Abstract:
A device for nucleic acid preparation includes an outer housing and an inner housing carried within the outer housing and rotatable with respect to the outer housing. The inner housing defines an inner chamber and includes inlet and outlet ports and a nucleic acid extraction substrate within the inner chamber. The outer housing includes a sample inlet port, wash fluid inlet and outlet ports, and elute fluid inlet and outlet ports, and the ports of the inner housing can be selectively aligned with ports of the outer housing by rotating the inner housing with respect to the outer housing. By aligning one or both ports of the inner housing with the appropriate ports of the outer housing, sample material can be introduced into the chamber and wash and elution fluids can be passed through the chamber. Nucleic acid from the sample introduced into the chamber is captured on the extraction substrate and is released from the extraction substrate by sonication prior to being eluted out of the chamber.

Description:
[0001]    This application claims the benefit of Provisional Patent Application Ser. No. 60/867,693, filed on Nov. 29, 2006, which is incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field Of The Invention 
         [0003]    The present invention relates to devices in which sample preparation procedures are performed, and, more specifically, sample preparation devices in which nucleic acid is extracted from biological samples. 
         [0004]    2. Discussion of Related Art 
         [0005]    Purified nucleic acids (DNA and RNA) from tissue samples such as human blood, serum, or saliva can be prepared by first lysing cells by chemicals (chaotropic salts, detergents, and/or strong base) or physical energy such as sonication, and then purifying the nucleic acids from other cellular components in the cell lysate. Historically, organic extraction (e.g., phenol:chloroform) followed by ethanol precipitation was used to purify the nucleic acids. This extraction method has been largely replaced by solid phase extraction methods that do not use phenol or chloroform. Various matrices have been used for solid phase extraction, most commonly silica particles. Nucleic acids bind to silica in the presence of high concentrations of chaotropic salts (Chen and Thomas, 1980; Marko et al. 1982; Boom et al. 1990). These salts are then removed with an alcohol-based wash and the DNA eluted in a low ionic strength solution such as TE buffer or water. The binding of DNA to silica seems to be driven by dehydration and hydrogen bond formation, which competes against weak electrostatic repulsion (Melzak et al. 1996). Hence, a high concentration of salt will help drive DNA adsorption onto silica, and a low concentration will release the DNA. 
         [0006]    Recently, new solid phase materials for DNA purification have been developed which take advantage of the negatively charged backbone of DNA to a positively charged solid substrate (under specific pH conditions), and eluting the DNA using a change in solvent pH, such as, for example, Invitrogen&#39;s charge switch technology (See United States Patent Application Publication No. 2006-0024712 A1). Whatman has an alternate technology (FTA paper, Whatman plc, Kent, UK) that utilizes a cellulose based solid substrate impregnated with a lysis material which lyses cells, inactivates proteins, and captures DNA in the cellulose fibers, where it is retained for use in downstream applications (See U.S. Pat. No. 6,322,983 B1). 
         [0007]    Typically, the above nucleic acid extraction methods are carried out in tubes or microwell plates, either by manual process or by automated processes. Pipetting devices are used to transfer the various liquids utilized in the process and centrifugation can be used to collect the solid phase materials. Alternatively, magnetized solid phase materials can be used, enabling the collection of the solid phase material by use of a magnet. 
         [0008]    Existing devices for automated solid phase extraction of nucleic acids are robotic pipetting or vacuum systems utilizing open tubes which are easily contaminated when processing multiple samples. Thus, there is a need for a device that integrates sample collection, storage, and processing within the same container to prevent contamination, carryover, and errors in sample handling throughout the multiple step process of nucleic acid purification. 
       SUMMARY OF THE INVENTION 
       [0009]    In one aspect, the present invention relates to a sample preparation device that integrates sample collection, storage, and nucleic acid extraction. The sample preparation device is designed to integrate with downstream processes for nucleic acid analysis, such as polymerase chain reaction (“PCR”). 
