Stabilization and isolation of extracellular nucleic acids

Patent metadata

Field	Value
Publication number	US-10144952-B2
Application number	US-201414777878-A
Country	US
Kind code	B2
Filing date	Mar 18, 2014
Priority date	Mar 18, 2013
Publication date	Dec 4, 2018
Grant date	Dec 4, 2018

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

The invention claimed is:

1. A method for stabilizing and treating an extracellular nucleic acid population comprised in a cell-containing biological sample, comprising:
a) stabilizing the cell-containing biological sample by contacting the cell-containing biological sample with butanamide; and
b) analyzing, detecting and/or further processing the extracellular nucleic acids either with or without previous isolation of the extracellular nucleic acids.

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

3. The method according to claim 1 , comprising additionally contacting the cell-containing biological sample with at least one compound according to formula 1

wherein R1 is a hydrogen residue or an 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.

4. The method according to claim 3 , wherein the compound according to formula 1 has one or both of the following characteristics:
(i) it is a N,N-dialkylpropanamide;
(ii) it is N,N-dimethylpropanamide.

5. The method according to claim 1 , wherein after contacting the cell-containing biological sample 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 claim 2 , comprising contacting for stabilization the cell-containing sample with
A) a stabilizing composition comprising:
a) butanamide;
b) at least one pancaspase inhibitor;
or
B) a stabilizing composition comprising:
a) butanamide;
b) at least one pancaspase inhibitor; and
c) at least one compound according to formula 1 and/or
d) an anticoagulant;
or
C) a stabilizing composition comprising:
a) butanamide;
b) at least one pancaspase inhibitor; and
c) a N,N-dialkylpropanamide and/or
d) EDTA.

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

8. The method according to claim 3 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 claim 1 , comprising additionally contacting the cell-containing biological sample with at least one poly(oxyethylene) polymer.

10. The method according to claim 9 , wherein:
a) the poly(oxyethylene) polymer is polyethylene glycol;
b) the poly(oxyethylene) polymer is a high molecular weight poly(oxyethylene) polymer having a molecular weight in a range of 1500 to 40000 or 3000 to 20000;
c) 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 or at least 500; and/or
d) the poly(oxyethylene) polymer is a high molecular weight poly(oxyethylene) polymer having a molecular weight in a range of 1500 to 40000 and the method comprises additionally contacting the cell-containing biological sample with a low molecular weight poly(oxyethylene) having a molecular weight in a range of 100 to 1000.

11. The method according to claim 1 , wherein after the stabilizing, 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 and the isolated nucleic acids are analysed and/or detected;
c) cells comprised in the stabilized sample are removed;
d) cells comprised in the stabilized sample are removed prior to performing an nucleic acid isolation, analysis and/or detection step;
e) 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;
f) (i) the stabilized sample, (ii) the stabilized sample from which cells have been removed and/or (iii) cells removed from the sample are stored;
g) cells are removed from the stabilized sample and are discarded; and/or
h) cells are removed from the stabilized sample and nucleic acids are isolated from cells that were removed from the stabilized sample;
i) 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.

12. The method according to claim 1 , comprising removing cells from the cell-containing biological sample between step a) and step b) to yield a cell-free or cell-depleted portion of the stabilized sample and wherein step b) comprises isolating extracellular nucleic acids from the cell-free or cell-depleted portion of the stabilized sample.

13. The method according to claim 1 , comprising isolating extracellular nucleic acids in step b) and processing and/or analyzing the isolated extracellular nucleic acids in a further step c).

14. A method for isolating extracellular nucleic acids from a cell-containing biological sample comprising the steps of
a) stabilizing the cell-containing biological sample by contacting the cell-containing biological sample with butanamide; and
b) isolating extracellular nucleic acids from the cell-containing biological sample stabilized in step a).

Assignees

• Qiagen Gmbh

Inventors

• Wyrich Ralf
• Voss Thorsten
• Grölz Daniel

Classifications

• 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

• C12Q1/6806 Primary

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

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2527/127

  the enzyme inhibitor or activator used · CPC title