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 kit for high through-put automated DNA template production comprising: 
 (a) reagents for preparing a host cell lysate;    (b) an aqueous solution of functional group-coated solid phase carriers; and    (c) at least one binding buffer formulated to comprise a suitable salt and a suitable nucleic acid precipitating reagent, wherein the salt and the nucleic acid precipitating reagent are each present at a concentration appropriate for binding a nucleic acid species characterized by a particular molecular size to the solid phase carriers or reagents for the formulation of a suitable buffer.    
     
     
         2 . The kit of  claim 1  wherein the kit additionally comprises reagents for the formulation of a wash buffer and an elution buffer, wherein the wash buffer dissolves impurities, but not nucleic acids bound to solid phase carriers and the elution buffer is a low ionic strength buffer.  
     
     
         3 . The kit of  claim 1  wherein the solid phase carriers are paramagnetic particles.  
     
     
         4 . The kit of  claim 3  wherein the kit further comprises a magnetic plate holder appropriate for applying a magnetic field of at least about 1000 Gauss to the wells of a microtiter plate, wherein said magnet comprises at least one N35 magnet.  
     
     
         5 . The kit of  claim 1  wherein the nucleic acid precipitating agent is selected from the group consisting of: polyalkylene glycol and alcohol.  
     
     
         6 . The kit of  claim 5  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.  
     
     
         7 . The kit of  claim 1  wherein the reagent for preparing a host cell lysate is a detergent.  
     
     
         8 . The kit 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.  
     
     
         9 . The kit 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.