COMPOSITIONS FOR STABILIZING DNA, RNA AND PROTEINS IN SALIVA AND OTHER BIOLOGICAL SAMPLES DURING SHIPPING AND STORAGE AT AMBIENT TEMPERATURES

• US 20130209997A1
• Filed: 07/26/2011
• Published: 08/15/2013
• Est. Priority Date: 07/26/2010
• Status: Active Grant

• Current Assignee: Biomatrica, Inc. (Exact Sciences Corporation)
• Original Assignee: Biomatrica, Inc. (Exact Sciences Corporation)
• Inventors: Whitney, Scott E., Muller, Rolf, Ghirmai, Senait
• Granted Patent: US 9,845,489 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

Compositions and methods are disclosed for substantially liquid, gel, suspension, slurry, semisolid and/or colloid storage of biological samples following admixture with the herein disclosed storage composition, permitting substantial recovery of biological activity following storage without refrigeration. In certain embodiments, unfractionated saliva samples may be stored without refrigeration for weeks, months or years in a form that permits recovery of intact DNA following the storage period.

Claims

1. A composition for substantially stable storage of nucleic acid and polypeptide molecules in a biological sample, comprising:

(a) one or more compounds of formula (I) or formula (II);

wherein formula (I) is;

2. The composition of claim 1 further comprising one or more of:

(a) a chelating agent,

(b) a reducing agent,

(c) a pH buffer, and

(d) water.

3. The composition of claim 1 or claim 2 wherein the pharmaceutically acceptable anion is selected from the group consisting of bromide, chloride, iodide, C₁-C₁₂alkylsulfonate, hexafluorophosphate, methylsulfate, ethylsulfate, tetrafluoroborate, trifluoromethanesulfonate and bis(trifluoromethylsulfonyl)imide.

4. The composition of any one of claims 1-3 wherein in formula (I):

A is —

C(R⁴)═

C(R⁴)—

;

R¹ is C₁-C₈alkyl;

R² is hydrogen or C₁-C₈alkyl; and

R³ is C₁-C₁₆alkyl, optionally substituted aryl or optionally substituted aralkyl;

each R⁴ is independently hydrogen, cyano, nitro, —

C(O)OR⁵, —

C(O)N(R⁵)₂, —

N(R⁵)₂, halo, haloalkyl, hydroxy, alkyl, alkoxy, haloalkoxy, alkoxyalkyl, alkenyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl or optionally substituted heteroarylalkyl; and

each R⁵ is independently hydrogen, alkyl, aryl or aralkyl.

5. The composition of any one of claims 1-5 wherein the compound of formula (I) is selected from the group consisting of:

1-methyl-3-carboxyethyl-imidazolium bromide,

1-hexyl-3-methylimidazolium bromide,

1-octyl-3-methylimidazolium bromide

1-decyl-3-methylimidazolium bromide, and

1-(2-hydroxyethyl)-3-methylimidazolium bromide.

6. The composition of claim 1 or claim 2 wherein the precipitating agent is selected from the group consisting of:

5-(4-dimethyl)amino benzylidene rhodanine,

sulfosalicylic acid,

lithium chloride, and

lithium hydroxide.

7. The composition of claim 1 or claim 2 wherein the lower alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, and isobutanol (2-methylpropan-1-ol).

8. The composition of claim 1 or claim 2 wherein the chaoptrope is selected from the group consisting of:

guanidine hydrochloride, guanidine thiocyanate, potassium thiocyanate, sodium thiocyanate and urea.

9. The composition of claim 1 or claim 2 wherein the chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), trans-1,2-diaminocyclohexane-N,N,N′

,N′

-tetraacetic acid (CDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′

,N′

-tetraacetic acid (BAPTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N-(2-hydroxyethyl)ethylenediamine-N,N′

,N′

-triacetic acid, and nitrilotriacetic acid (NTA).

10. The composition of claim 1 or claim 2 wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, thiosulfate, TCEP (tris-(2-carboxyethyl) phosphine), dithiothreitol and dithioerythritol.

11. The composition of claim 1 or claim 2 wherein the pH buffer is selected from the group consisting of citric acid, tartaric acid, malic acid, sulfosalicylic acid, sulfoisophtalic acid, oxalic acid, borate, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid), TAPS (N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), bicine (N,N-Bis(2-hydroxyethyl)glycine), tricine (N-[Tris(hydroxymethyl)methyl]glycine), tris (tris(hydroxymethyl)aminomethane) and bis-tris (2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol).

12. The composition of claim 1 or claim 2 which further comprises a surfactant or a detergent.

13. The composition of claim 11 wherein the surfactant or detergent is selected from Triton®

X-100, Nonidet®

P40 and a Brij®

detergent.

14. The composition of claim 1 or claim 2 wherein the nucleic acid molecules comprise one or more of DNA molecules and RNA molecules.

