Abstract:
This relates to filter columns for isolating nucleic acids, particularly at small elution volumes. The filter column is adapted for stable placement within the upper portion of standard plastic collection tubes of various sizes. The body of the filter column has a number of surfaces to accommodate placement within variously sized collection tubes. The filter column contains nucleic acid-specific filter which can be located at alternate regions within the filter column, providing different filter surface areas and loading volume capacities using the same column body. The filter column has an opening on an upper end adapted to be sealed by a cap. A method for recovering nucleic acids using such filter column is also provided.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application is a continuation-in-part of U.S. Ser. No. 10/209,508, filed Jul. 30, 2002, which is a continuation-in-part of U.S. Ser. No. 09/882,530, filed Jun. 15, 2001, the entire contents of both of these patent applications are hereby incorporated herein by reference as if fully set forth herein. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     1. Field of the Invention  
         [0003]     This invention relates to filtration filter columns and methods of filtering therewith. More specifically, this invention relates to system and methods for isolating nucleic acids such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) from other materials such as enzymes, salts, buffers, small molecules, and cellular debris.  
         [0004]     2. Discussion of the Related Art  
         [0005]     Isolation and purification of nucleic acids play a central role in modern molecular biology, and increasingly in medicine. Both laboratory and diagnostic research require the use of nucleic acids in gene cloning and genetic analysis. Many of these techniques require keeping ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) pure and free of contamination. In many instances, the availability of small amounts of starting sample material poses a problem during isolation of the nucleic acid. The limited amount of sample material makes the need to limit loss of the sample material a critical concern.  
         [0006]     A known method for isolating nucleic acids from a small amount of starting material includes the use of a spin filter column (“filter column”) that contains a nucleic acid binding material (i.e., a filter). Examples of binding material/filters include silicas like glass powder, silica particles, glass microfibers, diatomaceous earth, etc. These filters are often associated with a “filter surface area.” This filter surface area is not limited to the surface area of a side of the filter. Instead, since the filters are usually comprised of microscopic fibers, particles, porous substances, etc., the filter surface area is actually defined by the surface area of the components, which comprise the filter. For example, a filter that comprises glass microfibers may have a filter surface area defined by the surface area of the microfibers within the filter (either all of the microfibers or a portion thereof).  
         [0007]     In some cases, filter columns may isolate nucleic acids directly from cells or biological tissue. In the first step a filter column is inserted into a microcentrifuge tube (e.g., a 1.5 ml tube) and a solution containing nucleic acids along with undesirable impurities is loaded into the top of the filter column. Depending upon the application, the starting material containing the nucleic acids is prepared from cells that have been treated with a disrupting solution causing the release of the nucleic acids. Alternatively, the nucleic acid solution is the product of an earlier reaction step. In either case, the nucleic acid binds to the filter column filter in the presence of a chaotropic agent. Then the filter column is centrifuged in a microcentrifuge. Centrifugation forces the solution through the filter column&#39;s filter and binds the nucleic acid to the filter. Next, the filter with the nucleic acids bound therein is washed by applying a washing solution to the top of the filter column and centrifuging again. After each wash the filter column can be removed from the collection tube so that the material in the collection tube may be removed. Finally, placement of an elution buffer (usually water having a specific pH) at the top of the column and applying centrifugation elutes the nucleic acid that is bound to the filter. Given the proper pH, the nucleic acid dissolves and elutes with the liquid into the collection tube. It is important to note that the volumes of the binding and wash solutions can be relatively large, thus necessitating the use of a larger (1.5-2.0 mL) tube. On the other hand, the volumes for elution are often smaller, making it desirable to have a smaller tube for the elution step. Eluting directly into a smaller tube allows one to proceed to the next reaction step in the small tube, rather than having to pipette out of the large tube. It is desirable to use a small tube in downstream processes. As discussed herein, use of a pipette is undesirable as it introduces the risk of loss of samples as well as contamination of the sample. Moreover, as described herein, there are additional benefits in keeping the sample solution in a smaller tube.  
         [0008]     Several companies provide kits that include filter columns designed to use this technique for isolating nucleic acids. QIAGEN, Promega, and Boehringer Mannheim GmbH offer filter columns based on the above-described principle. However, existing filter columns cannot be used interchangeably with collection tubes of different sizes. Instead, these previously known filter columns only fit into a single size collection tube (e.g., a standard 1.5-2.0 mL microcentrifuge tube.)  
         [0009]     This limitation presents a problem as many applications may benefit if a single filter column could be interchanged with collection tubes of multiple sizes. For example, given a small amount of nucleic acid in the starting material, it is best to elute the purified nucleic acid into a very small volume of fluid so that nucleic acid does not become too dilute in the fluid. Obviously, the resulting combination of elution buffer and nucleic acid will occupy a small volume. Many applications that require processing of nucleic acid may benefit when storing this small volume of material in a smaller sized collection tube. For instance, an application such as amplification of the purified DNA by polymerase chain reaction (PCR) requires placement of the nucleic acid into a thin-walled 0.5 mL or 0.2 mL microcentrifuge tube.  
         [0010]     Accordingly, it may seem ideal to use a smaller filter column, which is specifically designed to fit a 0.5 mL or 0.2 mL microcentrifuge tube. However, a significant drawback is that these smaller filter columns limit the amount of wash and binding solution that can be passed through the column with each bind/wash. This limitation necessitates additional wash steps and increased handling of the filter column and microcentrifuge tube, thus presenting an undesirable increased risk of contamination. Also, as discussed above, the requirement of large volumes of binding and wash solutions often necessitate the use of a larger collection tubes (e.g., a 1.5-2.0 mL microcentrifuge tube.)  
         [0011]     The remaining alternative is to use a filter column specifically designed to fit into a larger collection tube (e.g., a 1.5 mL-2.0 mL microcentrifuge tube.) While this alternative minimizes the additional wash steps and increased handling discussed above, the alternative presents additional problems. For instance, after purification, the nucleic acid solution must be eluted from the filter column into an appropriately sized 1.5 mL-2.0 mL microcentrifuge tube. As discussed above, many applications may benefit by storing the elution buffer and nucleic acid in a smaller sized microcentrifuge tube. Consequently, the elution buffer and nucleic acid must then be transferred (e.g., by aspiration) into a smaller (e.g. 0.5 mL) tube. Again, this extra transfer step introduces the undesirable potentials of contamination and loss of some of the nucleic acid.  
