Patent Publication Number: US-2004043701-A1

Title: Plant-material sampling and grinding apparatus

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001] Nil  
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002] Not Applicable  
       BACKGROUND OF INVENTION  
       [0003] This invention is in the general area of ‘disposable laboratory plastic-ware.’ The invention is used for the sampling of a disc of plant material (predominantly leaf tissue) and subsequent processing for the preparation of DNA template for the Polymerase Chain Reaction (PCR). There is currently no product that facilitates the organized collection of plant material through to the production of PCR-ready DNA-template. Currently, there are systems for the efficient production of PCR-ready DNA-extract, but none incorporate sample collection and involve the purchase of grinding mills worth thousands of dollars.  
       BRIEF SUMMARY OF INVENTION  
       [0004] The invention, The Plant-material Sampling and Grinding Apparatus, facilitates the collection of plant material, through to the production of PCR-ready DNA-template. The invention facilitates the sampling and processing of the tissue within the one apparatus. The separate components of the invention include the upper and lower units. A piece of flat plant tissue is placed between the upper and lower units. Insertion of the upper unit into the lower unit cuts a disc of tissue. An extraction buffer can be introduced through the top of the upper unit and is contained by the well of the lower unit. Repeated manipulations involving the full insertion of the upper unit into the lower unit followed by incomplete separation of the two units, facilitates the grinding of the tissue in the extraction solution. Further solutions, such as neutralizing solutions, can be added through the top of the upper unit for the preparation of PCR-ready template.  
     
    
    
     BRIEF DESCRIPTION OF DRAWINGS  
     [0005]FIG. 1: Upper (A) and Lower (B) units of the Plant Material Sampling and Grinding Apparatus. Side view. Page 1/2.  
     [0006] 1) Top of upper unit possessing an opening to a cylindrical cavity.  
     [0007] 2) An indentation of variable shape in the top of the upper unit, for the introduction of a portion of a non-specified tool in order to secure the upper unit. The non-specified tool will facilitate insertion of the upper unit into the lower unit and relative movements between the upper and lower units.  
     [0008] 3) A rib with a sharp edge to aid in the removal of excess plant material.  
     [0009] 4) Lower cylindrical base of the upper unit that inserts into the upper cylindrical well of the lower unit.  
     [0010] 5) Holes of unspecified size, number, and location in the bottom of the cylindrical base of the upper unit.  
     [0011] 6) The bottom of the cylindrical base of the upper unit shown in conical form. Not all hidden detail is shown in order to reduce the complexity of drawing.  
     [0012] 7) A cutting edge on the bottom of the cylindrical base of the upper unit. Not all hidden detail is shown in order to reduce the complexity of drawing.  
     [0013] 8) Direction of insertion of the upper unit into the lower unit.  
     [0014] 9) Beveled edge on the upper cylindrical well of the lower unit.  
     [0015] 10) Outer wall of the upper cylindrical well of the lower unit.  
     [0016] 11) Inner base of the upper cylindrical well of the lower unit.  
     [0017] 12) Indentation to accommodate the cutting edge on the bottom of the cylindrical base of the upper unit.  
     [0018] 13) An indentation of variable shape in the middle section of the lower unit, for the introduction of a portion of a non-specified tool in order to secure the lower unit. The non-specified tool will facilitate insertion of the upper unit into the lower unit and relative movements between the upper and lower units.  
     [0019] 14) Cylindrical base of the lower unit.  
     [0020]FIG. 2: Plan view of the Upper unit of the Plant Material Sampling and Grinding Apparatus as illustrated in figure IA except a cutting edge on the bottom of the cylindrical base of the upper unit is not included in this plan. Page 1/2.  
     [0021] 2) An indentation of variable shape in the top of the upper unit, for the introduction of a portion of a non-specified tool in order to secure the upper unit. The non-specified tool will facilitate insertion of the upper unit into the lower unit and relative movements between the upper and lower units.  
     [0022] 3) A rib with a sharp edge to aid in the removal of excess plant material.  
     [0023] 4) Lower cylindrical base of the upper unit that inserts into the upper cylindrical well of the lower unit.  
     [0024] 5) Holes of unspecified size, number, and location in the bottom of the cylindrical base of the upper unit.  
     [0025] 6) The bottom of the cylindrical base of the upper unit shown in conical form. Not all hidden detail is shown in order to reduce the complexity of drawing.  
