US 2014/0154693 A1 · REAGENT USABLE FOR THE ISOLATION AND/OR PURIFICATION OF NUCLEIC ACIDS

Abstract

The present invention provides a special reagent for the pretreatment of sample materials. The pretreatment with the reagent according to the invention enables the isolation of nucleic acids by a uniform method from very diverse sample materials, in particular different bioprocess samples, even if the nucleic acids are only present in small amounts in the sample materials. The reagent used for the pretreatment comprises, as key component, at least one compound comprising an amino group. The invention further relates to methods of purification of nucleic acids, in which the sample material is pretreated correspondingly, and suitable kits.

Claims74

Dependent claims

-31. (canceled)

A method of isolation and/or purification of nucleic acids from a sample material, wherein the sample material is a bioprocess sample, the bioprocess sample is a purification fraction obtained in the purification process for a biotechnological product, and the nucleic acids to be isolated represent a contamination of the bioprocess sample, comprising:

(a) adding a reagent to the sample material, wherein the reagent comprises one or more compounds comprising an amino group selected from one or more of the following groups:
(i) polar-neutral proteinogenic α-aminocarboxylic acids,
(ii) nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids,
(iii) basic α-aminocarboxylic acids,
(iv) non-proteinogenic aminocarboxylic acids selected from the group consisting of β-alanine, D-alanine, L-homoserine and D-valine,
(v) aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vi) peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vii) a compound of the general formula R3R4N—CR1R2—COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen,
(viii) a compound of the general formula R3R4N—CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl,
(b) optionally mixing this reagent with the sample material,
(c) isolating and/or purifying nucleic acid(s) from the sample material that has been contacted with and/or mixed with the reagent, and
(d) optionally quantitatively and/or qualitatively detecting at least a portion of the isolated nucleic acid(s).

9 more elements

A method for preparing a bioprocess sample for subsequent nucleic acid isolation and/or purification, wherein the bioprocess sample is a purification fraction obtained in a purification process for a biotechnologically produced product and wherein the target nucleic acid represents a contamination of the bioprocess sample, comprising:

(a) pretreating the bioprocess sample with a reagent, wherein the reagent comprises at least one or more compounds with an amino group selected from one or more of the following groups:
a. (i) polar-neutral proteinogenic α-aminocarboxylic acids,
(ii) nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids,
(iii) basic α-aminocarboxylic acids,
(iv) non-proteinogenic aminocarboxylic acids selected from the group comprising β-alanine, D-alanine, L-homoserine and D-valine,
(v) aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vi) peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vii) a compound of the general formula R3R4N-CR1R2-COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, and wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen, and
(viii) a compound of the general formula R3R4N-CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl,
and optionally one or more of the following components:
b. at least one detergent,
c. at least one complexing agent,
d. at least one protein.

10 more elements

A reagent for isolation and/or purification of nucleic acids from a sample material, in particular for preparation of the sample material, wherein the sample material is a bioprocess sample and wherein the bioprocess sample is a purification fraction obtained in a purification process for a biotechnologically produced product and wherein the nucleic acids to be isolated represent a contamination of the bioprocess sample, which comprises the following components:

a. at least one or more compounds comprising an amino group, selected from one or more of the following groups:
i. polar-neutral proteinogenic α-aminocarboxylic acids, preferably asparagine, cysteine, glutamine, glycine, threonine and serine,
ii. nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids, preferably alanine, leucine, isoleucine, methionine, proline and valine,
iii. basic α-aminocarboxylic acids, preferably arginine, lysine and ornithine,
iv. non-proteinogenic aminocarboxylic acids, selected from the group comprising β-alanine, D-alanine, L-homoserine and D-valine,
v. aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably consisting of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably formed from identical, two or several different aminocarboxylic acid monomers, preferably in conjunction with halides, preferably selected from the group poly-L-lysine, poly-L-lysine hydrobromide, poly-L-lysine hydrochloride, poly(L-glutamine, L-alanine), poly(L-glutamine, L-lysine) hydrobromide, poly-L-ornithine,
vi. peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably consisting of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably selected from identical or different aminocarboxylic acids mentioned in (i.) to (iv.), preferably glycine dimers, glycine trimers and glycine tetramers,
vii. a compound of the general formula R3R4N—CR1R2—COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, and wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen, preferably selected from the group N-(tri(hydroxymethyl)methyl)glycine(tricine) and N,N-bis(2-hydroxyethyl)glycine (bicine),
viii. a compound of the general formula R3R4N—CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl, wherein preferably at least one of R5, R6 and R7 is a hydroxyalkyl, and wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, wherein the compound is preferably Tris(hydroxymethyl)-aminomethane (Tris), and
b. at least one detergent or mixtures of detergents, preferably selected from the group of non-ionic surfactants comprising alkylglucosides and/or polyoxyethylene-alkylphenyl ethers,
c. optionally at least one complexing agent or mixtures of complexing agents, and
d. at least one protein or mixtures of proteins, wherein the protein is selected from the group of albumins and/or globulins.

9 more elements

A method for detecting at least one target nucleic acid(s) from different bioprocess samples obtained from a purification process for a biotechnological product, wherein the different bioprocess samples are purification fractions obtained in the purification process, and wherein the at least one target nucleic acid(s) to be detected represents a contamination of the different bioprocess samples, comprising:

(a) pretreating the different bioprocess samples with a reagent, wherein the reagent comprises at least one or more compounds with an amino group selected from one or more of the following groups:
(i) polar-neutral proteinogenic α-aminocarboxylic acids,
(ii) nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids,
(iii) basic α-aminocarboxylic acids,
(iv) non-proteinogenic aminocarboxylic acids selected from the group comprising β-alanine, D-alanine, L-homoserine and D-valine,
(v) aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vi) peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vii) a compound of the general formula R3R4N-CR1R2-COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, and wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen, and
(viii) a compound of the general formula R3R4N-CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl;
(b) optionally mixing the reagent with the different bioprocess samples;
(c) isolating and/or purifying the at least one target nucleic acid(s) from the different bioprocess samples that are contacted with and/or mixed with the reagent;
and
(d) quantitatively and/or qualitatively detecting at least a portion of the isolated or purified target nucleic acid(s).

10 more elements

Dependencies are inferred from the claim text.

Reagent usable for the isolation and/or purification of nucleic acids

Life of this patent

Reagent usable for the isolation and/or purification of nucleic acids

Life of this patent

Abstract

Claims74