         [0012]     The present invention involves the elution of purified nucleic acids into small volumes (generally less than 20 microliters). The filters of the filtration columns described are adapted to optimally function with small elution volumes by minimizing fluidic holdup during the purification and elution procedures, and by tailoring the filter characteristics.  
         [0013]     In view of the above, there remains a need to use different-sized collection tubes with a single filter column. The ability to use different-sized collection tubes with a single filter column, particularly when dealing with small elution volumes, overcomes the problems associated with the existing art.  
         [0014]     The invention described herein addresses the problems discussed above. Moreover, the invention described herein allows centrifugation from one filter column into at least two distinct sizes of collection tubes. The disclosed invention may be used with commercially available collection tubes  
       BRIEF SUMMARY OF THE INVENTION  
       [0015]     According to one aspect of the invention, there is provided a filter column for isolating nucleic acids from a liquid sample. The filter column includes a body having a passageway extending therethrough. The body has a first end, a second end, an outer surface, an inner surface and a longitudinal axis. The outer surface has at least one bearing surface for seating on at least one collection tube. The inner surface has at least one angled surface located proximate or above the bearing surface. The angled surface forms an angle with the longitudinal axis that is less than approximately 90 degrees. A filter is disposed within the passageway.  
         [0016]     According to another aspect of the invention, there is provided a filter column that includes a body having a passageway extending therethrough. The body has an outer surface, an inner surface and a longitudinal axis. A filter is disposed within the passageway and the filter has a perimeter. A retainer is disposed within the passageway above the filter. The retainer has an outer edge, an inner edge, and an upper surface that is beveled.  
         [0017]     According to another aspect of the invention, there is provided a filter column that includes a body having a passageway extending therethrough. The body has an outer surface, an inner surface, and a longitudinal axis. A filter is supported within the passageway and the filter has a perimeter. At least a portion of the filter proximate to the perimeter is compressed relative to a portion of the filter that is inward from the perimeter.  
         [0018]     According to another aspect of the invention, there is provided a filter column that includes a body having a passageway extending therethrough. The body has an outer surface, an inner surface, and a longitudinal axis. A filter is disposed within the passageway and the filter has a perimeter. At least a portion of the filter has a greater density relative to another portion of the filter.  
         [0019]     According to another aspect of the invention, there is provided a filter column for isolating nucleic acids into solution of a preselected elution volume. The filter column includes a body having a passageway extending therethrough. The body of the filter column has a first end, a second end, an outer surface and an inner surface. At least one filter is disposed within the passageway. The filter has a wetting capacity that is approximately equal to the preselected elution volume.  
         [0020]     According to another aspect of the invention, there is provided a filter column for isolating nucleic acids into solution of a preselected elution volume. The filter column includes a body having a passageway extending therethrough. The body of the filter column has a first end, a second end, an outer surface and an inner surface. At least one filter is disposed within the passageway. The filter has a wetting capacity that is less than the preselected elution volume.  
         [0021]     According to yet another aspect of the invention, there is provided a method for isolating nucleic acid material. The method includes the step of providing a filter column that is comprised of a body having a passageway extending therethrough. The body has an outer surface and an inner surface. The filter column also includes at least one filter disposed within the passageway. The filter has a wetting capacity. The method also includes the step of providing a solution containing nucleic acid material. The solution containing nucleic acid is transferred to the filter of the filter column. The solution is transferred from the filter column. Elution buffer is added to the filter in an amount substantially equal to the wetting capacity of the filter. Nucleic acid is eluted from the filter column.  
         [0022]     According to another aspect of the invention, there is provided a filter column for isolating nucleic acids into solution of a preselected elution volume. The filter column includes a body having a passageway extending therethrough. The body of the filter column has a first end, a second end, an outer surface and an inner surface. A filter is disposed within the passageway. The filter has a shape that defines a volume. The filter is configured such that the volume defined by the shape of the filter is substantially equal to the preselected elution volume.  
         [0023]     According to yet another aspect of the invention, there is provided a method for isolating nucleic acid material. The method includes the step of providing a filter column that is comprised of a body having a passageway extending therethrough. The body has an outer surface and an inner surface. The filter column also includes a filter disposed within the passageway. The filter has a shape that defines a volume. The method also includes the step of providing a solution containing nucleic acid material. The solution containing nucleic acid is transferred to the filter. The solution is transferred from the filter column. Elution buffer is added to the filter in an amount substantially equal to the volume defined by the shape of the filter. Nucleic acid is eluted from the filter column.  
         [0024]     According to yet another aspect of the invention, there is provided a method for isolating nucleic acid material. The method includes the step of providing a filter column that is comprised of a body having a passageway extending therethrough. The body has an outer surface and an inner surface. The filter column also includes a filter disposed within the passageway. The method also includes the step of providing a solution containing nucleic acid material. The filter column is located in fluid communication with a first collection vessel. The solution containing nucleic acid is transferred to the filter. The solution is transferred from the filter column at a first speed followed by a second speed wherein the first speed is slower than the second speed. Elution buffer is added to the filter. Nucleic acid is eluted from the filter column.  
         [0025]     According to yet another aspect of the invention, there is provided a method for isolating nucleic acid material. The method includes the step of providing a filter column that is comprised of a body having a passageway extending therethrough. The body has an outer surface and an inner surface. The filter column also includes a filter disposed within the passageway. The method also includes the step of providing a solution containing nucleic acid material. The filter column is located in fluid communication with a first collection vessel. The solution containing nucleic acid is transferred to the filter of the filter column. The solution is transferred from the filter column. Elution buffer is added to the filter. Nucleic acid is eluted from the filter column at a first speed followed by a second speed wherein the first speed is slower than the second speed.  
         [0026]     According to another aspect of the invention there is provided a filter column. The filter column includes a body having a passageway extending therethrough. The body has a first end, a second end, an outer surface, and an inner surface. The outer surface includes at least two bearing surfaces for seating the filter column on collection vessels of at least two different sizes. A filter is disposed within the passageway and the cross-sectional area of the passageway is substantially constant. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0027]     A clear conception of the advantages and features constituting the invention, and of the components and operation of model systems provided with the invention, will become more readily apparent by referring to the exemplary, and therefore nonlimiting, embodiments illustrated in the drawings accompanying and forming a part of this specification, wherein like reference numerals (if they occur in more than one view) designate the same elements. Consequently, the claims are to be given the broadest interpretation that is consistent with the specification and the drawings. It should be noted that the features illustrated in the drawings are not necessarily drawn to scale.  
