Solid phase technique for selectively isolating nucleic acids

Abstract

A method of isolating target nucleic acid molecules from a solution comprising a mixture of different size nucleic acid molecules, in the presence or absence of other biomolecules, by selectively facilitating the adsorption of a particular species of nucleic acid molecule to the functional group-coated surface of magnetically responsive paramagnetic microparticles is disclosed. Separation is accomplished by manipulating the ionic strength and polyalkylene glycol concentration of the solution to selectively precipitate, and reversibly adsorb, the target species of nucleic acid molecule, characterized by a particular molecular size, to paramagnetic microparticles, the surfaces of which act as a bioaffinity adsorbent for the nucleic acids. The target nucleic acid is isolated from the starting mixture based on molecular size and through the removal of magnetic beads to which the target nucleic acid molecules have been adsorbed. The disclosed method provides a simple, robust and readily automatable means of nucleic acid isolation and purification which produces high quality nucleic acid molecules suitable for: capillary electrophoresis, nucleotide sequencing, reverse transcription cloning the transfection, transduction or microinjection of mammalian cells, gene therapy protocols, the in vitro synthesis of RNA probes, cDNA library construction and PCR amplification.

1 . A method of selectively isolating high molecular weight species of nucleic acid molecules present in a mixture comprising high molecular weight and low molecular weight species of nucleic acid molecules, wherein the molecular size of the high molecular weight species and the molecular size of the low molecular weight species differ by a factor of two, said method comprising the steps of:
 (a) preparing a combination comprising a mixture of nucleic acid molecules, a nucleic acid precipitating reagent, salt, and solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules, wherein the nucleic acid precipitating reagent and the salt are present in sufficient concentrations to selectively precipitate the high molecular weight species of nucleic acid molecule but not the low molecular weight species of nucleic acid molecules, thereby producing a first combination;   (b) maintaining the first combination under conditions appropriate for adsorption of the precipitated nucleic acid molecules to the functional group-coated surface of the solid phase carriers, thereby producing solid phase carriers having bound thereto the high molecular weight species of nucleic acid molecules but not the low molecular weight species of nucleic acid molecules; and   (c) removing the carriers having bound thereto the high molecular weight species of nucleic acid molecules from the first combination, thereby isolating the target species of nucleic acid molecules from the mixture and producing a second mixture.   
   
   
       2 . The method of  claim 1  further comprising the steps of:
 (d) eluting the high molecular weight species of nucleic acid molecules from the solid phase carriers, thereby producing a solution comprising the high molecular weight species of nucleic acid molecules; and   (e) removing the solid phase carriers, whereby the high molecular weight species of nucleic acid molecules is isolated from the mixture.   
   
   
       3 . The method of  claim 1  wherein the mixture is selected from the group consisting of:
 (a) a lysed host cell suspension prepared using cells obtained from a mammalian tissue, a mammalian body fluid or a plant cell;   (b) a lysed host cell suspension prepared from a cultured cell which has been transduced or transfected;   (c) a solution resulting from a PCR amplification procedure;   (d) a solution resulting from a post-DNA size shearing procedure;   (e) a solution resulting from a nucleotide sequencing reaction;   (f) a solution resulting from a restriction enzyme digestion comprising a mixture of nucleic acid molecule fragments;   (g) an agarose solution containing nucleic acid; and   (h) a solution resulting from an extension reaction.   
   
   
       4 . The method of  claim 1  wherein the nucleic acid precipitating reagent is selected from the group consisting of: polyalkylene glycol, alcohol and combinations thereof. 
   
   
       5 . The method of  claim 4  wherein the polyalkylene glycol is selected from the group consisting of: polyethylene glycol and polypropylene glycol, and the alcohol is selected from the group consisting of: ethanol and isopropanol. 
   
   
       6 . The method of  claim 1  wherein the solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules are selected from the group consisting of: amine-coated, carboxyl-coated and encapsulated carboxyl group-coated solid phase carriers. 
   
   
       7 . The method of  claim 6  wherein the solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules are magnetically responsive solid phase carriers having a functional group-coated surface selected from the group consisting of: amine-coated, carboxyl-coated and encapsulated carboxyl group-coated paramagnetic microparticles. 
   
