Cell concentration and lysate clearance using paramagnetic particles

Abstract

Methods are disclosed for using paramagnetic particles to concentrate or harvest cells. Methods are also disclosed for clearing a solution of disrupted biological material, such as a lysate of cells or a homogenate of mammalian tissue. Methods are also disclosed for using paramagnetic particles to isolate target nucleic acids, such as RNA or DNA, from a solution cleared of disrupted biological material using the same type or a different type of paramagnetic particle. Kits are also disclosed for use with the various methods of the present invention. Nucleic acids isolated according to the present methods and using the present kits are suitable for immediate use in downstream processing, without further purification.

1 . A method for preparing a cleared lysate solution comprising: 
 (a) contacting a material comprising cells with first magnetic particles under conditions wherein the cells selectively adsorb to the first magnetic particles;    (b) isolating the first magnetic particles by applying magnetic force;    (c) disrupting the cells to obtain a preparation comprising disrupted biological material and target nucleic acids;    (d) combining the preparation of step (c) with second magnetic particles under conditions wherein the disrupted biological material other than the target nucleic acids selectively adsorbs to the second magnetic particles; and    (e) separating the second magnetic particles of step (d) by applying magnetic force to obtain a cleared lysate solution.    
   
   
       2 . The method of  claim 1 , wherein the first magnetic particles and the second magnetic particles have a particle size of about 1 to about 15 μm.  
   
   
       3 . The method of  claim 1 , wherein the first magnetic particles are pH dependent ion exchange magnetic particles.  
   
   
       4 . The method of  claim 3 , wherein the pH dependent ion exchange magnetic particles are selected from the group consisting of glycidyl-histidine modified silica magnetic particles and glycidyl-alanine modified silica magnetic particles.  
   
   
       5 . The method of  claim 1 , wherein the first magnetic particles, the second magnetic particles, or both the first magnetic particles and the second magnetic particles, are silica magnetic particles.  
   
   
       6 . The method of  claim 5 , wherein the silica magnetic particles consist essentially of a magnetic core coated with a siliceous oxide having a hydrous siliceous oxide adsorptive surface.  
   
   
       7 . The method of  claim 1 , wherein the target nucleic acids are selected from the group consisting of plasmid DNA, total RNA, mRNA, and genomic DNA.  
   
   
       8 . The method of  claim 1 , wherein the disrupted biological material is a bacterial cell lysate.  
   
   
       9 . The method of  claim 1 , wherein the disrupted biological material is a lysate of blood.  
   
   
       10 . The method of  claim 1 , wherein the disrupted biological material is a homogenate of mammalian tissue.  
   
   
       11 . A method of isolating a target nucleic acid from a disrupted biological material comprising the target nucleic acid, a first non-target material, and a second non-target material, comprising the steps of: 
 (a) contacting the disrupted biological material with first magnetic particles under conditions wherein the first non-target material adsorbs to the first magnetic particles;    (b) separating the first magnetic particles by applying magnetic force, forming a preparation comprising the target nucleic acid and the second non-target material;    (c) combining the preparation of step (b) with second magnetic particles under conditions wherein the target nucleic acid adsorbs to the second magnetic particles; and    (d) isolating the second magnetic particles from the preparation of step (c).    
   
   
       12 . The method of  claim 11 , further comprising combining the second magnetic particles of step (d) with an elution solution under conditions wherein the target nucleic acid is desorbed from the second magnetic particles.  
   
   
       13 . The method of  claim 12 , further comprising washing the second magnetic particles of step (d) with a wash solution and separating the second magnetic particles from the wash solution by applying magnetic force prior to combining the second magnetic particles with the elution solution.  
   
   
       14 . The method of  claim 11 , wherein the first magnetic particles are selected from the group consisting of silica magnetic particles and pH dependent ion exchange magnetic particles.  
   
   
       15 . The method of  claim 11 , wherein the first non-target material comprises cell debris or homogenized tissue and a precipitate, wherein the precipitate is comprised of material selected from the group consisting of proteins, non-target nucleic acids, and lipids.  
   
   
       16 . The method of  claim 11 , wherein the second non-target material remains in the preparation of step (b) when the target nucleic acid is adsorbed to the second magnetic particles in step (c).  
   
   
       17 . The method of  claim 11 , wherein the target nucleic acid is selected from the group consisting of plasmid DNA, total RNA, mRNA, and genomic DNA.  
   
   
       18 . A kit for isolating a target nucleic acid from a disrupted biological material comprising a first non-target material and the target nucleic acid, comprising at least one type of magnetic particle having the capacity to selectively adsorb (1) the first non-target material under conditions that promote the adsorption of the first non-target material to the magnetic particles, and (2) the target nucleic acid under conditions that promote the adsorption of the target nucleic acid to the magnetic particles.  
   
   
       19 . The kit of  claim 18 , wherein the at least one type of magnetic particle is a silica magnetic particle.  
   
   
       20 . The kit of  claim 18 , wherein the at least one type of magnetic particle is a pH dependent ion exchange magnetic particle.  
   
   
       21 . The kit of  claim 18 , further comprising a wash solution configured to wash the magnetic particles prior to desorption of the target nucleic acid from the magnetic particles.  
   
   
       22 . The kit of  claim 18 , further comprising an elution solution configured to promote desorption of the target nucleic acid from the magnetic particles.