutter member  38  to excise the selected spots, bands or plaques from the gel  34 . 
     The control device  58  is programmed to “know” where the gel  34 , cutting tip rack  30  and container  32  are located. Because the support platform  24  has specific locations for placement of the gel  34 , rack  30  and receiving container  32 , the relative distance between locations on those items and a starting point can be determined. The program recognizes starting, or home, X, Y coordinates. The program “knows” where the individual cutting tips  36  in the rack  30  and the individual wells in the container  32  are located by use of delta values to represent the distance between the starting X, Y coordinates and the X, Y coordinates of the consecutive cutting tips  36  and positions in the receiving container area. For example, the positions of the center of each well in a standard sized container are preprogrammed. Similarly, the distance between cutter tips in the rack, the location of differently sized tips and the rows of tips or other alignment of tips in the rack can be determined and preprogrammed. The delta values used to determine the X, Y coordinates of the selected gel sample may be, for example, based on pixels in the image of the gel. When a selection of a portion of the image representative of a sample on the gel is made, the distance in pixels in the image translates into discrete distance units on the actual gel and is converted into corresponding X, Y coordinates to pin the location of the sample on the support platform. The computer  58  then directs the movement of the platform  24  to the designated X, Y coordinates. The relative heights of the items on the platform  24  along the Z axis and the Z′ axis and the delta values between a withdrawn position and an engaged position are also preprogrammed. 
     The sample retrieval apparatus  10  operates as follows. First, the user places the gel  34 , cutting tip rack  30  and receiving container, for example, the multi-well processing plate  32 , on the support platform  24 . The gel  34  will usually be a polyacrylamide electrophoresis gel but can also be an agar plate used to grow microorganisms or any other kind of gel or material of similar consistency. Next, the user images the gel  34  in multiple wavelengths using the gel imaging system  20 . To determine the proper exposure conditions, the support platform  24  is moved to predefined X, Y coordinates to position the gel  34  under the camera. When the conditions suitable for creating acceptable images are determined, the camera and filter are set to the appropriate settings. Because the CCD field-of-view is much smaller than the area of the gel, a composite set of tiled images is recorded so that the entire gel is imaged. In the tiling routine, the CCD camera scans back and forth across the gel  34  starting from one corner and moving systematically across and down the entire gel to the diagonal corner until images of the entire gel have been taken and stored. The tiled images are computationally assembled to create one complete image of the gel  34 . One composite image is created for each dye-labeled sample on the gel  34 . The gel&#39;s image is displayed on the display device  50  and the user can select a portion of the sample, such as the spots, bands or plaques of interest, based on visual inspection using one of the input devices  52 ,  54  or  56 . The selection can also be based on the results of commercially available automated protein difference detection software. 
     As shown in FIGS. 1 and 2, in order to engage a cutting tip  36 , the control device  58  directs the movement of the support platform  24  from its home position to a first X, Y coordinate position such that the tip holding sleeve  66  is aligned above the first available cutting tip  36  in the rack  30 . The tip holding sleeve  66  is then lowered and the linear stepping motor  60  drives the drive shaft  72  which then moves the spring-loaded coupling shaft  74  downward to engage the plunger  70 . The assembly is then raised to remove the cutting tip  36  from the rack  30 . The spring-loaded coupling shaft  74  then moves the plunger  70  down the passage  44  of the cutting tip  36  to give a 1 to 2 mm gap between the end of the plunger  70  and the tip of the cutting tip  36 . 
     Next, the control device  58  directs the movement of the support platform  24  to another X, Y coordinate to position the selected spot, band or plaque of the sample in alignment with the cutter member  38 . At this time, the user may wish to inspect the position of the cutting tip  36  relative to the selected sample by using the video camera. If the alignment was not correct, adjustments can be made by means of a manual adjustment of the support platform  24  or, preferably, by adjusting the origin parameters through the computer program control. If positioned correctly, the cutter member  38  with tip  36  attached is then lowered along the Z axis until the disposable cutting tip  36  rests on the gel  34 . The cutter member  38  operates as shown in FIG. 3 to use one tip  36  to make one cut in a gel  34  per sample. The weight of the cutter member  38  causes the motor mount sleeve  64  to slip against the motor mount  62  so that the cutting tip  36  slices into the gel  34 . The cutting tip  36  has a negative bevel to allow for easier excision of spots, bands or plaques from the gel  34 . After the tip  36  slices into the gel  34 , the plunger  70  is withdrawn along the Z′ axis enough to create a suction to hold the excised gel section within the passage  44 . The cutter member  38  is then raised to remove the selected spot, band or plaque from the gel  34 . 
     The control device  58  then directs the movement of the support platform  24  to third X and Y coordinates to position the cutter member  38  above the first available well in the multi-well processing plate  32  or other receiving container for transfer of the selected sample. The wells in the multi-well processing plate  32  are preferably pre-filled with solution required for processing of the sample. The cutter member  38  is then lowered along the Z axis to position the cutting tip  36  over the surface of the processing solution and the spring-loaded coupling shaft  74  extends the plunger  70  along the Z′ axis within the passage of the cutting tip  36  to eject the selected sample in the gel fragment into the processing solution. Following ejection of the sample, the cutter member  38  is raised. 
     Next, the control device  58  directs the movement of the support platform  24  to the first X, Y coordinates to position the cutting tip  36  above the location in the rack  30  from which it was originally taken. The cutter member  38  is then lowered and deposits the tip  36  back into the rack  30  before being raised to its initial position. The control device  58  then directs the movement of the support platform  24  to its original starting position. The foregoing sample retrieval procedure is repeated as many times as desired. As a result, the sample retrieval apparatus  10  is able to retrieve multiple samples from molecular biology gels without cross-contamination from previously excised samples. 
     Another embodiment of the invention allows for single cuts to be made in a gel  34  using a plurality of different size cutting tips  36  in order to excise different size spots, bands or plaques in the gel  34 . A schematic of the gel imaging and cutting routine used in this embodiment is shown in FIG.  6 . The different size cutting tips  36  are all designed with the same size proximal engaging ends  46  to fit on the same tip holding sleeve  66 . The difference between this embodiment and the general embodiment shown in FIG. 4 is that this embodiment has the additional step of allowing the user to select the size of the cutting tip  36  using the input device  52 ,  54  or  56 . Preferably, however, the size of the cutting tip  36  will be automatically calculated by the computer  58  based on the size of the sample selected. There may be several pre-set sizes for circles surrounding a selected sample defining for example, small, medium and large or extra large protein spots. Each circle size corresponds to a different size cutting tip  36 . The tip rack  30  is filled with the different sized tips  36  in predefined positions within rack  30 . 
     A further embodiment of the invention allows for a single cutting tip  36  to make several cuts in a large band or plaque in order to excise the entire band or plaque before exchanging tips and subsequently excising a new band or plaque. A schematic of the gel imaging and cutting routine used in this embodiment is shown in FIG.  5 . The difference between this embodiment and the general embodiment shown in FIG. 4 is that this embodiment has the additional step of calculating the number of cuts (n) needed to excise the entire spot, band or plaque and then repeating the cutting process n times before ejecting the cutter tip  36 . 
     Although the foregoing invention has been described in detail using illustrations and specific commercial components for clarity, it will be apparent to persons having ordinary skill in the art in light of the detailed description and drawings, that certain changes and modifications may be made thereto without departing from the spirit and scope of the invention.