         [0010]    The device includes an inner housing with a chamber containing a solid nucleic acid extraction substrate having inlet and outlet ports and an outer housing with a sample port for delivering a liquid tissue sample, optionally a hypodermic needle protruding from the sample port attached to a capillary from the needle to the interior of the chamber, a set of two ports for flowing wash buffer through the chamber containing the solid substrate, and a set of two ports for eluting the nucleic acid sample from the extraction substrate. 
         [0011]    In a second aspect, the present invention provides a method of using the device that includes aligning one of the chamber ports of the inner housing with the sample input port and then introducing a sample material into the inner chamber under conditions that will allow nucleic acid within the sample to bind to the extraction substrate. The chamber inner ports are then aligned with wash ports and wash fluid is introduced into and removed from the chamber through the wash ports. Ultrasonic energy is then applied to release nucleic acid from the extraction substrate. The chamber inner ports are then aligned with the elute ports and elution fluid is introduced into and removed from the chamber through the elute ports. 
         [0012]    Thus, the device is a closed system for sample preparation. After sample is introduced, all processes occur within the cartridge until the purified nucleic acid extract is expelled. The advantage is that there is less chance of crossover, contamination, and errors in sample identification. 
         [0013]    In a third aspect, a genomic isolation chamber is provided which comprises a housing, a chamber within the housing, a first set of channels, wherein the first set of channels is configured to deliver a first medium to the chamber and extract a second medium from the chamber. The housing also includes a second set of channels that is configured to deliver a third medium to the chamber and extract a fourth medium from the chamber. An input channel is also provided that is configured to deliver a sample to the chamber. The genomic isolation chamber further comprises an adsorption substrate that is configured to adsorb at least a portion of nucleic acid in the sample. The genomic isolation chamber is configured so that a combination of flowing the first, second, third, and fourth mediums through or from the chamber isolates the adsorbed nucleic acid in the fourth medium. The nucleic acid is released from the adsorption substrate by using either sonication from a sonic emitter inserted through the input channel or by noncontact sonication. 
         [0014]    Other aspects of the present invention, including the methods of operation and the function and interrelation of the elements of structure, will become more apparent upon consideration of the following description and the appended claims, with reference to the accompanying drawings, all of which form a part of this disclosure, wherein like reference numerals designate corresponding parts in the various figures. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]      FIG. 1  is perspective view of a device for nucleic acid preparation embodying aspects of the present invention; 
           [0016]      FIG. 2  is a cross-sectional view of the device along the line  2 - 2  in  FIG. 1 ; 
           [0017]      FIG. 3  is a cross-sectional view of the device along the line  3 - 3  in  FIG. 1 ; and 
           [0018]      FIG. 4  is a flow chart illustrating a nucleic acid preparation procedure embodying aspects of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0019]    A device for nucleic acid preparation embodying aspects of the present invention is shown in  FIGS. 1-3  and includes a cartridge designated by reference number  10 . Cartridge  10  includes an outer housing  12  and an inner housing  30 . Inner housing  30  is circular and fits within a circular opening  24  formed in the outer housing  12 . The inner housing  30  is mounted so as to be rotatable about its axis within the opening  24  of the outer housing  12 . 
         [0020]    Inner housing  30  defines an inner chamber  32  surrounded by the outer periphery of the inner housing  30  and radial sidewalls  34  and  36 . The inner chamber  32  is preferably circular with first and second ports  38 ,  40  formed preferably at 180 degrees from each other and communicating with the inner chamber  32 . One of the ports  38 ,  40  will function as an inlet port for introducing materials into the chamber  32 , and the other port will function as an outlet port. A solid nucleic acid extraction substrate, indicated by reference number  42 , is disposed within the inner chamber  32 . In one non-limiting embodiment, the nucleic acid extraction device  42  is disposed against the sidewall  34  of the inner housing  30 , as illustrated in  FIG. 2 . 
