Stabilization and isolation of extracellular nucleic acids

Patent metadata

Field	Value
Publication number	US-2016281132-A1
Application number	US-201414777878-A
Country	US
Kind code	A1
Filing date	Mar 18, 2014
Priority date	Mar 18, 2013
Publication date	Sep 29, 2016
Grant date	—

Abstract

The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using butanamide.

First claim

1 . A method for stabilizing an extracellular nucleic acid population comprised in a cell-containing biological sample by contacting the cell-containing biological sample with butanamide.

2 . The method according to claim 1 , wherein the cell-containing biological sample is additionally contacted with a caspase inhibitor.

3 . The method according to claim 1 or 2 , wherein the cell-containing biological sample is additionally contacted with at least one compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, a C1-C4 alkyl residue or a C1-C3 alkyl residue, more preferred a C1-C2 alkyl residue, R2 and R3 are identical or different and are selected from a hydrogen residue and a hydrocarbon residue with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen, sulphur or selenium residue, preferably R4 is oxygen.

4 . The method according to claim 3 , wherein the compound according to formula 1 is a N,N-dialkylpropanamide, preferably N,N-dimethylpropanamide.

5 . The method according to one or more of claims 1 to 4 , in particular claim 2 , wherein after the cell-containing biological sample has been contacted with butanamide and optionally further additives used for stabilization, the resulting mixture comprises butanamide in a concentration range of 0.25% (w/v) to 15% (w/v), 0.5% (w/v) to 12.5% (w/v), 0.75% (w/v) to 10% (w/v), 1% (w/v) to 9% (w/v), 1.25% (w/v) to 8% (w/v), 1.5% (w/v) to 7% (w/v), 1.75% (w/v) to 6% (w/v), 1.8% (w/v) to 5.5% (w/v), 1.9% (w/v) to 5.25% (w/v), 2% (w/v) to 5% (w/v), 2.1% (w/v) to 4.75% (w/v), 2.2% (w/v) to 4.5% (w/v), 2.3% (w/v) to 4.25% (w/v), 2.4% (w/v) to 4% (w/v), 2.5% (w/v) to 3.75% (w/v) or 0.75% (w/v) to 2% (w/v).

6 . The method according to one or more of claims 2 to 5 , wherein for stabilization, the cell-containing sample is contacted with a stabilizing composition comprising:
a) butanamide;
b) at least one pancaspase inhibitor;
c) optionally at least one compound according to formula 1, preferably a N,N-dialkyl-carboxylic acid amide, more preferred a N,N-dialkylpropanamide; and
d) optionally an anticoagulant, preferably a chelating agent, more preferably EDTA.

7 . The method according to one or more of claims 1 to 6 , wherein the stabilization (i) does not involve the use of additives in a concentration wherein said additives would induce or promote lysis of nucleated cells, (ii) wherein the stabilization does not involve the use of a cross-linking agent that induces protein-nucleic acid and/or protein-protein crosslinks and/or (iii) wherein the stabilization does not involve the use of toxic agents.

8 . The method according to one or more of claims 3 to 7 , in particular one or more of claims 7 to 25 , for stabilizing an extracellular nucleic acid population comprised in a blood sample, comprising contacting the blood sample with butanamide, at least one caspase inhibitor, at least one compound according to formula 1 and an anticoagulant, wherein the release of genomic DNA from cells contained in the blood sample into the cell-free portion of the blood sample is reduced.

9 . The method according to one or more of claims 1 to 8 , wherein the cell-containing biological sample is additionally contacted with at least one poly(oxyethylene) polymer.

10 . The method according to claim 9 , wherein the poly(oxyethylene) polymer is polyethylene glycol.

11 . The method according to claim 9 or 10 , wherein the poly(oxyethylene) polymer is a high molecular weight poly(oxyethylene) polymer having a molecular weight of at least 1500, preferably in a range selected from 2000 to 40000, 2500 to 30000, 3000 to 20000, 3500 to 15000, 4000 to 10000, 4500 to 9000, 5000 to 8000 and 5500 to 7000.

12 . The method according to one or more of claims 9 to 11 , wherein at least two poly(oxyethylene) polymers are used for stabilization which differ in their molecular weight, wherein the difference in the molecular weight is at least 100, preferably at least 200, at least 300, at least 400 or at least 500.

13 . The method according to claim 11 , wherein the cell-containing biological sample is additionally contacted with a low molecular weight poly(oxyethylene) having a molecular weight of 1000 or less, preferably in the range selected from 100 to 800, 150 to 700, 200 to 600 and 200 to 500.

14 . The method according to one or more of claims 1 to 13 , wherein after the stabilization period, the method comprises one or more of the following
a) the stabilized sample is subjected to a nucleic acid analysis and/or detection method;
b) extracellular nucleic acids are isolated from the stabilized sample;
c) extracellular nucleic acids are isolated from the stabilized sample and the isolated nucleic acids are analysed and/or detected;
d) cells comprised in the stabilized sample are removed;
e) cells comprised in the stabilized sample are removed prior to performing an nucleic acid isolation, analysis and/or detection step;
f) cells are removed from the stabilized sample and extracellular nucleic acids are isolated from the cell-free or cell-depleted portion of the stabilized sample;
g) (i) the stabilized sample, (ii) the stabilized sample from which cells have been removed and/or (iii) cells removed from the sample are stored;
h) cells are removed from the stabilized sample and are discarded; and/or
i) cells are removed from the stabilized sample and nucleic acids are isolated from cells that were removed from the stabilized sample;
j) cells are removed from the stabilized sample and extracellular nucleic acids are isolated from the cell-free or cell-depleted portion of the stabilized sample using a size selective nucleic acid isolation method.

15 . A method for isolating nucleic acids from a stabilized cell-containing biological sample comprising the steps of
a) stabilizing the cell-containing biological sample according to the method defined in one or more of claims 1 to 14 ; and b) isolating nucleic acids, in particular extracellular nucleic acids.

16 . The method according to claim 15 , comprising removing cells from the cell-containing biological sample between step a) and step b) and wherein step b) comprises isolating extracellular nucleic acids from the cell-free or cell-depleted portion of the stabilized sample.

17 . The method according to claim 15 or 16 , wherein the isolated extracellular nucleic acids are in a further step c) processed and/or analysed and preferably are
i) modified;
ii) contacted with at least one enzyme;
iii) amplified;
iv) reverse transcribed;
v) cloned;
vi) sequenced;
vii) contacted with a probe;
viii) detected;
ix) quantified; and/or
x) identified.

18 . A composition suitable for stabilizing a cell-containing biological sample wherein the composition comprises butanamide and at least one further additive selected from the group consisting of an apoptosis inhibitor, an anticoagulant and a compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, a C1-C4 alkyl residue or a C1-C3 alkyl residue, preferably a C1-C2 alkyl residue, R2 and R3 are identical or different and are selected from a hydrogen residue and a hydrocarbon residue with a length of the carbon chain of 1-20 at

Assignees

• Qiagen Gmbh

Inventors

• Wyrich Ralf
• Voss Thorsten
• Grölz Daniel

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title