Method for isolating nucleic acids

Abstract

It was found that adding a chelating agent during resuspension considerably increases the nucleic acid yield as the formation of precipitates which irreversibly adhere to the container wall is considerably reduced.

1 . A method for isolating nucleic acids from a sample, preferably a blood sample, comprising the following steps:
 a) obtaining a sample which has been stabilised by the use of at least one cationic detergent, wherein the cationic detergent has formed complexes with the nucleic acids;   b) obtaining the complexes optionally together with other sample components from the stabilised sample, wherein said complexes comprise the nucleic acids to be isolated;   c) resuspending the complexes and optionally adding one or more additives before, during and/or after resuspension, thereby obtaining a resuspended sample comprising at least   i) the nucleic acid to be isolated;   ii) at least one chaotropic agent; and   iii) at least one chelating agent; and   d) isolating nucleic acids from the resuspended sample.   
     
     
         2 . The method according to  claim 1 , wherein the resuspended sample is put on hold between step c) and step d) for at least 0.2 h, at least 0.3 h, at least 0.4 h, at least 0.5 h, at least 0.75 h or at least 1 h and/or for a time period of 0.5 h to 12 h, 1 h to 10 h, 1.5 h to 8 h, 2 h to 7 h or 3 h to 6 h. 
     
     
         3 . The method according to  claim 1 , wherein the resuspension performed in step c) has one or more of the following characteristics:
 a) a resuspension solution is added wherein said resuspension solution comprises a non-chaotropic salt, preferably an ammonium salt;   b) the chelating agent is added prior, during or after resuspension;   c) the chelating agent is added separately from the resuspension solution;   d) the chelating agent is comprised in a resuspension solution;   e) the chelating agent is added in a concentration so that the resuspended sample comprises the chelating agent in a concentration selected from 0.5 mM to 75 mM, 1 mM to 50 mM, 2.5 mM to 25 mM and 5 to 15 mM;   f) the chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA) and/or   g) at least one additive is added before, during and/or after resuspension which is selected from the group consisting of chaotropic agents, protein-degrading compounds and buffering agents and thereby is comprised in the resuspended sample.   
     
     
         4 . The method according to  claim 1 , wherein the chelating agent present in the resuspended sample
 a) reduces binding of the precipitated sample to the container comprising the sample;   b) increases the yield of the isolated nucleic acid; and/or   c) reduces variations in the nucleic acid isolation efficiency or quantity attributable to different holding times between step c) and d).   
     
     
         5 . The method according to  claim 1 , wherein said method has with respect to the chaotropic agent comprised in the resuspended sample one or more of the following characteristics:
 a) the concentration of the chaotropic agent in the resuspended sample is selected from the group consisting of 0.1 M to 4 M, 0.5 M to 3 M and 0.75 M to 2.5 M and preferably is at least 1 M;   b) the chaotropic agent is added in step c) in form of a separate solution;   c) the chaotropic agent is added in step c) after resuspension of the complexes and thereby is comprised in the resuspended sample; and/or   d) the chaotropic agent present in step c) is selected from the group consisting of chaotropic salts, guanidinium hydrochloride, guanidinium thiocyanate, guanidinium isothiocyanate, sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluroacetate, urea and preferably is GTC or GITC.   
     
     
         6 . The method according to  claim 1 , wherein a protein degrading compound is added in step c). 
     
     
         7 . The method according to  claim 6 , wherein said method has with respect to the protein degrading compound that is added in step c) one or more of the following characteristics:
 a) the protein degrading compound is a proteolytic enzyme; and/or   b) the protein degrading compound is a proteolytic enzyme selected from the group consisting of proteinases, proteases, subtilisins and subtilases, and preferably is proteinase K.   
     
     
         8 . The method according to  claim 1 , wherein step c) comprises
 aa) a resuspension solution is added which comprises a non-chaotropic salt and a chelating agent and which does not comprise a chaotropic agent and the complexes are resuspended,   bb) a chaotropic agent is added after resuspension of the complexes and thereby becomes comprised in the resuspended sample wherein preferably, the chaotropic agent is added to the resuspended complexes in form of an aqueous solution,   cc) optionally a proteolytic enzyme is added after resuspension of the complexes and thereby is included in the resuspended sample.   
     
     
         9 . The method according to  claim 1 , wherein the isolation performed in step d) comprises the following steps:
 i) digesting and/or denaturing the resuspended sample, preferably by heating and/or agitating the resuspended sample in the presence of a proteolytic enzyme;   ii) binding the nucleic acids to a solid phase using appropriate binding conditions, and   iii) optionally washing the nucleic acids;   iv) optionally eluting the nucleic acids.   
     
     
         10 . The method according to  claim 1 , wherein the sample is a blood sample and in step a) the blood sample is stabilised by contacting the blood sample with a stabilizing composition comprising
 i) a cationic compound of the general formula:
   Y + R 1 R 2 R 3 R 4 X −   
   wherein Y represents nitrogen or phosphor, preferably nitrogen   R 1 R 2 R 3  and R 4  independently, represent a branched or unbranched C 1 -C 20 -alkyl group, a C 6 -C 20 -aryl group and/or a C 6 -C 26  aralkyl group;   X −  represents an anion of an inorganic or organic, mono- or polybasic acid; and   ii) at least one proton donor.   
     