         [0021]    The outer housing  12  includes a sample input port  14 , first and second wash ports  16 ,  18  formed preferably at 180 degrees from each other, and first and second elute ports  20 ,  22  formed preferably at 180 degrees from each other. Outer housing  12  may include a hypodermic needle (not shown) protruding from the sample input port  14  attached to a capillary from the needle to the interior of the inner chamber  32 . The inner housing  30  fits inside the outer housing  12  and is able to rotate so as to selectively align the first and second inner ports  38 ,  40  with ports formed in the outer housing. In  FIG. 2 , inner housing  30  is shown positioned so that first and second inner ports  38 ,  40  are aligned with first and second wash ports  16 ,  18 . Alternatively, the inner housing  30  can be held fixed and the outer housing  12  rotated (or both inner housing  30  and outer housing  12  can be rotated) to effect selective alignment of inner and outer housing ports. 
         [0022]    It will be appreciated that ports  16  and  18 ,  20  and  22 , and  38  and  40  need not necessarily be 180 degrees from each other. It is merely necessary that ports  16  and  18  and ports  20  and  22  of the outer housing  12  be angularly spaced by the same amount as ports  38  and  40  of the inner housing  30 , so that the ports of the inner housing  30  can be aligned with the ports of the outer housing  12 . Also, it is not necessary for the implementation of the invention that first and second wash ports  16 ,  18  and the first and second elute ports  20 ,  22  be aligned along mutually orthogonal directions as shown in  FIG. 2 . 
         [0023]    The sample input port  14  can be used to deliver a sample material to the inner chamber  32  when one of the first and second ports  38 ,  40  of the inner housing  30  is aligned with the sample input port  14 . The first and second wash ports  16 ,  18  can be used to flow a wash fluid through the inner chamber  32  when the first and second ports  38 ,  40  of the inner housing  30  are aligned with the first and second wash ports  16 ,  18 . Finally, the first and second elute ports  20 ,  22  can be used to flow an elution fluid through the inner chamber  32  when the first and second inner ports  38 ,  40  of the inner housing  30  are aligned with the first and second elute ports  20 ,  22 . 
         [0024]    Cartridge  10  is preferably disposable and may be incorporated within a closed system or instrument, thereby decreasing the likelihood of carryover, errors, or contamination which can occur during the multi-step process of nucleic acid purification. Thus, one of the first and second wash ports  16 ,  18  may be connected to a source (e.g., a pump, chamber, compartment, or container) of a wash fluid, and the other wash port may be connected to a waste chamber. Similarly, one of the first and second elute ports  20 ,  22  may be connected to a source (e.g., a pump, chamber, compartment or container) of an elution fluid, and the other elute port may be connected to a compartment for storing the nucleic acid or to other downstream processing. Alternatively, compartments for storage of wash fluid, elution fluid, wastes, and extracted nucleic acid can be formed in the cartridge itself, for example in the outer housing. Rotation of the inner housing  30  (or, alternatively, of the outer housing  12 ) can be effected by any suitable means, such as mechanical, electromechanical, pneumatic, magnetic, piezoelectric, or other actuator means. Furthermore, the outer shape of the outer housing  12  shown in  FIGS. 1-3  is for illustration only; the outer housing  12  may have any outer shape so as to conform to a system or instrument in which it is incorporated. 
         [0025]    As mentioned, the inner chamber  32  contains a nucleic acid extraction substrate  42  for capturing the components of the sample to be lysed. Suitable substrates generally include filters, beads, fibers, membranes, glass wool, filter paper, polymers, and gels. The substrate may capture the desired sample components through physical retention, e.g., adsorption, size exclusion, through affinity retention, or through chemical interaction. Suitable filter materials include glass, fiberglass, nylon, nylon derivatives, cellulose, cellulose derivatives, and other polymers. In an alternative embodiment, the substrate comprises polystyrene, silica, agarose, cellulose, or acrylamide beads. In the presently preferred embodiment, the substrate comprises a membrane or filter formed from FTA paper (Whatman plc, Kent, UK). FTA paper, as described in U.S. Pat. No. 6,322,983, the disclosure of which is incorporated by reference, utilizes a cellulose based solid substrate impregnated with a lysis material which lyses cells, inactivates proteins, and captures nucleic acid in the cellulose fibers. 