         [0028]      FIGS. 1A, 1B ,  1 C and  1 D illustrate variations of filter columns of the present invention appropriate for mating with collection tubes of varying sizes.  
         [0029]      FIG. 2A  illustrates a sectional view of a variation according to the invention.  FIG. 2B  illustrates a sectional view  200  of  FIG. 2A .  
         [0030]      FIGS. 3A and 3B  show perspective and cross-sectional views, respectively, of a variation of the retaining ring according to the invention.  FIG. 3C  shows an exploded view of the assembly of a filter, ring and support within the filter column of the invention.  
         [0031]      FIG. 4  illustrates a cut-away perspective view of a variation of a filter column of the present invention containing a filter, ring and support.  
         [0032]      FIG. 5A  illustrates a cross-sectional view of a filter column of the present invention removably seated in a first collection tube.  FIG. 5B  illustrates a sectional view  500  of  FIG. 5A .  
         [0033]      FIG. 6  illustrates a cross-sectional view of the filter column illustrated in  FIG. 5  removably seated in a second collection tube being of a different size than the first collection tube of  FIG. 5 .  
         [0034]      FIGS. 7A and 7B  illustrate additional aspects of the invention that may be combined with any variation of the invention singly or in combination.  
         [0035]      FIGS. 8, 9  and  10  illustrate a method of the present invention.  
         [0036]      FIG. 11  illustrates a filter column of the present invention for use with a carrier device.  
         [0037]      FIG. 12  illustrates a perspective view of the filter column of the present invention having vents located in the lid. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0038]     The following discussion of the variations of the invention and the reference to the attached drawings are for explanatory purposes and do not exhaustively represent the possible combinations and variations of the invention. Those skilled in the art will readily appreciate that many variations may be derived using the following description. The following examples are intended to convey certain principles of the invention. These examples are not intended to limit the scope of the claims to any particular example. It is understood that the claims are to be given their broadest reasonable interpretation in view of the description herein, any prior art, and the knowledge of those of ordinary skill in the field.  
         [0039]      FIG. 1A  shows a profile of a variation of the present invention. In this variation, the filter column  1  includes a passageway extending therethrough and a body portion that includes a first body portion  3 , a second body portion  5 , and a third body portion  7 . The filter column  1  also contains a first bearing surface  9  and a second bearing surface  11  located between body portions  3 ,  5 , and  7  as illustrated. The bearing surfaces are intended to permit placement of the filter column  1  in various collection vessels or tubes of at least two different sizes (e.g., see  FIGS. 5 and 6 ). Accordingly, the present invention contemplates that the bearing surfaces, either alone, or along with body portion(s) adjacent to a bearing surface, serve to provide stability of a filter column when seated within a collection tube. Such stable placement is necessary for the intended use of the filter column (e.g., during centrifugation, vacuum filtering, handling, adding/removing material, etc.) Furthermore, although bearing surfaces  9 ,  11  are located at the intersection of the respective body portions, in other variations of the invention the bearing surfaces are located anywhere along the various body portions. As illustrated in  FIG. 1B , the bearing surfaces  10 ,  12  are located along respective body portions of a filter column  1 . Additionally, while bearing surfaces  9 ,  11 , are illustrated as being tapered, the invention is not limited as such.  
         [0040]     Variations of the invention also include filter columns with more than three body portions as well as filter columns with one or more bearing surfaces. The variation of the spin-column depicted in  FIG. 1  further includes an outer rim  17  adjacent to a top  15  of the filter column  1 . The outer rim  17  may also provide an additional bearing surface  19  and may also aid in grasping and manipulating the filter column  1 . This rim is also used for mating with upstream devices that transfer solution to the invention such as an extraction device. The bottom of the filter column may be reduced in diameter  13  to assist in retaining a filter (not shown) within the filter column  1 .  
         [0041]     Another variation is shown in  FIGS. 1C and 1D . The filter column  100  includes an inner surface  102  and an outer surface  104 . A first body portion  103  and a second body portion  105  may also be defined. The filter column  100  includes at least one bearing surface  109  located between first body portion  103  and second body portion  105 . A second bearing surface  111  is located between the second body portion  105  and a rim  117  adjacent to the top  115  of the filter column  100 . As shown, the bearing surfaces  109  and  111  are formed in the outer surface  104 . The inner surface  102  does not have any angled surfaces. The inner surface  104  forms a passageway  121  that extends throughout the length, from the bottom  113  to the top  115  of the filter column  100 . The passageway  121  has a cross-sectional area that is substantially constant throughout the length of the passageway  121 ; however, the invention is not so limited. The inner passageway  121  may be tapered, include angled surfaces, and basically have a cross-sectional area that is not constant along the length of the passageway. As shown in  FIG. 1C , the filter column  100  is formed in one piece.  
         [0042]      FIG. 1D , illustrates a filter column  100  that is formed from two pieces-an inner portion  119  and an outer portion  120 . The outer portion  120  may be separable form the inner portion  119  in one variation. Together, the inner portion  119  and the outer portion  120  provide an outer surface  104  that includes at least one bearing surface. At least two bearing surfaces are included such that the filter column is adapted to seat on collection vessels of at least two different sizes. The filter column  100  of  FIG. 1D  includes a first body portion  103 , a second body portion  105  and a third body portion  107 . The filter column  100  includes at least one bearing surface  109  located between the first body portion  103  and the third body portion  107 . A second bearing surface  111  is located between the second body portion  105  and the rim  117  adjacent to the top  115  of the filter column  100 . A third bearing surface  121  is located between the third body portion  107  and the second body portion  105 . Of course, any number of body portions and/or bearing surfaces may be formed with the bearing surfaces being located anywhere along any body portion.  
         [0043]     It is noted that the body of the filter column may be adapted as required to accommodate any particular filtration process for example, centrifugation, vacuum filtering, or any known filtering process. For example, if a filter column of the present invention is intended for use with vacuum filtering, the filter column may also include a manifold to accommodate the vacuum. Such modifications are well known to those familiar with filter columns and methods of using such devices.  