     
         11 . The method according to  claim 1 , wherein the sample is a blood sample and in step a) the blood sample is stabilised by contacting the blood sample with a stabilizing composition comprising
 (i) an amino surfactant having the following formula (2):
   R1R2R3N(O)x (2) 
   wherein,   R1 and R2 each independently is H, C1-C6 alkyl residue, C6-C12 aryl residue or C6-C12 aralkyl residue,   R3 is C1-C20 alkyl group, C6-C26 aryl residue or C6-C26 aralkyl residue,   X is an integer of 0 and 1 and   (ii) an acid or acid salt.   
     
     
         12 . A method according to  claim 1 , wherein a plurality of samples is processed and wherein the holding time between step c) and step d) differs at least between some of the resuspended samples. 
     
     
         13 . The method according to  claim 1 , having one or more of the following characteristics:
 a) the nucleic acid is RNA;   b) a plurality of samples is prepared according to steps a) to c) thereby providing a plurality of resuspended samples, wherein the resuspended samples are divided into batches and the nucleic acids are isolated from the batches according to step d) and wherein the holding time between step c) and d) varies at least between two batches;   c) step d) is performed using an automated system;   d) wherein a plurality of samples is processed manually up to step c) thereby providing a plurality of resuspended samples and wherein preferably the resuspended samples are processed using an automated system for isolating the nucleic acids in step d); and/or   e) at least RNA is isolated from a sample comprising at least RNA and DNA and wherein isolation step d) comprises the following steps   obtaining the resuspended sample comprising a proteolytic enzyme and continuing the digestion of the resuspended sample preferably by incubating the resuspended sample for at least 5 min above room temperature preferably above 50° C.;   removing at least a portion of the DNA from the resuspended and digested sample, by binding DNA to a first solid phase and separating the DNA bound to said first solid phase from the remaining sample comprising the RNA,   binding the RNA to a second solid phase, wherein at least one chaotropic agent and at least one alcohol in a concentration>30% v/v is used during this RNA binding step,   optionally performing at least one washing step for washing the RNA bound to said second solid phase, and   optionally performing a DNase digest and/or a digest using a proteolytic enzyme,   optionally eluting the RNA.   
     
     
         14 . A method according to  claim 1 , for isolating RNA from a blood sample, comprising the following steps:
 a) obtaining a sample which has been stabilised by the use of at least one cationic detergent, wherein the cationic detergent has formed complexes with the nucleic acids and wherein preferably, a cationic detergent as defined in  claim 10  or  11  was used for stabilisation;   b) obtaining the complexes optionally together with other sample components from the stabilised sample, wherein said complexes comprise the nucleic acids to be isolated;   c) resuspending the complexes and optionally adding one or more additives before, during and/or after resuspension, wherein step c) comprises:   aa) adding a resuspension solution which comprises a non-chaotropic salt and a chelating agent wherein the resuspension solution does not comprise a chaotropic salt and resuspending the complexes,   bb) adding a chaotropic agent after resuspension of the complexes wherein preferably, the chaotropic agent is added to the resuspended complexes in form of an aqueous solution,   cc) optionally adding a proteolytic enzyme after resuspension of the complexes,   thereby obtaining a resuspended sample comprising at least   i) the nucleic acid to be isolated;   ii) at least one chaotropic agent; and   iii) at least one chelating agent; and   iv) optionally a proteolytic enzyme;   and   d) isolating RNA from the resuspended sample, wherein preferably, the RNA is isolated according to the features of  claims 13 e) and wherein preferably, magnetic silica particles are used as solid phase for binding the RNA and wherein preferably, an automated system is used for RNA isolation, and wherein a plurality of samples is prepared according to steps a) to c) thereby providing a plurality of resuspended samples, wherein the resuspended samples are divided into batches and the nucleic acids are isolated from the batches according to step d) and wherein the holding time between step c) and d) varies at least between two batches.   
     
     
         15 . A method for isolating nucleic acids from a sample, preferably a blood sample, wherein the nucleic acids are isolated from a plurality of samples and wherein variations in yield and quality of the nucleic acids that are isolated from said plurality of samples which result from that the plurality of samples prepared for isolation have diverging holding times before the nucleic acids are isolated from the prepared samples are thereby reduced that the samples prepared for isolation comprise at least one chaotropic agent and at least one chelating agent. 
     
     
         16 . The method according to  claim 15 , wherein the nucleic acids are isolated according to a method that comprises the following steps:
 a) obtaining a sample which has been stabilised by the use of at least one cationic detergent, wherein the cationic detergent has formed complexes with the nucleic acids;   b) obtaining the complexes optionally together with other sample components from the stabilised sample, wherein said complexes comprise the nucleic acids to be isolated;   c) resuspending the complexes and optionally adding one or more additives before during and/or after resuspension, thereby obtaining a resuspended sample comprising at least   i) the nucleic acid to be isolated;   ii) at least one chaotropic agent; and   iii) at least one chelating agent; and   d) isolating nucleic adds from the resuspended sample.   
     
     
         17 . Use of a chelating agent in order to prevent or reduce the formation of a precipitate that attaches to the container wall of a sample comprising at least one chaotropic agent and nucleic acids. 
     
     
         18 . The use according to  claim 17 , wherein the sample comprises at least one ingredient selected from the group of a cationic detergent, a protein-degrading compound, a salt and/or a buffer.