         [0026]    In one embodiment, the nucleic acid preparation procedure performed in accordance with the present invention includes the use of suitable energy applied to the nucleic acid extraction substrate  42  to release the captured nucleic acid from the nucleic acid extraction substrate  42 . For example, any mechanical energy suitable to dislodge the captured nucleic acid from the nucleic acid extraction substrate  42  can be used, and such energy can be generated by any suitable energy-emitting device. Preferably, the mechanical energy comprises pressure waves emitted by a pressure wave-emitting device. The pressure wave emitting device may comprise an acoustic energy emitting device. The acoustic energy emitting device produces acoustic energy that is used to release genetic material from a solid support. Any device that generates sound waves can be used as a source of acoustic energy. Such devices include, but are not limited to, ultrasonic transducers, piezoelectric transducers, magnorestrictive transducers, and electrostatic transducers. Suitable devices are well known in the art and include such commercially available devices as the Sonicator 4000 (Misonix, Inc., Farmingdale, N.Y., USA), Microson® Sonicator Microprobe or Micro Cup Horn (Kimble/Kontes, Vineland, N.J., USA) and Covaris™ Adaptive Focused Acoustics (Nexus Biosystems, Poway, Calif., USA). Other suitable devices are described in U.S. Pat. Nos. 6,881,541 and 6,878,540 and in U.S. Patent Application Publication No. 2007/0170812. 
         [0027]    Thus, according to one embodiment, the device includes, or is used in conjunction with, an ultrasonic transducer, such as an ultrasonic horn for non-contact sonication. In another aspect, this embodiment includes, or is used in connection with, an ultrasonic probe for contact sonication that is coupled to the cartridge  10  for transferring ultrasonic energy to the components captured on the substrate  42 . Contact sonication is performed by introducing a probe sonicator (ultrasonic probe) into the inner chamber  32  through the sample input port  14  and one of the first or second inner ports  38 ,  40  aligned with port  14 . Alternatively, non-contact sonication is performed by placing a non-contacting sonicator (e.g., an ultrasonic horn) adjacent the side wall  34  of the inner housing  30  that is in close proximity to the substrate  42 . To aid in the transfer of ultrasonic energy to the sample components if non-contact sonication is to be employed with the cartridge  10 , it is preferred that side wall  34  be a relatively thin film or membrane, preferably having a thickness in the range of 0.01 to 0.5 mm, and more preferably have a thickness of about 0.05 mm. 
         [0028]    The cartridge  12  may be fabricated of any suitable polymer and may be fabricated using conventional techniques. In particular, the inner housing  30  and the outer housing  12  preferably comprise molded plastic. Examples of suitable plastic materials for the inner housing  30  and the outer housing  12  (including side wall  34  if it comprises a thin film or membrane for non-contact sonication) include, e.g., polycarbonate, polystyrene, polypropylene, polyethylene, acrylic, and commercial polymers. Substrate  42  may optionally be heat sealed within the inner chamber  32  of the inner housing  30 . 
         [0029]      FIG. 4  is a flow chart illustrating a process for nucleic acid extraction in accordance with the present invention using the cartridge  10 . In Step  50 , inner housing  30  is moved (rotated) to align the first or the second inner port  38 ,  40  of the inner housing  30  with the sample input port  14  of the outer housing  12  to open inner chamber  32 . In Step  52 , sample material (e.g., whole blood, saliva, cerebro spinal fluid, amniotic fluid, serum, urine, fluid from a vaginal or buccal swab, or tissue sample) is introduced into inner chamber  32  through sample input port  14  and the first or second inner port  38 ,  40  with which the sample input port  14  is aligned. In Step  54 , the sample material temperature is allowed stabilized within inner chamber  32  at ambient temperature, and the sample material is allowed to bind to substrate  42 . 
