Nucleic acid purification

Abstract

Methods and composition for nucleic acid isolation are provided. In one embodiment, a method is provided for nucleic acid purification from biological samples, such as whole blood samples, extracted with phenol-based denaturing solvents, which does not require phase separation or nucleic acid precipitation. Methods according to the invention may also be used for of small RNAs (e.g., siRNAs or miRNAs) purification and are amenable to automation.

1 - 59 . (canceled) 
     
     
         60 . A method for binding a nucleic acid molecule to a substrate comprising:
 (a) obtaining a sample comprising a nucleic acid molecule and phenol; and   (b) contacting the sample to magnetic silica beads in the presence of a binding agent comprising a chaotropic salt, an alcohol or a combination thereof, thereby binding the nucleic acid molecule to the silica magnetic silica beads, wherein one or more steps (a)-(b) is automated.   
     
     
         61 . A method for binding a small RNA to a silica substrate for purification comprising:
 (a) contacting a nucleic acid sample with a denaturing solvent comprising phenol;   (b) adding a binding agent to the sample, wherein the binding agent comprises a chaotropic salt, an alcohol or a mixture thereof; and   (c) contacting the sample with a silica substrate thereby binding the nucleic acid to the silica substrate,   wherein the sample comprises an aqueous and an organic phase after the addition of the denaturing solvent and wherein the aqueous and organic phases are not separated prior to binding the nucleic acid to the silica substrate.   
     
     
         62 . The method of  claim 61 , wherein one or more of steps (a)-(c) is automated. 
     
     
         63 . (canceled) 
     
     
         64 . The method of  claim 61 , wherein the denaturing solvent comprises at least one additional agent selected from the group consisting of a chaotropic salt, an antioxidant, a chelating agent, a phase separating agent and a buffer. 
     
     
         65 - 66 . (canceled) 
     
     
         67 . The method of  claim 61 , wherein the denaturing solvent has a pH of between about 6.0, 5.5, 5.0, 4.5 or 4.0 and about 2.0. 
     
     
         68 . The method of  claim 67 , wherein the denaturing solvent has a pH of less than about 4.0. 
     
     
         69 - 74 . (canceled) 
     
     
         75 . The method of  claim 61 , wherein the binding agent comprises an alcohol, wherein the alcohol is a lower alcohol. 
     
     
         76 . The method of  claim 75 , wherein the lower alcohol is methanol, ethanol, isopropanol, butanol, or a combination thereof. 
     
     
         77 . The method of  claim 61 , wherein the binding agent comprises an alcohol, wherein the total alcohol content of the sample is at least about 40% after the binding agent is added. 
     
     
         78 . The method of  claim 61 , wherein the binding agent is an aqueous solution comprising a chaotropic salt or mixture of chaotropic salts. 
     
     
         79 . The method of  claim 78 , wherein the chaotropic salt is guanidinium thiocyanate, guanidinium chloride, sodium iodide, sodium perchlorate, urea or thiourea. 
     
     
         80 - 81 . (canceled) 
     
     
         82 . The method of  claim 61 , wherein the small RNA is miRNA, siRNA, piRNA or an RNA oligonucleotide. 
     
     
         83 . The method of  claim 61 , wherein the nucleic acid sample comprises small RNA molecules and is essentially free of RNA molecules over 200 nucleotides in length. 
     
     
         84 . The method of  claim 61 , wherein the silica substrate is in the form of beads, fibers or a porous matrix. 
     
     
         85 . The method of  claim 61 , wherein the silica substrate is a (para)magnetic bead. 
     
     
         86 . The method of  claim 61 , wherein the silica substrate is immobilized. 
     
     
         87 - 90 . (canceled) 
     
     
         91 . A kit for nucleic acid purification comprising: a denaturing solvent comprising phenol; a binding agent comprising a chaotropic salt, an alcohol or a combination thereof; and a silica substrate. 
     
     
         92 . The kit of  claim 91 , wherein the silica substrate comprises magnetic beads. 
     
     
         93 . The kit of  claim 91 , further comprising one or more of the following: an affinity purification column; a further organic solvent; a detergent; a wash solution; a magnetic bead; an elution buffer; or instructions for using the kit. 
     
     
         94 . The kit of  claim 93 , wherein the purification column comprises an array of a plurality of columns.