# US 8,785,120 B2 · Method for the treatment of a sample containing biomolecules > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,785,120 B2 - Document ID: US8785120B2 - Status: Granted - Kind: B2 - Application number: 12/862,156 - Filed: 2010-08-24 - Published: 2010-12-23 - Granted: 2014-07-22 - Earliest claimed: 2005-04-01 - Assignees: Qiagen GmbH - Applicants: Christian Korfhage; Friederike Wilmer; Dirk Loffert; Ralf Himmelreich; Claudia Fritz; Kathleen Rieske - Inventors: Christian Korfhage; Friederike Wilmer; Dirk Loffert; Ralf Himmelreich; Claudia Fritz; Kathleen Rieske - Web page: https://patentgrep.com/patent/US8785120B2 ## CPC - C12N 1/06: Lysis of microorganisms - C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor} - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The invention generally provides a method for the sample preparation for a subsequent preparation, processing or analysis method of a sample containing an at least one species of nucleic acid and/or one species of protein, whereby the method comprises the following steps: A) providing a sample which contains at least one species of a nucleic acid and/or of a protein, B) contacting the sample with a fluid or solid composition to produce a fluid sample preparation, whereby the composition contains at least a nitrogenous compound, which is selected from the group consisting of a) polyamines, b) amino acids, and oligo- and polypeptides, c) nitrogenous heterocyclic compounds, including homo-oder heteropolymeres, which comprise these nitrogenous compounds, d) amines of the type R1R2NR3, whereby R1, R2 and R3 are chosen independently from one another from the group consisting of H, C1-C5-alkyl groups and aryl groups, whereby R1, R2 and R3 are not H simultaneously, e) carbonxylic acid amides, f) inorganic ammonium salts, g) ammonium groups containing inner salt compounds, h) antibiotica binding nucleic acid, i) compounds which bind in the small cavity of the DNA, and mixtures of two or more of these compounds. The invention provides in particular a method for the lysis of a biological sample, and methods for the stabilization of biomolecules, a method for the decrease of inhibiting effects in a sample containing biomolecules and a differential masking method. ## Claims ### Claim 1 A method for stabilizing at least one species of nucleic acid and species of protein comprised in a sample, said method comprising mixing the sample with a fluid preparation to form a sample preparation comprising the at least one species of nucleic acid and the at least one species of the protein, and wherein the fluid preparation comprises at least one nitrogenous compound which is at least one selected from the group consisting of: a) polyamines; b) amino acids, and oligo and polypeptides; c) nitrogenous heterocyclic compounds including homo or hetero polymers which comprise at least one nitrogenous compound; d) amines of the type R1R2NR3, whereby R1, R2 and R3 are chosen independently from one another and are selected from the group consisting of H, C1-C5 alkyl groups and aryl groups, whereby R1, R2 and R3 are not H simultaneously; e) carboxylic acid amides; f) inorganic ammonium salts; g) inner salt compounds containing ammonium groups; h) antibiotics binding nucleic acid; and i) compounds which bind in the small cavity of DNA; wherein an amount of the nitrogenous compound is chosen so as to minimize precipitation of the nucleic acid and protein present during and after said mixing, wherein the ratio of concentration of the at least one species of nucleic acid and protein in the sample preparation is the same as the ratio of concentration of the at least one species of nucleic acid and protein in the sample, and wherein the species of nucleic acid and species of protein in the sample are not encapsulated by a cell membrane. ### Claim 2 (depends on 1) A method of claim 1, wherein at least one of said species of nucleic acid or protein is immobilized on a solid carrier before, during and/or after said mixing of said sample with said fluid preparation. ### Claim 3 (depends on 1) A method of claim 1, wherein at least one of said species of nucleic acid and/or protein in said sample is dissolved and/or suspended after said mixing with said fluid preparation. ### Claim 4 A method for stabilizing at least one species of nucleic acid and species of protein comprised in a sample, said method comprising mixing the sample with a fluid preparation to form a sample preparation comprising the at least one species of nucleic acid and the at least one species of the protein, and wherein the fluid preparation comprises at least one nitrogenous compound which is at least one selected from the group consisting of: a) polyamines; b) amino acids, and oligo and polypeptides; c) nitrogenous heterocyclic compounds including homo or hetero polymers which comprise at least one nitrogenous compound; d) amines of the type R1R2NR3, whereby R1, R2 and R3 are chosen independently from one another and are selected from the group consisting of H, C1-C5 alkyl groups and aryl groups, whereby R1, R2 and R3 are not H simultaneously; e) carboxylic acid amides; f) inorganic ammonium salts; g) inner salt compounds containing ammonium groups; h) antibiotics binding nucleic acid; and i) compounds which bind in the small cavity of DNA; wherein an amount of the nitrogenous compound