   
       8 . The method of  claim 1  wherein the solid phase carriers are removed from the combination or nucleic acid solution using a method selected from the group of methods consisting of: applying a magnetic field, applying vacuum filtration and centrifugation. 
   
   
       9 . The method of  claim 1  wherein the salt is selected from the group consisting of: sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride. 
   
   
       10 - 11 . (canceled) 
   
   
       12 . A method of separating host cell DNA from exogenous DNA present in a host cell wherein the host cell DNA and the exogenous DNA differ in molecular size by at least a factor of two comprising the steps of:
 (a) combining a lysed host cell suspension with: (1) solid phase carriers having a functional group-coated surface, (2) a nucleic acid precipitating reagent and (3) salt, wherein the nucleic acid precipitating reagent and the salt are present in concentrations which result in binding of host cell DNA to the solid phase carriers, but not in binding of exogenous DNA to the solid phase carriers, thereby producing a first combination, which comprises host cell DNA bound to the solid phase carriers and unbound exogenous DNA;   (b) separating the solid phase carriers from the first combination, thereby producing a second combination;   (c) adding solid phase carriers with a functional group-coated surface and sufficient nucleic acid precipitating reagent to the second combination to increase the nucleic acid precipitating reagent concentration of the combination to a concentration that results in binding of exogenous DNA to the solid phase carriers, thereby producing a third combination, which comprises exogenous DNA bound to the solid phase carriers; and   (d) separating the solid phase carriers from the third combination, whereby exogenous DNA is separated from host cell DNA.   
   
   
       13 . The method of  claim 12  wherein the host cell is a mammalian cell and the exogenous nucleic acid is bacterial DNA, viral DNA, viral RNA or replicative form DNA. 
   
   
       14 . (canceled) 
   
   
       15 . The method of  claim 12  wherein the nucleic acid precipitating agent is selected from the group consisting of: polyalkylene glycol and alcohol. 
   
   
       16 . The method of  claim 15  wherein the polyalkylene glycol is selected from the group consisting of: polyethylene glycol and polypropylene glycol, and the alcohol is selected from the group consisting of: ethanol and isopropanol. 
   
   
       17 . The method of  claim 12  wherein the solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules are selected from the group consisting of: amine-coated, carboxyl-coated and encapsulated carboxyl group-coated solid phase carriers. 
   
   
       18 . The method of  claim 12  wherein the salt is selected from the group consisting of: sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride. 
   
   
       19 - 32 . (canceled) 
   
   
       33 . A method of selectively isolating extension products from a sequencing reaction mixture, comprising the steps of:
 (a) preparing a combination comprising (1) an aliquot of labeled dye terminator or dye primer nucleotide sequencing reaction products comprising extension products; (2) functional group-coated solid phase carriers and (3) a nucleic acid precipitating agent, wherein the precipitating reagent is present in sufficient concentration to selectively precipitate the extension products but not other nucleic acid molecules having a molecular size which is smaller than the molecular size of the extension products by a factor of two in the reaction mixture;   (b) maintaining the combination under conditions appropriate for adsorption of the precipitated extension products to the functional group-coated surface of the solid phase carriers, thereby producing solid phase carriers having the extension product, but not other nucleic acid molecules having a molecular size which is less than the molecular size of the extension product bound thereto;   (c) removing the extension product-coated solid phase carriers from the combination;   (d) eluting the extension products from the solid phase carriers; and   (e) removing the solid phase carriers from the solution of (e), whereby the extension products of a sequencing reaction mixture are selectively isolated.   
   
   
       34 . (canceled) 
   
   
       35 . The method of  claim 33 , wherein the nucleic acid precipitating reagent is selected from the group consisting of: polyalkylene glycol and alcohol. 
   
   
       36 . The method of  claim 35  wherein the polyalkylene glycol is selected from the group consisting of polypropylene glycol and polyethylene glycol, and wherein the alcohol is selected from the group consisting of: ethanol and isopropanol. 
   
   
       37 . The method of  claim 33  wherein the solid phase carriers having a functional group-coated surface that reversibly binds nucleic acid molecules are selected from the group consisting of: amine-coated, carboxyl-coated and encapsulated carboxyl group-coated solid phase carriers. 
   
   
       38 . The method of  claim 33  wherein the salt is selected from the group consisting of: sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride. 
   
   
       39 - 76 . (canceled)