Methods and compositions for purification of nucleic acid from a host cell

Abstract

Methods and compositions are provided for gently lysing and solubilizing cells. Methods and compositions are further provided for quickly purifying high quality low molecular weight nucleic acid from host cells. Target cells are treated with a pre-chilled lysis solution having a zwitterionic detergent, for example n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and a brief room-temperature incubation. Where nucleic acid purification is required, the lysis solution-treated cells are contacted with a nucleic acid capture matrix having an average pore size of at least about 1 μm.

What is claimed is:  
     
         1 . A lysis composition for lysing a host cell comprising: 
 a buffering agent; and    a zwitterionic detergent;    wherein at least a portion of protein in the host cell is solubilized by the zwitterionic detergent.    
     
     
         2 . The composition of  claim 1  further comprising lysozyme.  
     
     
         3 . The composition of  claim 2  further comprising RNase.  
     
     
         4 . The composition of  claim 2  further comprising DNase.  
     
     
         5 . The composition of  claim 2  further comprising polyethylene glycol.  
     
     
         6 . The composition of  claim 1  wherein said zwitterionic detergent is selected from the group consisting of n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and mixtures thereof.  
     
     
         7 . The composition of  claim 6  wherein said zwitterionic detergent is at a final concentration of from about 1% to about 5%.  
     
     
         8 . The composition of  claim 1  further comprising a chaotropic salt.  
     
     
         9 . The composition of  claim 1  wherein the composition has a final pH of from about 7.9 to about 8.4.  
     
     
         10 . A kit for purifying low molecular weight nucleic acid from a host cell, the kit comprising: 
 a lysis solution for purifying low molecular weight nucleic acid from host cells, the lysis solution comprising a buffering agent and a zwitterionic detergent; and    a nucleic acid capture matrix for capturing low molecular weight nucleic acid.    
     
     
         11 . The kit of  claim 10  wherein said nucleic acid capture matrix comprises a spin column comprising a capture matrix material.  
     
     
         12 . The kit of  claim 11  wherein said capture matrix material comprises a glass fiber having an average pore size of at least 1 μm.  
     
     
         13 . The kit of  claim 11  wherein said capture matrix material comprises a glass fiber having an average pore size of at least 3 μm.  
     
     
         14 . The kit of  claim 13  further comprising a frit for support of the capture matrix material.  
     
     
         15 . The kit of  claim 10  further comprising: 
 a wash buffer for removal of contaminants from captured low molecular weight nucleic acid; and  
 an elution buffer for elution of captured low molecular weight nucleic acid from the nucleic acid capture matrix.  
 
     
     
         16 . The kit of  claim 10  wherein said zwitterionic detergent is selected from the group consisting of n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and mixtures thereof.  
     
     
         17 . The kit of  claim 16  wherein said zwitterionic detergent is at a final concentration of from about 0.2% to about 6%.  
     
     
         18 . The kit of  claim 10  further comprising lysozyme.  
     
     
         19 . The kit of  claim 10  further comprising RNase.  
     
     
         20 . The kit of  claim 10  further comprising DNase.  
     
     
         21 . The kit of  claim 10  further comprising a tube for culturing cells.  
     
     
         22 . The kit of  claim 21  wherein said tube for culturing cells is a lid-bac tube.  
     
     
         23 . The kit of  claim 10  wherein said nucleic acid capture matrix is integrated into at least one well of a ninety-six well plate, wherein the ninety-six well plate is included in the kit.  
     
     
         24 . A method for purifying a low molecular weight nucleic acid from a host cell, the method comprising: 
 adding a lysing amount of lysis solution to host cells for the release of the low molecular weight nucleic acid from the host cells, said lysis solution comprising a zwitterionic detergent;    combining the host cells treated with the lysis solution with a nucleic acid capture matrix, the nucleic acid capture matrix having at least one layer of capture matrix material having an average pore size of at least 1 μm for the capture of the low molecular weight nucleic acid; and    eluting the low molecular weight nucleic acid from the nucleic acid capture matrix.    
     