         [0030]    Next, in Step  56 , inner housing  30  is moved (rotated) to align the first and second inner ports  38 ,  40  of the inner housing  30  with first and second wash ports  16 ,  18 , respectively, of the outer housing  12 . In Step  58 , wash fluid (e.g., water) is introduced from a wash fluid source (e.g., a pump, compartment, chamber, or container (not shown)) into the inner chamber  32  through first wash port  16  and aligned first inner port  38 , and, in Step  60 , the wash fluid is removed from inner chamber  32  through second wash port  18  and aligned second inner port  40 . The removed wash fluid may be transmitted to a waste chamber or container (not shown). The direction of flow of the wash fluid can be reversed, that is, from second wash port  18  to first wash port  16 . 
         [0031]    In Step  62 , inner housing  30  is moved (rotated) to align the first or second inner port  38 ,  40  with sample input port  14  to open inner chamber  32 . Then, in Step  64 , a probe sonicator is introduced into inner chamber  32  through sample input port  14  and aligned first or second inner port  38 ,  40 . The probe is introduced into fluid contained within inner chamber  32 , and, in Step  66 , the sample is sonicated to release nucleic acid from substrate  42  in a procedure referred to as contact sonication. 
         [0032]    As an alternative to contact sonication, nucleic acid may be released from substrate  42  by non-contact sonication. A non-contact sonication procedure is performed by first, in Step  68 , moving inner housing  30  so the first and second inner port  38 ,  40  are not aligned with any ports of the outer housing  12 , to thereby seal off the inner chamber  32 . In Step  70 , a non-contact sonicator (e.g., an ultrasonic horn) is positioned adjacent chamber side wall  34 , which, to enable such a process, and as described above, is made of a thin membrane material constructed and arranged to enable the transmission of sufficient sonic energy to release nucleic acid from the substrate  42 . In Step  72 , the sample is sonicated by the non-contact sonicator to release nucleic acid from substrate  42 . In a non-limiting example, a frequency of approximately 20 kH can be used for a duration of approximately 15 seconds to 60 seconds to achieve the desired release of at least a portion of nucleic acid. Of course, other sonical frequencies and duration of sonication can be used as well. 
         [0033]    Following sonication by the contact or non-contact procedures described above, in Step  74 , the inner housing  30  is moved to align the first and second inner ports  38 ,  40  of the inner housing  30  with first and second elute ports  20 ,  22 , respectively, of the outer housing  12 . In Step  76 , elution fluid (e.g., an elution fluid that is compatible for downstream processing and/or storage) is introduced from an elution fluid source (e.g., a pump, compartment, chamber, or container (not shown)) into the inner chamber  32  through first elute port  20  and aligned first inner port  38 . In Step  78 , the elution fluid is removed from inner chamber  32  through second elute port  22  and aligned second inner port  40 , to remove the released nucleic acid from the chamber. The nucleic acid is then transmitted to a compartment for storage or transmitted downstream for further processing. In another embodiment, the direction of flow of the elution fluid can be reversed, that is, from second elute port  22  to first elute port  20 . 
         [0034]    In another aspect of the invention, a genomic isolation chamber is disclosed which comprises a housing  12 , a chamber  42  within the housing, a first set of channels  16 ,  18 , wherein the first set of channels is configured to deliver a first medium (e.g. a wash medium) to the chamber and extract a second medium (e.g. wash medium plus contaminants) from the chamber. See  FIGS. 1-3 . The housing also includes a second set of channels  20 ,  22  that is configured to deliver a third medium (e.g. an elution medium) to the chamber and extract a fourth medium (elution medium plus nucleic acid) from the chamber. An input channel  14  is also provided that is configured to deliver a sample to the chamber. The genomic isolation chamber further comprises an adsorption substrate  42  that is configured to adsorb at least a portion of nucleic acid in the sample. The genomic isolation chamber is configured so that a combination of flowing the first, second, third, and fourth mediums through or from the chamber isolates the adsorbed nucleic acid in the fourth medium. The nucleic acid is released from the adsorption substrate by using either sonication from a sonic emitter inserted through the input channel or by noncontact sonication. 
         [0035]    The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. 
         [0036]    It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.