         [0044]     The dimensions of the body portions  3 ,  5 ,  7 ,  103 ,  105 ,  107  are selected so that the filter column  1  may fit into various collection tubes that are sized for the respective body portion and bearing surface. Variations of the invention include sizing of a filter column to include body portions and bearing surfaces that accommodate both a 1.5-2.0 mL and a 0.5 mL microcentrifuge tube. An example of such tubes includes PGC Scientifics No. 16-8105-52 (1.5 mL) supplied by PGC Scientifics Corporation of Maryland, Eppendorf No. 22 36 430-8 (0.5 mL) supplied by Eppendorf AG of Germany, Gene Amp No. N801-0611 (0.5 mL) supplied by Perkin-Elmer Life Science of Massachusetts, Ciro Manufacturing Corporation of Pompano, Fla. (2.0 ml) and MJ Research, Inc. of Waltham, Mass. (0.5 ml). For example, the opening diameter for a 1.5 mL-2.0 mL microcentrifuge tube may range from 0.32-0.37 in. The opening diameter for a 0.5 mL microcentrifuge tube may range from 0.25-0.27 in. It is intended that, where appropriate, the definition of diameter of the filter column may include any such external feature as crush ribs, buttress, collar, or any other feature that may be present on a portion of a filter column body that is intended for insertion into a collection tube. The invention is not limited to filter columns or collection tubes having circular cross-sections.  
         [0045]     While the invention may be suited for use with the tubes described above, the invention is not limited to compatibility with such tubes. Moreover, although variations of the inventive device described herein are discussed for use with existing microcentrifuge tubes ranging in size from 0.5 mL capacity to 1.5-1.5 mL capacity, the invention is not limited as such. Instead, the inventive device may be applied to collection tubes as described herein, centrifuge tubes of any size, or any type of collection tube where a benefit from the improvements of the current invention are desirable. Furthermore, the invention may be adapted to accommodate any number of combinations of large and small collection vessels, including, but not limited to a filter column adapted to accommodate a large 0.5 mL tube and a small 0.2 mL tube, or a filter column adapted to accommodate a large 1.5 mL tube and a small 0.2 mL tube.  
         [0046]     The height of the filter columns of the present invention is selected so that the filter column along with the particular collection tube used will fit within the centrifuge apparatus (e.g., an Eppendorf 5415C centrifuge supplied by Eppendorf AG of Germany.) For example, for a 0.5 mL microcentrifuge tube to fit in the centrifuge previously listed, the height which protrudes from the 0.5 mL microcentrifuge tube (i.e., referring to  FIG. 1A , the height from the top of bearing surface  11  to the top of the device) must be below 0.625 in. preferably below 0.5 in. Of course, height is not a constraint when used with vacuum filtering.  
         [0047]     The filter columns of the present invention may be fabricated from materials readily known to those familiar with existing filter columns. Such materials include, but are not limited to polypropylene, polycarbonate, polyethylene, polyethylene terephthalate, fluoropolymers such as polytetrafluoroethylene and polyvinylidine flouride, polyarylene ether ketones, co-polymers and any thermoplastic or other commonly used material. It is often desirable to use a material, which is thermoplastic to allow molding of the columns. The columns can also be machined out of appropriate materials. In some cases, it is desirable to choose materials for the filter column, which permit sterilization and/or the removal of contaminants and harmful agents including the removal of nucleic acids and nucleases, thereby, allowing the filter column, filter, and sample to be nuclease-free.  
         [0048]      FIG. 2  illustrates the internal body structure of a variation of a filter column of the present invention. In particular  FIG. 2A , illustrates a cross-sectional view of a filter column  1  comprising a body having a passageway  21  extending therethrough. The body portion having an inner surface, an outer surface, a top end and a bottom end. In the variation shown in  FIG. 2A , the filter column  1  includes a first body portion  3 , a second body portion  5 , and a third body portion  7 . The body portions  3 ,  5 , and  7  are interconnected in a telescoping fashion such that the width of the first body portion  3  is greater than the width of the second body portion  5  which is greater than the third body portion  7 . As can be seen, body portions of different widths form bearing surfaces therebetween. The body portions together with the bearing surfaces enable the filter column to seat on collection vessels of different sizes. Still referencing  FIG. 2A , for example, the filter column mates with a larger first collection vessel which seats against the bearing surface located between the first body portion  3  and the second body portion  5  such that the second body portion  5  is securely located inside the larger second collection vessel in a friction-fit engagement. A snap-fit engagement, or adhesive may also be employed to secure the filter column to the collection vessel. The filter column also mates with a smaller second collection vessel which seats against the bearing surface located between the second body portion  5  and the third body portion  7  such that the third body portion  7  is located inside the smaller first collection vessel in a friction-fit engagement. Of course, the filter column can mate with a relatively large collection vessel which seats against the bearing surface located between the rim and first body portion  3  such that the first body portion  3  is securely located inside the collection vessel. The filter column may be secured to the collection vessel using any means known to one skilled in the art. Also, in one variation, the cross-sectional area of the passageway  21  at the first body portion  3  is greater than the cross-sectional area of the second body portion  5  which is greater than the cross-sectional area of the third body portion  7 .  
         [0049]     Liquid solutions may be loaded into the top of the filter column  15  and a lid may also be used. Typically, liquid solutions include the nucleotide-containing solution, wash or rinsing solutions and an elution buffer (e.g. water or tris/ethylenediamine-tetra-acetate (TE)). The nucleic acid solution can be from a lysate (e.g. isolated directly from cells) or nucleic acids from a reaction mixture. The nucleic acids from reaction mixtures could be from reactions such as PCR, DNA or RNA polymerization, reverse transcription, etc. Before being loaded into the filter column the nucleic acid solution is usually combined with a binding buffer containing a chaotropic agent to aid in binding the nucleic acid to the filter. The compositions of binding buffers, wash or rinsing solutions and elution buffers are well known in the field and can be found in U.S. Pat. No. 5,075,430 to Little, U.S. Pat. No. 5,808,041 to Padhye, et al., and U.S. Pat. No. 5,652,141 to Henco, et al., the entirety of each is hereby incorporated by reference herein.  