     [0026]FIG. 3: Plan view of the Lower unit of the Plant Material Sampling and Grinding Apparatus as illustraed in FIG. 1B except an indentation to accommodate a cutting edge on the bottom of the cylindrical base of the upper unit, is not included in this plan. Page 1/2.  
     [0027] 9) Beveled edge on the upper cylindrical well of the lower unit.  
     [0028] 10) Outer wall of the upper cylindrical well of the lower unit.  
     [0029] 11) Inner base of the upper cylindrical well of the lower unit.  
     [0030] 13) An indentation of variable shape in the middle section of the lower unit, for the introduction of a portion of a non-specified tool in order to secure the lower unit. The non-specified tool will facilitate insertion of the upper unit into the lower unit and relative movements between the upper and lower units.  
     [0031] 14) Cylindrical base of the lower unit.  
     [0032]FIG. 4: Upper (A) and Lower (B) units of the Plant Material Sampling and Grinding Apparatus as illustrated in FIG. 4A. Only basic features are included in this figure. Side view. Page 2/2.  
     [0033] 1) Top of upper unit possessing an opening to a cylindrical cavity.  
     [0034] 2) An indentation of variable shape in the top of the upper unit, for the introduction of a portion of a non-specified tool in order to secure the upper unit. The non-specified tool will facilitate insertion of the upper unit into the lower unit and relative movements between the upper and lower units.  
     [0035] 4) Lower cylindrical base of the upper unit that inserts into the upper cylindrical well of the lower unit.  
     [0036] 5) Holes of unspecified size, number, and location in the bottom of the cylindrical base of the upper unit.  
     [0037] 6) The flat bottom of the cylindrical base of the upper unit.  
     [0038] 8) Direction of insertion of the upper unit into the lower unit.  
     [0039] 10) Outer wall of the upper cylindrical well of the lower unit.  
     [0040] 11) Inner base of the upper cylindrical well of the lower unit.  
     [0041] 13) An indentation of variable shape in the middle section of the lower unit, for the introduction of a portion of a non-specified tool in order to secure the lower unit. The non-specified tool will facilitate insertion of the upper unit into the lower unit and relative movements between the upper and lower units.  
     [0042] 14) Cylindrical base of the lower unit.  
     [0043]FIG. 5: Plan view of the Upper unit of the Plant Material Sampling and Grinding Apparatus showing basic features only. Page 2/2.  
     [0044] 2) An indentation of variable shape in the top of the upper unit, for the introduction of a portion of a non-specified tool in order to secure the upper unit. The non-specified tool will facilitate insertion of the upper unit into the lower unit and relative movements between the upper and lower units.  
     [0045] 4) Lower cylindrical base of the upper unit that inserts into the upper cylindrical well of the lower unit.  
     [0046] 5) Holes of unspecified size, number, and location in the bottom of the cylindrical base of the upper unit.  
     [0047] 6) The flat bottom of the cylindrical base of the upper unit.  
     [0048]FIG. 6: Plan view of the Lower unit of the Plant Material Sampling and Grinding Apparatus showing basic features only. Page 2/2.  
     [0049] 10) Outer wail of the upper cylindrical well of the lower unit.  
     [0050] 11) Inner base of the upper cylindrical well of the lower unit.  
     [0051] 13) An indentation of variable shape in the middle section of the lower unit, for the introduction of a portion of a non-specified tool, in order to secure the lower unit, and facilitate relative movements between the upper and lower units through the action of the non specified tool.  
     [0052] 14) Cylindrical base of the lower unit.  
    
    
     DETAILED DESCRIPTION OF INVENTION  
     [0053] There is currently no product that facilitates the organized collection of plant material through to the production of Polymerase-Chain-Reaction (PCR) ready DNA template. Currently, there are systems for the efficient production of Polymerase-Chain-Reaction (PCR) ready DNA extract, but none incorporate sample collection and involve he purchase of grinding mills worth thousands of dollars. The system described here facilitates sampling and processing of the tissue within the one apparatus and does not require the purchase of expensive equipment.  
     [0054] The invention is the Plant-material Sampling and Grinding Apparatus. A description of the invention follows. The components of the invention include the upper and lower units (FIGS.  1  to  6 ). The dimensions and relative dimensions between sections of the invention are variable, however further comments regarding dimensions and relative dimensions will occur throughout the detailed description when important to unction or efficient use. The construction material is not specified but is likely to be a plastic that is rigid yet possessing some elastic qualities (flexible and returns to original shape after deformation) (e.g. polypropylene).  