is chosen so as to minimize precipitation of the nucleic acid and protein present during and after said mixing, wherein the ratio of concentration of the at least one species of nucleic acid and protein in the sample preparation is the same as the ratio of concentration of the at least one species of nucleic acid and protein in the sample, wherein the species of nucleic acid and species of protein in the sample are not encapsulated by a cell membrane, and wherein said sample comprises a biological sample which was subjected to lysis and optionally subjected to at least one further purification step, before said mixing with said fluid preparation. ### Claim 5 (depends on 1) A method of claim 1, wherein said sample comprises a biological sample which comprises at least one species of nucleic acid and at least one species of protein and said biological sample is contacted with said fluid preparation to obtain a lysate. ### Claim 6 (depends on 1) A method of claim 1, wherein DNA and/or RNA is dissolved, suspended, or immobilized on a carrier in said sample preparation. ### Claim 7 (depends on 1) A method of claim 1, wherein the nitrogenous compound is selected from the group of a) polyamines, b) polar, alkaline and acidic amino acids, c) heterocyclic compounds, and d) antibiotics binding nucleic acid selected from the group consisting of distamycines, mitomycines, norfloxacins, streptozocine, duocarmycines, actinomycines, and aminoglycisides. ### Claim 8 (depends on 7) A method of claim 7, wherein said polyamines are selected from the group consisting of unbranched polyamines with 2 to 6 amino groups, said amino acids are selected from the group consisting of lysine, arginine, histidine, and glutamic acid, the heterocyclic compounds are selected from the group consisting of imidazole, 2,3-dimethylpyrazine, pyrimidine and guanine. ### Claim 9 (depends on 1) A method of claim 1, wherein said nitrogenous compound comprises spermidine. ### Claim 10 (depends on 4) A method of claim 4, wherein said lysis is conducted using a detergent and/or protease. ### Claim 11 (depends on 10) A method of claim 10, wherein said lysis is conducted using proteinase K. ### Claim 12 (depends on 4) A method of claim 4, wherein said mixing is conducted after an enzymatic degradation by a nuclease. ### Claim 13 A method for stabilization of at least one biomolecule contained in a sample comprising at least one species of nucleic acid and at least one species of protein, the method comprising mixing the sample with a fluid preparation to form a sample preparation, and wherein the fluid preparation comprises at least one nitrogenous compound selected from the group consisting of arginine, imidazole, proline, lysine, spermine, spermidine, glutamic acid, indazole, thymine, thymidine, guanine, guanosine, adenine, histidine, tryptophane, and ammonium sulphate, wherein the species of nucleic acid and species of protein in the sample are not encapsulated by a cell membrane. ### Claim 14 (depends on 1) A method of claim 1, wherein by employing said at least one nitrogenous compound, direct separation of species comprised in the sample preparation is not necessary and/or can take place at a later time. ### Claim 15 (depends on 13) The method of claim 13, wherein an amount of the nitrogenous compound is chosen so as to minimize precipitation of the biomolecule during and after said mixing, and wherein the ratio of concentration of the at least one biomolecule in the sample preparation is the same as the ratio of concentration of the biomolecule in the sample. ### Claim 16 (depends on 13) The method of claim 13, wherein said sample comprises a biological sample which was subjected to lysis and optionally subjected to at least one purification step before said mixing with said fluid preparation. ### Claim 17 (depends on 13) The method of claim 13, wherein the sample preparation is suitable for use directly for analysis of the biomolecule without any further reconditioning step. ### Claim 18 (depends on 1) The method of claim 1, wherein the concentration of the nitrogenous compound in the sample preparation is from 0.001 mM to 1 M. ### Claim 19 (depends on 18) The method of claim 18, wherein: (a) the nitrogenous compound is a polyamine and the concentration of the polyamine in the sample preparation is from 0.001 mM and 15 mM; or (b) the nitrogenous compound is an amino acid and the concentration of the amino acid in the sample preparation is from 0.001 mM to 20 mM; or (c) the nitrogenous compound is a nitrogenous heterocycle and the concentration of the nitrogenous heterocycle in the sample preparation is from 0.001 to 20 mM; or (e) the nitrogenous compound is a carboxylic acid amines and the concentration of the carboxylic acid amines in the sample preparation is from 0.001 to 15 mM; or (f) the nitrogenous compound is an inorganic ammonium compound and the concentration of the inorganic ammonium compound in the sample preparation is from 0.001 mM to 100 mM; or (g) the nitrogenous compound is an inner salt compound containing ammonium groups and the concentration of the inner salt compound containing ammonium groups in the sample preparation is from 0.001 mM to 300 mM. ### Claim 20 (depends on 1) The method of claim 1, wherein the sample preparation is suitable for analysis of the nucleic acid or protein without any further reconditioning step.