     
         25 . The method of  claim 24  wherein said zwitterionic detergent in said lysis solution is at from about 0.2% to about 6%.  
     
     
         26 . The method of  claim 25  wherein said zwitterionic detergent in said lysis solution is at from about 1% to about 5%.  
     
     
         27 . The method of  claim 24  wherein said zwitterionic detergent in said lysis solution is selected from the group consisting of n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and mixtures thereof.  
     
     
         28 . The method of  claim 24  wherein said lysis solution is chilled to a temperature below room temperature before addition to the host cells.  
     
     
         29 . The method of  claim 28  further comprising: 
 incubating the lysis solution on the host cells at room temperature for at least three minutes prior to combining with the nucleic acid capture matrix.  
 
     
     
         30 . The method of  claim 29  wherein the incubation is for at least five minutes.  
     
     
         31 . The method of  claim 24  further comprising: 
 washing the nucleic acid capture matrix having the captured low molecular weight nucleic acid with a wash buffer prior to eluting.  
 
     
     
         32 . The method of  claim 24  wherein said lysis solution further comprises polyethylene glycol.  
     
     
         33 . The method of  claim 32  wherein said polyethylene glycol is at a final concentration of from about 2% to about 20%.  
     
     
         34 . The method of  claim 24  wherein said lysis solution further comprises lysozyme.  
     
     
         35 . The method of  claim 24  wherein said lysis solution further comprises RNase.  
     
     
         36 . The method of  claim 24  wherein said lysis solution further comprises DNase.  
     
     
         37 . The method of  claim 24  wherein said lysis solution further comprises a chaotropic salt.  
     
     
         38 . The method of  claim 24  wherein said low molecular weight nucleic acid comprises a plasmid DNA.  
     
     
         39 . A composition for releasing extrachromosomal DNA from a host cell comprising a solution having from about 0.2% to about 6% of a zwitterionic detergent.  
     
     
         40 . The composition of  claim 39  wherein said zwitterionic detergent is selected from the group consisting of n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and mixtures thereof.  
     
     
         41 . The composition of  claim 40  further comprising lysozyme.  
     
     
         42 . The composition of  claim 41  further comprising RNase.  
     
     
         43 . The composition of  claim 41  further comprising polyethylene glycol.  
     
     
         44 . The composition of  claim 41  further comprising chaotropic salt.  
     
     
         45 . A composition for solubilizing cellular protein during the separation of cellular protein from nucleic acid comprising a detergent is selected from the group consisting of n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and mixtures thereof.  
     
     
         46 . The composition of  claim 45  wherein said detergent is at a final concentration of from about 0.2% to about 6%.  
     
     
         47 . The composition of  claim 46  wherein said detergent is at a final concentration of from about 1% to about 5%.  
     
     
         48 . A lysis composition for isolating nucleic acid from a cleared lysate comprising: 
 a buffering agent;    polyethylene glycol; and    a salt;    wherein the lysis composition is added to the cleared lysate, and the combination of cleared lysate and lysis composition is added to a silica fiber for capture of the nucleic acid on the silica fiber.    
     
     
         49 . A method for purifying a low molecular weight nucleic acid from a host cell in a cell culture, the method comprising: 
 adding a lysing amount of lysis solution to the cell culture for release of the low molecular weight nucleic acid from the host cell;    combining the host cells treated with the lysis solution with a nucleic acid capture matrix, the nucleic acid capture matrix having at least one layer of capture matrix material having an average pore size of at least 1 μm for the capture of the low molecular weight nucleic acid; and    eluting the low molecular weight nucleic acid from the nucleic acid capture matrix.    
     
     
         50 . The method of  claim 49  wherein the lysis solution is added to the cell culture at a ratio of lysis solution to volume of cell culture of about 1:2 to about 3:1.