     [0055] The lower unit consists of a lower cylinder (FIGS. 1B, 3,  4 B, and  6 ; point  14 ), a middle section possessing an indentation suitable for insertion of a non-specified holding and manipulating device (FIGS. 1B, 3,  4 B, and  6 ; point  13 ), and an upper portion that possesses an open cylindrical well (FIGS. 1B, 3,  4 B, and  6 ; point  10 ).  
     [0056] The upper section of the Plant-material Sampling and Grinding Apparatus (FIGS. 1A, 2,  4 A, and  5 ) consists of a partially closed cylindrical base (FIGS. 1A, 2, A, and  5 ; point  4 ) and an upper section possessing an indentation suitable for insertion of a non-specified holding and manipulating device (FIGS. 1A, 2,  4 A, and  5 ; point  2 ).  
     [0057] The cylindrical base of the upper unit (FIGS. 1A, 2,  4 A, and  5 ; point  4 ) inserts into the open cylindrical well of the lower unit (FIGS. 1B, 3,  4 B, and  6 ; point  10 ) as indicated in FIGS. 1 and 4 point  8 . The tightness of fit should create a seal but not so tight as to impede a smooth insertion or extraction of the upper unit.  
     [0058] A holding and manipulating device or grinding tool will facilitate the insertion of the base of the upper section into the open well of the lower section, and the extraction, or partial extraction, of the base of the upper section from the open well of the lower section. The tool is generally unspecified but could possess upper and lower two pronged forks that will insert into the indentations of the upper and lower sections of the Plant-material Sampling and Grinding Apparatus (FIGS.  1  to  6  points  2  and  13 ). The indentations could be one sided (semi-circular or possessing multiple curves); two sided (of equal or unequal sides), three sided as indicated in FIGS. 1B, 3,  4 B, and  6  (point  13 ) of equal or unequal size; or have multiple sides and angles. The forks of the tool should fit tightly enough to firmly hold the two units and ensure appropriate alignment for insertion, but be loose enough to also allow smooth removal of the joined units from the tool.  
     [0059] A disc of plant tissue will be cut by placing the tissue (e.g. leaf) between the upper and lower units, while each unit is held by the tool, followed by the insertion of the upper unit into the lower unit of the Plant-material Sampling and Grinding Apparatus. The insertion is facilitated by the action of the tool. Beveled edges could be included on the upper lip of the lower unit of the Plant-material Sampling and Grinding Apparatus to create a sharp edge to improve the cutting of the sample disc (FIGS. 1B and 3 point  9 ). The disc of plant material will be cut by the combined action of the cutting edges of the lower unit and the downward force of the base of the upper section (FIGS. 1A, 2,  4 A and  5 ; point  6 ) on the tissue sample (e.g. leaf).  
     [0060] Grinding or extraction solutions will be introduced through the opening of the upper unit (FIGS. 1A and 4A; point  1 ), freely move through the holes in the bottom of the upper unit (FIGS. 1A, 2,  4 A and  5 ; point  5 ), and be contained within the upper cylindrical well (FIGS. 1B, 3,  4 B and  6 ; point  10 ) of the lower unit.  
     [0061] Complete insertion of the upper section into the lower section grinds the sample between the base of the upper unit (FIGS. 1A, 2,  4 A, and  5 , point  6 ) and the base of the well of the lower unit (FIGS. 1B, 3,  4 B, and  6 , point  11 ). Several cycles of partial extraction and complete insertion would follow to complete the grinding process.  
     [0062] The grinding tool could have the general action of secateurs or forceps. The use of a grinding tool with secateur-like handles would be less strenuous when many samples are to be processed and allow more pressure to be applied. Options, for the grinding tool, could include;  
     [0063] i) Spring return handles;  
     [0064] ii) Pronged forks lower and parallel to main shaft to allow better access to Sampling and Grinding Apparatus when in a supporting plate;  
     [0065] iii) Multiple head tool;  
     [0066] iv) Locking device to ensure upper and lower sections remain together when grinding;  
     [0067] v) A tool where the focus of the arc of travel for the upper and lower components of the apparatus are behind the hand, thus reducing the curvature of travel.  
     [0068] The bottom of the upper section of the Sampling and Grinding Apparatus could be flat (FIGS.  4 A, and  5 ; point  6 ) or otherwise, such as angled away from the sides of the cylindrical wall to form a shallow cone (FIGS. 1A and 2; point  6 ). When an angled and flexible bottom of the upper section is lowered onto a disc of leaf material positioned on the bottom of the upper compartment of the lower section of the Sampling and Grinding Apparatus (FIGS. 1B, 3,  4 B and  6 ; point  11 ), the central region of the cone will make contact with the tissue first. As more pressure is applied the flexible base will be pushed flat, and (increasingly) more of the tissue will be squashed by the base of the upper-section. This process will facilitate more uniform grinding and better mixing with the grinding solution.  