         [0050]     The variation of the invention depicted in  FIG. 2A  also illustrates a vent  29 . The vent  29  may be located along an interior surface of a passageway  21  of a filter column  1  but will be placed in fluid communication with an exterior of the filter column  1 . The vent  29  permits venting of pressure within the passageway  21  during placement of a lid (not shown) in the top opening  15  of the filter column  1 . Without a vent, the placement of a lid could increase pressure within the passageway  21  such that sample material is forced out of the bottom  13  of the filter column  1 . Although such displacement of the material may not have an effect on the function of a device of the present invention, such an occurrence may be undesirable. The device  1  may have any number of vents. These vents may be placed randomly or spaced evenly apart on a wall of a passageway. In one variation (not shown) four vents are placed at intervals of 90 degrees along the walls of the passageway. In another variation, a vent is placed in another location in the filter column  1  or even within a lid itself as described with respect to  FIG. 12 . In another variation, no vents are employed.  
         [0051]      FIG. 12  illustrates a perspective view of a filter column  130  according to the present invention wherein one or more vents  131  are located in the lid  132 . Vents  131  permit venting of air and prevent buffer, wash or elution fluid, for example, from being inadvertently pushed through the filter when the lid  132  is closed down onto the column. As shown in  FIG. 12 , the vent  131  has a curved surface  133 . Also, the vent  131  is tapered such that the vent  131  is narrower towards the top  134  of the lid  132 . These scalloped vents  131  that taper towards the top  134  of the lid  132  provide a more robust air and fluid barrier when the lid  132  is closed preventing fluid from spraying from, around, or through the lid  132 . This variation allows no fluid to exit the column after the lid  132  closes with a snap-fit bead or ridge  135  located on the passageway of the column  130 . Also, this variation makes manufacture of the filter column easier and less expensive because the injection molding tool does not have to accommodate a vent and a bead in the same location as required when the vent is formed in the passageway of the column. Therefore, the molding tool is easier to fabricate. Although four vents  131  are shown in the lid  132 , the invention is not so limited and any number of vents  131  can be included in the lid. Similarly, the invention is not limited to scalloped or tapered vents.  
         [0052]      FIG. 2B  shows greater detail  200  of the juncture between two body portions. The wall  201  of the second body portion  5  connects to the wall of the third body portion  7  by an intermediary wall  204 . The intermediary wall  204  has an outer surface  206  and an inner surface  210 . The outer surface  206  of this intermediary wall  204  forms a bearing surface  11  as previously described. The inner surface  210  of this intermediary wall  204  is angled with respect to body portions  5  and  7  such that fluid in the filter column does not pool on the inner surface  210  while the filter column is in a stationary vertical position or while the filter column is undergoing centrifugation. An angled surface within the passageway need not be located at the intermediary wall. And, as shown above with respect to  FIGS. 1C and 1D , an angled surface is not a necessary characteristic of the invention, as internal components, such as the filter and filter support, as well as external mating parts can be supported and/or retained by other methods such as with adhesive.  
         [0053]     A funnel angle for the angled surface  210  is defined as the angle between the longitudinal axis A of the filter column and the angled surface  210  in question. The funnel angle of the angled surface is less than 90 degrees. In another variation, not only the juncture  200  of body portions  5  and  7  form a funnel angle that is less than 90 degrees, but also, the funnel angle of all junctures of the filter column passageway through which fluid must flow is less than 90 degrees whether or not the angled surface is located at the intermediary wall. This design advantageously prevents the creation of a shelf that is a potential source of fluid holdup when force is applied to the filter column. If the funnel angle of any angled surface is 90 degrees or more, fluid can be trapped on the angled surface. The funnel angle of a surface indicates whether fluid will collect on the surface. Fluid holdup should be avoided because it reduces the efficacy of the filter column purification. Thus, the inner passageway is designed to prevent fluid holdup within the filter column.  
         [0054]      FIG. 2B  shows the funnel angle  202  for the inner surface  210  when the direction of force applied is parallel to the wall  201  of the second body portion  5 . As shown, the funnel angle  202  is approximately 45 degrees. This angle is less than 90 degrees required to prevent fluid holdup on this surface.  FIG. 2B  represents the case, of a filter column adapted for spinning in a microcentrifuge with a swinging-arm rotor so that the centrifugal force applied is substantially parallel to the walls of the second body portion. Alternatively, it would also apply if a vacuum or gravity were used to apply force to an upright filter column. Generally, the primary adaptation of the filter column is for it to be employed in a microcentrifuge with a rotor that holds the column at an angle of 45 degrees with respect to the direction of centrifugal force, and the depicted embodiment is designed for that case.  
         [0055]     Preventing fluid holdup is particularly critical when eluting from the filter column into a small volume (e.g., less than 20 microliters). Many microcentrifuges have fixed-angle rotors where the tubes are held at a forty-five degree angle. In this example, the angle between the angled inner surface  210  and the wall  201  of the second body portion  5  is less than 45 degrees, resulting in a funnel angle that is less than 90 degrees. For other microcentrifuges that do not hold the tubes at 45 degrees, the surfaces of the passageway through the filter column could be designed so that the funnel angle remains less than 90 degrees throughout the passageway of the filter column. In another variation, the funnel angle  202  is approximately 30 degrees at one or more junctures.  
         [0056]     As illustrated in  FIG. 2A , the filter column  1  of the present invention also allows for varying placement of a filter (not shown) within a passageway  21  of the filter column  1 . For example, placement of a filter in a bottom  23  of the second body portion  5  permits the filter column  1  to provide a certain volume capacity. The volume capacity of the filter column  1  can increase by changing the location of the filter to a bottom  25  of the third body portion  7 . Accordingly, the same filter column  1  body may be used for varying application and more than one filter may be disposed within the filter column. To maximize the volume of fluid that can be added to a filter column of the present invention, a filter can be located in the lowest chamber. Moreover, locating the filter towards a middle chamber reduces the volume of fluid that may be added but permits a larger filter surface area as the diameter of the middle chamber may be greater than the diameter of a lower chamber. For the filter, it is desirable to have a high surface area glass fiber filter (e.g., a borosilicate glass). The filter may be one that is adapted to isolate nucleic acids from a liquid sample by, for example, centrifugation, vacuum filtering, or any other filtering method. Surface area refers to the total surface area of all the fibers and not just the area of the disk. Moreover, filter columns of the present invention may have a microliter capacity greater than 200 microliters. Another variation of the filter column having a microliter capacity ranging between 50 microliters and 1000 microliters. The microliter capacity is defined by the volume within a passageway of the filter column that is above the filter and not occupied by other components such as a lid.  
