Use of tde for isolation of nucleic acids

Abstract

The invention provides the use of tetraethylene glycol dimethyl ether for adsorbing nucleic acids to solid phases such as those with silica surfaces. To this end, the invention also provides compositions comprising TDE. Methods are disclosed and claimed to purify nucleic acids from samples, as well as kits useful for performing these methods. Particularly, the invention encompasses methods for the purification of nucleic acids with low molecular weight. The nucleic acids purified by a method of the invention are suited for assays aiming at the detection of a target nucleic acid.

1 - 24 . (canceled) 
     
     
         25 . A composition effective for isolating nucleic acids, the composition comprising between about 10% and 75% tetraethylene glycol dimethyl ether (TDE) by volume, an aqueous buffer, and a chaotropic agent, with the proviso that acetate at a pH of from 1 to 6 and a concentration of acetate from 5 mM to 200 mM are excluded. 
     
     
         26 . The composition according to  claim 25 , wherein the nucleic acids comprise 150 or more nucleotides and the composition comprises between about 10% and 40% TDE by volume. 
     
     
         27 . The composition according to  claim 25 , wherein the nucleic acids comprise less than 150 nucleotides and the composition comprises between about 40% and 75% TDE by volume. 
     
     
         28 . The composition of  claim 26 , wherein the concentration of TDE in the composition is about 20% by volume. 
     
     
         29 . The composition of  claim 25 , further comprising a compound selected from the group consisting of 1,3-dioxolan, 1,3-dioxan, 5-hydroxy-1,3-dioxane, and 4-hydroxymethyl-1,3-dioxolane. 
     
     
         30 . The composition of  claim 25 , further comprising a detergent. 
     
     
         31 . The composition of  claim 30 , wherein the detergent is selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, cetyltrimethylammoniumbromide, deoxycholic acid, sodium lauroyl sarcosine, TRITON-X100, TWEEN 20, octyl beta-D-glucoside, Nonidet P40, BRIJ 35P, and Sulfobetaine 14. 
     
     
         32 . The composition of  claim 25 , wherein the pH is between about 4 and 7.5. 
     
     
         33 . The composition of  claim 25 , wherein the chaotropic agent is selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, urea, sodium acetate, an alkali perchlorate, and an alkali halogenide. 
     
     
         34 . The composition of  claim 25 , wherein the chaotropic agent has a concentration between about 0.5 M and 10 M. 
     
     
         35 . A method for adsorbing a nucleic acid onto a solid phase, the method comprising the steps of:
 providing a sample comprising the nucleic acid,   dissolving the sample in a liquid composition comprising between about 10% and 75% tetraethylene glycol dimethyl ether (TDE) by volume, an aqueous buffer, and a chaotropic agent,   contacting the liquid composition containing the dissolved sample with a solid phase whereby the nucleic acid is adsorbed onto the solid phase.   
     
     
         36 . The method according to  claim 35 , wherein the nucleic acid comprises 150 or more nucleotides and the composition comprises between about 10% and 40% TDE by volume. 
     
     
         37 . The method according to  claim 35 , wherein the nucleic acid comprises less than 150 nucleotides and the composition comprises between about 40% and 75% TDE by volume. 
     
     
         38 . The method of  claim 35 , wherein the solid phase comprises a porous or non-porous silica substrate. 
     
     
         39 . The method of  claim 35 , wherein the solid phase comprises a substrate selected from the group consisting of glass fibers and quartz fibers. 
     
     
         40 . The method of  claim 35 , wherein the solid phase comprises magnetic particles with a silica surface. 
     
     
         41 . The method of  claim 35 , further comprising the steps of:
 separating the solid phase with the adsorbed nucleic acid from the liquid composition,   contacting the solid phase with the adsorbed nucleic acid with a desorption solution, thereby desorbing the nucleic acid from the solid phase and dissolving the nucleic acid in the solution,   separating the solution with the nucleic acid from the solid phase, thereby purifying the nucleic acid, and optionally,   precipitating the nucleic acid from the solution and isolating the precipitated nucleic acid, thereby further purifying the nucleic acid.   
     