     [0069] A sharp protruding lip could be included on the base of the lower cylinder of the upper unit of the Sampling and Grinding Apparatus to aid in the cutting of the plant tissue (FIG. 1A point  7 ). The lip would fit into a corresponding recess in the base of the well of the lower unit of the Plant-material Sampling and Grinding Apparatus (FIG. 1B point  12 ).  
     [0070] Sharp ribs (FIGS. 1A and 2; point  3 ) could be included on the upper section of the Plant-material Sampling and Grinding Apparatus, to help in the removal of excess plant material.  
     [0071] The suggested method of use is represented by the following points;  
     [0072] i) 96 Plant-material Sampling and Grinding Apparatus (upper section inserted into lower section) designed to fit into the wells of one or more commercially available, standard, 96-well microtiter-plates (apparatus supported by the insertion of the lower cylindrical base of the lower unit (FIGS. 1B, 3,  4 B and  6 ; point  14 ) into a well of the plate, the base of the lower unit should fit tightly into the wells but not so tight as to impede a smooth extraction, individual apparatus should not be touching, and most likely constructed of a rigid, yet possessing some elastic qualities, (flexible and returns to original shape after deformation) plastic (e.g. polypropylene)).  
     [0073] ii) 96 Plant-material Sampling and Grinding units (upper section inserted into the lower section) purchased pre-packaged and supported in a 96 well sampling plate (not a standard 96 well microtiter plate) by the insertion of the lower cylindrical base of the lower unit (FIGS. 1B, 3,  4 B and  6 ; point  14 ) into a well of the sampling plate. The base of the lower unit should fit tightly into the wells but not so tight as to impede a smooth extraction. The wells of the sampling plate could be sufficiently separated to allow easy access of the grinding tool to the Sampling and Grinding units. The sampling plate will have a lid. The plate with lid could slot onto a lower container. The three components would constitute the Sampling Container. The lower container could be filled with ice or a cold freezer block thus facilitating reduced sample deterioration. Alternatively, the Plant-material Sampling and Grinding units (upper section inserted into the lower section) could be supplied loose or in cassettes that could be stored in a holding rack for subsequent loading of the Sampling and Grinding units into the Sampling Plate and subsequent utilization of the Sampling Container.  
     [0074] iii) Take labeled Sampling and Grinding units, contained within the cooled Sampling Container, to the experimental plants. Sample leaf discs, and return apparatus to the wells of the sampling container. Return to the laboratory.  
     [0075] iv) An adaptor will be used to facilitate the addition, by multi-channel pipettor, of, for example, 200 micro liters of extraction solution (e.g. 0.25 N NaOH), into each Sampling and Grinding Apparatus. The adaptor will be placed on top of the sampling tray containing 96 apparatus. Tubes or tunnels passing through the adapter will insert into the opening in the upper unit (FIGS. 1A and 4A; point  1 ). The tubes or tunnels will lead from the openings in the top of the adapter to the apparatus in the sampling plate, maintaining spatial relationships. The openings will have the configuration of a standard 96-well microtiter-plate. Remove adaptor.  
     [0076] v) Each leaf sample will then be ground, with the aid of the grinding tool, until the solution has turned to a pale green color. The number of units ground simultaneously will depend on the number of holding forks on the head of the grinding tool.  
     [0077] vi) After grinding, Plant-material Sampling and Grinding units could then be transferred to a standard, commercially available, 96-well microtiter-plate, supported by the cylindrical base of the lower unit (FIGS. 1B, 3,  4 B and  6 ; point  14 ) within the wells of the plate.  
     [0078] vii) 50 micro liters, for example, of extract solution would then be removed from each unit and placed into the well of a standard, commercially available, 96 well microtiter plate that already contains, for example, 200 micro liters of neutralization buffer (e.g Tris buffer pH 7). Multi channel pipettors should be used if available.  
     [0079] viii) 2 micro liters of the neutralized extract could be used as template for a 20 micro liter PCR amplification.  
     [0080] ix) Alternatively, continuing from step 5, neutralization solution could be added individually to the original apparatus, either directly, or indirectly through the adaptor. It this case, PCR ready template would be produced in the original sampling apparatus.