         [0057]     The filter chosen can be silica or other types such as polymeric membranes, and may also contain other functional groups for purification of the nucleic acid such as ion exchange groups or groups which would specifically bind nucleic acid sequences. The structure and thickness of the filter will determine the filter wetting capacity. Wetting capacity is the amount of solution that can occupy the filter. For example, a filter made of glass fibers will have a wetting capacity approximately defined by the volume of space between the glass fibers. Thus, a rough approximation of the wetting capacity of a filter made of glass fiber could be determined by subtracting the volume of glass fiber from the total volume of the filter. The volume of the glass fiber can be determined by dividing the mass of the glass fiber filter by the specific gravity of the glass fiber. The wetting capacity can be determined by empirically measuring the amount of fluid a volume of filter material can hold. The wetting capacity is also called the void volume and other ways of measuring the void volume can be used to determine the wetting capacity. The quantitative definition of maximum wetting capacity is the void space within the filter. The void space within the filter can be calculated by taking the total space, both solid and void, that is circumscribed by the outermost fibers of the filter minus the solid space occupied by the filter fibers themselves. The space occupied by the filter fibers can be calculated by dividing the mass of the filter by the specific gravity of the fiber material. Other techniques known to one skilled in the art could also be used to determine wetting capacity and is within the scope of the present invention.  
         [0058]     Wetting capacity can be used to optimize the dimensions of the filter used in the filter column, particularly for low-volume purification. A mismatch between the wetting volume and the volume of solution applied to the filter can decrease the yield of recovery of purification using a filter. For example, eluting purified nucleic acid into a preselected small volume (20 mL or less) of elution buffer when the wetting capacity of the filter is greater than the elution volume could result in incomplete recovery of the purified nucleic acid material. Thus, the elution buffer will not contact all parts of the filter that have bound nucleic acid, and therefore not all of the bound nucleic acid will be eluted. Additionally, if the elution volume is much larger than the wetting capacity, the effective in-filter residence time of the fluid is decreased from 100%, down to the ratio of wetted fluid to total volume. It is desirable to substantially match the wetting capacity of the filter chosen to the elution volume that is selected for the processes to be performed. To recover purified material into a low-volume of elution buffer (e.g., 20 microliters) using the inventive filter column, the filter has a similarly low wetting capacity. In one variation, the wetting capacity of the filter is substantially equal to the volume of elution buffer that is employed. A precise match between elution volume and wetting capacity is not required as long as it is plus or minus 10% of the elution volume. In one variation, the filter is configured such that the volume, that includes the space of both void and solid fiber as defined by the shape of the filter, is substantially equal to the preselected elution volume. In yet another variation, the wetting capacity is between approximately 8 microliters and 12 microliters.  
         [0059]     In another variation, the wetting capacity is slightly less than the volume of elution buffer employed. In another variation the wetting capacity is approximately 66 percent to 100 percent of the elution volume. In another variation, the wetting capacity is approximately 50 percent to 100 percent of the elution volume. The wetting capacity of the filter column is easily modified by changing the dimensions of the filter, e.g. filter thickness, diameter, etc. If the filter is compressed, the wetting capacity is the resulting wetting capacity of the compressed filter.  
         [0060]     At low volumes it may also be difficult to completely wet the filter because the surface tension may inhibit absorption into the filter. This is particularly critical when using low volumes of elution buffer. One solution is to include surface tension reducing agents, such as detergents, in the elution buffer. For example, addition of 0.1% of the detergent Triton X-100 could be included in the elution buffer to help wet the filter when eluting purified nucleic acid.  
         [0061]     Securing of the filter may be accomplished, for example, by placement of a disk of porous substrate material (not shown) at approximately position  25  of  FIG. 2A . A filter membrane (not shown) is placed on top of the substrate material. Optionally, a retainer (not shown) is added such that there is an interference or adhesion or interlocking between the retainer and the wall of the passageway. Accordingly, the retainer secures the filter on the porous substrate. Another means of securing the filter is to mold an integral grating within the passageway to seat the filter.  FIG. 3C  illustrates placement of a filter using the porous substrate and a retainer in the shape of a ring.  
         [0062]      FIG. 3A  shows a perspective view of one embodiment of the retainer  33  of the present invention. The retainer  33  is shaped to substantially conform to the shape of the passageway. The retaining ring  33  includes an inner edge  302  and an outer edge  304 . The inner edge  302  of the retaining ring  33  is beveled. This beveling creates a funnel angle that reduces fluid holdup.  FIG. 3B  shows a side cross-sectional view of the retaining ring  33  of  FIG. 3A . The outer edge of the filter  304  is not beveled, and is designed to fit snugly against the walls of the passageway of the filter column. Alternately, the outer edge  304  may loosely fit and be attached to the inner wall of the passageway using an adhesive or other attachment means. As shown in  FIG. 3B , the inner edge  302  includes a lower surface  306  and an upper surface  308 . Both the lower  306  and upper  308  surfaces of the inner edge  302  of the retaining ring  33  are beveled in  FIG. 3B  such that the thickness of the retaining ring  33  decreases with distance from the outer edge towards the inner edge. The angle as well as the extent of this beveling can be varied resulting in a different funnel angle for the upper surface  308  of the retaining ring, or a different compression profile of the filter from the lower portion of the retaining ring. In one variation, the entire upper surface  308  of the retaining ring  33  is beveled, eliminating any shelf-like protrusion on which solution could collect while stationary or while undergoing centrifugation or vacuum filtering. The lower surface  306  may also have a similar beveled profile. In yet another variation, the funnel angle of the upper surface  308  is less than 90 degrees. Also, either one of the lower or upper surfaces may be beveled.  
