Nucleic Acid Purification

Abstract

A self-contained apparatus for isolating nucleic acid, cell lysates and cell suspensions from unprocessed samples apparatus, to be used with an instrument, includes at least one input, and: (i) a macrofluidic component, including a chamber for receiving an unprocessed sample from a collection device and at least one filled liquid purification reagent storage reservoir; and (ii) a microfluidic component in communication with the macrofluidic component through at least one microfluidic element, the microfluidic component further comprising at least one nucleic acid purification matrix; and (iii) at least one interface port to a drive mechanism on the instrument for driving said liquid purification reagent, through the microfluidic element and the nucleic acid purification matrix, wherein the only inputs to the apparatus are through the chamber and the interface port to the drive mechanism.

We claim: 
     
         1 . A self-contained apparatus for isolating nucleic acid from at least one unprocessed sample, said apparatus to be used with an instrument, said apparatus consisting of a macrofluidic component, a microfluidic component, at least one drive line, and two inputs, a first input for receiving unprocessed sample or samples from a collection device, and a second input for interfacing at least one drive mechanism on said instrument at, at least one port:
 (i) said macrofluidic component consisting of: (a) at least one sample chamber, each of said at least one sample chamber composed of said first input for receiving said at least one unprocessed sample from a collection device; (b) at least one, but no less than all reagent storage chambers necessary for isolating nucleic acid from said at least one unprocessed sample, each of said reagent storage chambers pre-filled with at least one necessary liquid reagent, including, at least two lysis reagent storage chambers pre-filled with lysis reagent; a wash reagent storage chamber pre-filled with wash reagent; and an elution reagent storage chamber pre-filled with elution reagent and (c) at least one empty holding chamber;   (ii) said microfluidic component composed of at least one microfluidic element and at least one nucleic acid purification matrix, said microfluidic component in communication with said macrofluidic component via said at least one microfluidic element, and said at least one drive line;   (iii) said second input composed of at least one drive mechanism interface port for connection to said drive mechanism on said instrument configured to drive said first and second lysis reagents, said wash reagent, and said elution reagent and combinations thereof sequentially through said microfluidic element and said nucleic acid purification matrix, and back into said holding chamber   (iv) said at least one drive line in communication with said second input for interfacing with said at least one drive mechanism on said instrument and with said at least one microfluidic element to supply controlled flow or controlled pressure or a controlled volumetric displacement of gas or liquid to the apparatus.   whereby in use, spent lysis, wash and elution reagents and combinations thereof are driven through said microfluidic element and back into said at least one sample chamber, now serving as a waste chamber for spent process reagents.   
     
     
         2 . The apparatus of  claim 1  wherein said collection device and/or chamber is labeled with a bar code or RFID. 
     
     
         3 . The apparatus of  claim 1  wherein said drive mechanism is pneumatic, mechanical, magnetic, or fluidic. 
     
     
         4 . The apparatus of  claim 1  wherein the unprocessed sample is selected from the group consisting of: i) a nasal swab, nasopharyngeal swab, buccal swab, oral fluid swab, stool swab, tonsil swab, vaginal swab, cervical swab, blood swab, wound swab, or tube containing blood, sputum, purulent material, or aspirates; (ii) a forensic swab, cutting, adhesive tape lift, or card; or (iii) an environmental air filter, water filter, and swab. 
     
     
         5 . The apparatus of  claim 1  wherein the nucleic acid purification matrix is selected from the group consisting of comprises silica membranes, silica beads, silica magnetic beads, ion exchange resins, and ion exchange beads. 
     
     
         6 . The apparatus of  claim 1  wherein said at least one microfluidic element is selected from the group consisting of: channels, reservoirs, active valves, passive valves, pneumatically actuated valves, reaction chambers, mixing chambers, venting elements, access holes, pumps, metering elements, mixing elements, heating elements, magnetic elements, reaction chambers, filtration elements, purification elements, drive lines, and actuation lines. 
     
     
         7 . The apparatus of  claim 1 , wherein the apparatus can be placed into or interfaces with another instrument that performs at least one of thermal cycling, capillary electrophoresis, microfluidic electrophoresis, nucleic acid fragment sizing, short tandem repeat (STR), Y-STR, and mini-STR, single nucleotide polymorphism, PCR, highly multiplexed PCR, Real-time-PCR, Reverse Transcription PCR, sequencing, hybridization, microarray, VNTR, immunoassays, mass spectroscopy and RFLP analyses. 
     
     
         8 . The apparatus of  claim 1 , wherein said first lysis reagent storage chamber of said at least two pre-filled lysis reagent storage chambers is pre-filled with guanidinum and said second lysis reagent storage chamber of said at least two pre-filled lysis reagent storage chambers is pre-filled with ethyl alcohol and said pre-filled wash reagent storage chamber is pre-filled with an ethanol based wash reagent. 
     
     
         9 . The apparatus of  claim 1 , wherein an aggregate fluid volume of the at least one sample chamber, the at least two pre-filled lysis reagent storage chambers, the pre-filled was reagent storage chamber, and the pre-filled elution reagent storage chamber is between about 1 and 1000 mL. 
     
     
         10 . A method for purifying nucleic acids from an unprocessed sample comprising,
 providing the apparatus of  claim 1 ;   providing said at least one unprocessed sample comprising nucleic acids to at least one sample chamber of said apparatus;   driving at least a portion of a first lysis reagent from a first of said at least two lysis reagent storage chambers into said at least one sample chamber to provide a first mixture;   bubbling a gas through the first mixture to provide a stirred first mixture;   driving at least a portion of said second lysis reagent from said second lysis reagent chamber into said at least one sample chamber to provide a second mixture; and   driving at least a portion of the stirred first mixture through said at least one nucleic acid purification matrix to provide a filtrate and a retentate, wherein the retentate comprises at least a portion of the nucleic acids;   driving at least a portion of said wash reagent from said wash reagent storage chamber and through said at least one purification matrix to provide a washed retentate and a waste;   optionally drying the washed retentate;   driving at least a portion of said elution reagent from said elution reagent storage chamber through said at least one purification matrix to provide an eluted nucleic acid solution; and   bubbling a gas through the eluted nucleic acid solution to provide a homogenized eluted nucleic acid solution   driving said homogenized eluted nucleic acid solution through said particulate filter to and into said at least one holding chamber to provide a lysate and ethanol mixture;   driving said lysate and ethanol mixture through said at least one nucleic acid purification matrix and back into a sample chamber, serving as a waste chamber.   
     
     
         11 . The method of  claim 10 , wherein the at least one unprocessed sample is selected from the group consisting of: nasal swab, nasopharyngeal swab, buccal swab, oral fluid swab, stool swab, tonsil swab, vaginal swab, cervical swab, blood swab, wound swab, tube containing blood, sputum, purulent material, or aspirates, forensic swab, cutting, adhesive tape lift, adhesive tape card, environmental air filter, water filter and an environmental swab.