     
         42 . The method of  claim 41 , further comprising the step of, after separating the solid phase with the adsorbed nucleic acid from the liquid composition, washing the solid phase with a washing solution comprising water and TDE whereby the nucleic acid remains bound to the solid phase. 
     
     
         43 . The method of  claim 42 , wherein the washing solution additionally comprises a salt. 
     
     
         44 . A composition comprising tetraethylene glycol dimethyl ether (TDE) and magnetic particles with a silica surface. 
     
     
         45 . A method for purifying a nucleic acid with low molecular weight comprising the steps of:
 providing a sample comprising the nucleic acid with low molecular weight,   dissolving the sample in a liquid composition comprising an aqueous buffer, tetraethylene glycol dimethyl ether (TDE) at a concentration between 10% and 75% by volume, and a chaotropic agent,   contacting the liquid composition containing the dissolved sample with a solid phase whereby the nucleic acid with low molecular weight is adsorbed onto the solid phase,   separating the solid phase with the adsorbed nucleic acid with low molecular weight from the liquid composition,   washing the solid phase with a washing solution comprising an organic solvent at a concentration between 40% and 100%,   contacting the solid phase with the adsorbed nucleic acid with low molecular weight with an aqueous desorption solution containing solutes in a lower concentration compared to the liquid composition, thereby desorbing the nucleic acid with low molecular weight from the solid phase and dissolving the nucleic acid with low molecular weight in the desorption solution,   separating the solution with the nucleic acid with low molecular weight from the solid phase, thereby purifying the nucleic acid with low molecular weight; and optionally   precipitating the nucleic acid with low molecular weight from the solution and isolating the precipitated nucleic acid with low molecular weight, thereby further purifying the nucleic acid with low molecular weight, wherein the nucleic acid with low molecular weight is selected from the group consisting of single-stranded nucleic acids having a size between 10 and 150 bases and double-stranded nucleic acids having a size between 5 and 75 bases.   
     
     
         46 . The method of  claim 45  wherein the concentration of TDE in the composition is about 40% by volume. 
     
     
         47 . A method for determining the presence of a nucleic acid in a sample comprising the steps of:
 dissolving a sample containing the nucleic acid in a liquid composition comprising, an aqueous buffer, a chaotropic agent, and about 10% to about 75% tetraethylene glycol dimethyl ether (TDE) by volume, whereby the sample is dissolved in the liquid composition,   contacting the liquid composition containing the dissolved sample with a solid phase, whereby the nucleic acid is adsorbed onto the solid phase,   separating the solid phase with the adsorbed nucleic acid from the liquid composition,   contacting the solid phase with the adsorbed nucleic acid with an aqueous desorption solution containing solutes in a lower concentration than the liquid composition, thereby desorbing the nucleic acid from the solid phase and dissolving the nucleic acid in the desorption solution,   separating the solution with the nucleic acid from the solid phase, thereby purifying the nucleic acid, and   detecting in the solution the presence of the nucleic acid, thereby determining the presence of the nucleic acid.   
     
     
         48 . The method of  claim 47  wherein the nucleic acid is RNA or DNA. 
     
     
         49 . The method of  claim 47  wherein the nucleic acid is RNA and the detection step comprises:
 reverse transcribing the RNA to form a cDNA, 
 amplifying, by means of a polymerase chain reaction, the cDNA, and 
 detecting the presence of the cDNA, thereby determining the presence of the nucleic acid. 
 
     
     
         50 . A kit of parts comprising an instruction manual, packaging material, containers, tetraethylene glycol dimethyl ether (TDE), a concentrated stock solution of a buffer salt and a chaotropic agent selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, an alkali perchlorate, and an alkali iodide, and chromatographic and filtering material comprising a material with a surface capable of interacting with the phosphate residues in the backbone of nucleic acids. 
     
     
         51 . A kit of parts comprising an instruction manual, packaging material, containers, a suspension of silica-coated magnetic particles in tetraethylene glycol dimethyl ether (TDE), and a concentrated stock solution of a buffer salt and a chaotropic agent selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, an alkali perchlorate, and an alkali iodide.