         [0063]      FIG. 3C  shows how the retaining ring  33  inserts into the column filter in order to secure the filter in place.  FIG. 3C  illustrates an exploded view of a retaining ring  33 , filter  29  and filter support  31  fitting into one embodiment of the filter column  1 . The filter  29  is sandwiched between the retaining ring  33  and the filter support  31 , and all three fit into the filter column. The filter support  31  and the retaining ring  33  match the diameter of the portion of the filter column into which they will be inserted. In one variation, when the retaining ring  33  is inserted, it abuts the filter  29  acting to compress at least a portion of the filter  29  that is proximate to the outer perimeter relative to a portion of the filter that is inward from the perimeter. In another variation, the insertion of the retainer  33  acts to increase the density of at least a portion of the filter relative to another portion of the filter. If a retainer  33  as shown in  FIG. 3  is employed, the at least a portion of the filter of greater density is proximate to the perimeter of the filter relative to a portion of the filter that is inward from the perimeter. In one case, the top surface  28  of the filter  29  adopts a surface profile that is substantially complementary to the surface profile of the lower surface  306  of the retaining ring  33 . A beveled lower surface  306  of the retaining ring  33  results in a gradient of compression across the filter  29  such that the middle region of the filter is uncompressed or only slightly compressed relative to the edge regions of the filter that are more compressed. The compression of the filter decreases the available surface area and wetting capacity in that region. While not bound by theory, this compression is believed to increase capillary affinity at the compressed circumference of the filter, and therefore, help draw fluid into the filter and toward the edges. A retainer need not be employed to compress at least a portion of the filter.  FIG. 4  shows a filter column with the filter secured.  
         [0064]      FIG. 4  illustrates a cut-away profile view of a variation of a filter column  1  of the present invention. The illustration shows a filter  29  held in the lower portion of the filter column  1 . In this variation, the filter  29  is held between a porous support membrane  31 , which has the same diameter as the interior of the lower portion of the spin column  1 . In this variation, the support membrane  31  is retained in place by the shoulder formed by the reduced diameter of the bottom  13  of the device. The filter  29  may be fixed against the filter support membrane  31  by a retaining ring  33 , which fits securely against the inner wall of the lower portion of the filter column  1 . The present invention is not limited to the previous illustration. It will be apparent to those skilled with previously known filter columns to provide other means of retaining the filter within a filter column. An example of a filter for use with the invention includes a borosilicate glass fiber filter. The device may also contain filters of other types such as polymeric membranes, and may also contain other functional groups for purification of the nucleic acid such as ion exchange groups or groups which would specifically bind nucleic acid sequences.  
         [0065]     The porous substrate could be comprised of a material such as a sintered polyethylene or polypropylene as supplied by Porex of Fairburn, Ga. or GenPore of Reading, Pa. In some variations of the invention it is desirable that the porous substrate is comprised of a hydrophobic material in order to minimize holdup of the aqueous solution and to prevent fluid intended for residence in the filter from exiting the filter prematurely. It was found that pore sizes in the range of 1 micron to 150 microns are useful. When the filter column is used for very low volumes of solution (e.g. elution buffer) it is preferred that the surface of the filter support that contacts the filter be substantially smooth and free from excessive pits or cavities. Although the filter support must be porous, gaps in the interface between the filter support and the filter create regions where fluid can leave the filter, reducing the amount of contact between the fluid and the filter fibers. Generally, it is better to have a more uniform and therefore more substantially smooth surface for the filter support. A smaller average pore size usually results in a more uniform filter support surface. The primary function of the porous material is support for the filter. It is also possible to have the porous support as integral to the device. The porous support can also be in the form of an open grating. It is also possible to have a filter that is strong enough to support itself or is materially combined with support material.  
         [0066]      FIG. 5A  illustrates a variation of a filter column  1  of the present invention that is mated with a standard collection tube  35  of a first size (e.g., a 1.5 mL tube.) Standard collection tubes contain an integral body made of polymers such as polypropylene and have a standard inner diameter. As shown in  FIG. 5A , the second body portion  5  of the filter column  1  may fit securely against an inner wall of the collection tube  35 . The first bearing surface  9  may rest against a shoulder formed by a rim  37  of the collection tube  35 . Accordingly, when the tube  35  and filter column  1  spin in the centrifuge, liquid placed into the filter column  1  is forced through a filter  29  and collects in the collection tube  35 . In this variation, the sizing of the first body portion  5  along with the bearing surface  9  permits stable placement of the filter column  1  within in the collection tube  35 . After centrifugation, the filter column can be easily removed from the used collection tube, and placed into a new collection tube or placed back into the original tube.  
         [0067]      FIG. 5B  illustrates a view of section  500  of  FIG. 5A . A retaining ring  33 , filter  29  and a filter support  31  are disposed within a body portion  501  of the filter column  1 . The body portion  501  has a volume. The filter  29  is located between the filter support  31  and the retaining ring  33 . The top surface  28  of the filter conforms to the lower surface  306  of the retaining ring  33  such that the outer portion  502  of the filter  29  is more compressed relative to the inner portion  504  of the filter  29 . As can be seen in  FIG. 5B , the compression profile of the filter corresponds to the surface profile of the lower surface  306  of the retaining ring  33 . In one variation, the filter has a compression profile such that the compression of the filter gradually increases with distance towards the outer edge. The compressed filter defines a volume. In one variation, the compressed wetting capacity of the filter  29  is selected to be approximately equal to or slightly greater than the elution volume.  
         [0068]      FIG. 6  illustrates the filter column  1  of  FIG. 5  placed within a collection tube  39  (e.g., a 0.5 mL tube) that is smaller than the tube illustrated in  FIG. 5 . As illustrated in  FIG. 6 , the third body portion  7  of the filter column  1  fits securely into the inner walls of the collection tube  39 . During centrifugation, a bearing surface  11  of the filter column  1  seats against a rim  41  of the collection tube  39 .  
         [0069]      FIG. 7A  shows another variation of a filter column  42  of the present invention having features, which may be applied to any variation of the invention disclosed herein. In this variation the filter column  42  includes a lid  45  having a portion  47  which may be removably secured within a first end  49  of the filter column  42 . The lid  45  may assist in preventing contamination of the filter column  42  and any solutions loaded therein and may provide a surface for labeling. As shown, this variation of the filter column  42  contains a vent  27 . Although this variation depicts the lid  45  as being integral with the filter column  42  via a hinge  51 , the invention is not limited as such. For example, the present invention also includes a lid as being discrete from a filter column.  
         [0070]     As illustrated in  FIG. 7A , the variation of the inventive filter column also may include one or more snap-fit beads or ridges  53  to assist in retention of a lid  45 . In this variation the ridge  53  is included in the in a portion of a passageway in a first body portion  3  of the filter column  42 . As depicted in  FIG. 7A , the lid  45  and hinge  51  may be attached to the underside of an outer rim  55  (e.g., subflush to the rim  55 ) of the filter column  42 . This placement allows the outer rim  55  to be mated to other devices as required (e.g., a ExtracSure.™. sample extraction device useful for laser capture microdissection supplied by Arcturus Engineering of Mountain View, Calif. or other sample carrier).  
         [0071]     Another aspect of the invention depicted in  FIG. 7A  is deformable ribs  57 , which may be located along an outside surface of the filter column  42 . The deformable ribs  57  assist in securing the filter column  42  in a slightly larger diameter tube (e.g., a 2.0 mL tube) by increasing a diameter of the filter column  42  for a friction-fit engagement, for example. Accordingly, these ribs deform upon insertion of the filter column  42  into a tube having a diameter slightly larger than the corresponding body portion upon which the ribs  57  are situated. The number and design of the deformation ribs  57  may vary as needed, however, the ribs should be placed on a portion of the filter column that accommodates the varying sizes of tubes. The invention also contemplates deformation ribs  57 , which are either plastically or elastically deformable, or exhibit a limited degree of plastic or elastic deformation.  
         [0072]      FIG. 7B  illustrates another aspect of the invention, in which a filter column  59  contains at least one protrusion  61 , which extends radially away from the filter column  59 . Such protrusions  61  may serve as “finger grips” to increase the ease with which the filter column  59  may be manipulated.  
         [0073]      FIGS. 8-10  represent a flow diagram illustrating isolation of nucleic acids such as DNA or RNA from a biological material  80  using a filter column  86  of the present invention.  FIG. 8  represents the first step of placing a lysate  82  of biological material  80  in a collection tube  84 . This lysate  82  is spun to remove large lytic debris, and the “cleared” lysate is then applied to the top of a filter column  86  of the present invention.  
         [0074]     Alternatively, the nucleic acid containing solution does not have to come directly from lysis of biological materials. This method can also purify nucleic acids following nucleic acid amplification, enzymatic restriction digestion, ligation, extension and virtually any solution containing significant nucleic acids.  
         [0075]     Where small amounts of nucleic acid solutions are used, it is desirable to pre-wet the filter column with binding buffer before applying the nucleic acid solution. Alternatively, or additionally, the solution containing nucleic acid is transferred to the filter (wherein the nucleic acid material binds to the filter). This transfer may be performed at a first speed or rate employing any method known to one skilled in the art such as centrifugation or via vacuum. This binding step also includes a second step, which is performed at a high speed or rate of centrifugation, for example. Hence, solution is transferred from the filter column at a first speed followed by a second speed wherein the first speed is slower than the second speed. In one variation in which centrifugation is employed, the first slow speed is approximately 500 g or less, 1000 g or less, 1500 g or less, or 2000 g or less. This first step at a slow speed advantageously increases the resident time that the solution containing the nucleic acid material is in contact with the filter. The second relatively fast speed is approximately 10,000 g or more.  
         [0076]      FIG. 9A  illustrates the solution passing though a filter of the filter column  86  after centrifugation, during which the nucleic acids bind to the filter. The flow-through solution (from which the nucleotides have been mostly extracted) is retained at the bottom of a collection tube  88 . Generally, tubes  88 ,  84  are discarded after use. Upon removal of the filter column  86  from the tube  88 , the binding buffer (flow through) is discarded, and the filter column is replaced in a collection vessel (either a new tube or the same tube).  FIG. 9B  represents repeated washing of the filter column  86  with a washing buffer and centrifuging after each addition of washing solution into the top of the filter column. Once again the flow-through accumulated at the bottom of the tube  88  is discarded.  FIG. 10  represents the last step of transferring the filter column  86  of the present invention into a smaller 0.5 mL tube  92  and the addition of a small volume of elution buffer into the filter column  86 . Elution buffer is added to the filter in an amount substantially equal to the wetting capacity of the filter. Additional elution buffer may also be added to ensure that filter is not under-wetted. In one variation, additional elution buffer is added to the filter column in an amount that is between zero and approximately fifty percent of the wetting capacity of the filter. Alternatively, additional elution volume is added in an amount that is between zero and approximately 66% of the wetting capacity of the filter. In another variation, a volume of elution buffer is added to the filter column in an amount substantially equal to the volume defined by the shape of the filter. The volume defined by the shape of the filter includes the space occupied by both the void and solid filter fibers in a space circumscribed by the outermost fibers of the filter or approximately the volume of fluid that can occupy the space bounded by the bottom surface of the filter, the top surface of the filter, and the edges of the filter and/or the filter column walls. As a result of the addition of the elution buffer, the nucleic acids release from the filter into the elution buffer, which is centrifuged into the bottom of the 0.5 mL tube  92 . When small volumes of elution buffer (e.g., 20 microliters or less) are used, the step of eluting the material is performed in two parts. First, the elution step is performed at a low-speed followed by a second step having a slow speed relative to the first slow-speed step. The elution step may be performed employing any method known to one skilled in the art including centrifugation or via vacuum. Hence, nucleic acid material is eluted from the filter column at a first speed followed by a second speed wherein the first speed is slower than the second speed. If centrifugation is employed, the first slow speed is approximately 500 g or less, 1000 g or less, 1500 g or less, or 2000 g or less. The first slow-speed step advantageously ensures that the filter is completely bathed in the elution buffer. A second spin at a higher speed (relative to the first spin) of approximately 10,000 g or more is done to allow collection of the maximum amount of elution buffer.  
         [0077]     As shown in  FIGS. 8-10 , the inventive filter column permits use of more than one collection tube. All of the centrifugation steps can take place in a standard bench-top microcentrifuge (for example, Eppendorf 5415C), usually at accelerations less than 20,000 g.  
         [0078]     The biological material used with the filter column may also be provided by laser capture microdissection extraction device as described in U.S. patent application Ser. No. 09/844,187, entitled “LASER CAPTURE MICRODIS SECTION (LCM) EXTRACTION DEVICE AND DEVICE CARRIER, AND METHOD FOR POST-LCM FLUID PROCESSING,” the entirety of which is hereby incorporated by reference. As shown in  FIG. 11 , a filter column  1  attached to a LCM extraction device flange interface  94  which seats a laser capture microdissection extraction device  96 . The biological material will be located on a bottom surface of the extraction device  96 .  
         [0079]     While the present invention has been described with reference to one or more particular variations, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the present invention. Each of these embodiments and obvious various thereof are contemplated as falling within the spirit and scope of the claimed invention, which is set forth in the claims.