15. The composition of claim 1 or claim 2 wherein the biological sample comprises vertebrate saliva or sputum.

16. The composition of claim 15 wherein the vertebrate saliva or sputum is unfractionated.

17. The composition of claim 1 or claim 2 wherein the biological sample comprises human saliva.

18. The composition of claim 17 wherein the human saliva is unfractionated.

19. The composition of claim 1 or claim 2 wherein the biological sample is selected from the group consisting of saliva, sputum, DNA, RNA, blood, buffy coat from blood, urine, feces, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, plasmid DNA, mRNA, tRNA, rRNA, siRNA, micro RNA, hnRNA, cDNA, a protein, a polypeptide, a lipid, a glycolipid, a glycoprotein, an oligosaccharide, a polysaccharide, a vaccine, a cell, a sorted or selected cell, a tissue, a cell lysate, homogenate or extract, a tissue lysate, homogenate or extract, a blood sample, biopsy specimen, tissue explant, organ culture and a biological fluid.

20. A substantially stably-stored nucleic acid molecule from a biological sample, comprising the biological sample admixed with the composition of any one of claims 1-19.

21. A substantially stably-stored nucleic acid molecule from a vertebrate saliva or sputum sample, comprising vertebrate saliva or sputum admixed with the composition of any one of claims 1-19.

22. A substantially stably-stored polypeptide molecule from a human saliva sample, comprising human saliva admixed with the composition of any one of claims 1-19.

23. The substantially stably-stored nucleic acid molecule of any one of claims 20-21 wherein the nucleic acid molecule comprises one or more of a DNA molecule and an RNA molecule.

24. The composition of any one of claims 1-19 which is capable, following admixture with a first sample of unfractionated human saliva to obtain a test mixture and storage of the test mixture without refrigeration for a time period of at least 60 days, of substantially preventing degradation of at least 70% of recoverable DNA from the test mixture, relative to DNA recoverable from a second sample of said unfractionated human saliva that is stored at −

20°

C. during said time period.

25. The composition of any one of claims 1-19 which is capable, following admixture with a first sample of unfractionated human saliva to obtain a test mixture and storage of the test mixture without refrigeration for a time period of at least 21 days, of substantially preventing degradation of at least 70% of recoverable RNA from the test mixture, relative to RNA recoverable from a second sample of said unfractionated human saliva that is stored at −

80°

C. during said time period.

26. A method for substantially stabilizing one or a plurality of nucleic acid or polypeptide molecules that are present in a biological sample, comprising:

(a) admixing the biological sample with the composition of any one of claims 1-19 to obtain a mixture; and

(b) maintaining the mixture without refrigeration for a time period of at least 7 days, and thereby substantially stabilizing said one or a plurality of nucleic acid or polypeptide molecules that are present in the biological sample, wherein either one, two or all three of;

(i) degradation is substantially prevented of at least 70% of recoverable DNA in the mixture, relative to an amount of DNA or polypeptide that is recoverable from the sample when stored for the time period at −

20°

C. without the composition of any one of claims 1-19,

(ii) degradation is substantially prevented of at least 70% of recoverable RNA in the mixture, relative to an amount of RNA that is recoverable from the sample when stored for the time period at −

80°

C. without the composition of any one of claims 1-18, and

(iii) degradation is substantially prevented of at least 70% of recoverable polypeptide molecules in the mixture, relative to an amount of polypeptide molecules that is recoverable from the sample when stored for the time period at −

20°

C. without the composition of any one of claims 1-19.

27. The method of claim 26 wherein the biological sample comprises vertebrate saliva or sputum.

28. The method of claim 27 wherein the vertebrate saliva or sputum is unfractionated.

29. The method of claim 26 wherein the biological sample comprises human saliva.

30. The method of claim 29 wherein the human saliva is unfractionated.

31. The method of claim 26 wherein the biological sample is selected from the group consisting of DNA, RNA, saliva, sputum, blood, blood buffy coat, urine, feces, serum, serosal fluid, plasma, lymph, cerebrospinal fluid, saliva, mucosal secretion, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, abdominal fluid, cell culture medium, organ culture medium, buccal cells, bacteria, viruses, yeast cells, plasmid DNA, mRNA, tRNA, rRNA, siRNA, micro RNA, hnRNA, cDNA, a protein, a polypeptide, a lipid, a glycolipid, a glycoprotein, an oligosaccharide, a polysaccharide, a vaccine, a cell, a sorted or selected cell, a tissue, a cell lysate, homogenate or extract, a tissue lysate, homogenate or extract, a blood sample, biopsy specimen, tissue explant, organ culture and a biological fluid.

32. The method of claim 26 wherein the step of maintaining comprises maintaining for a time period of at least 10, 20, 30, 40, 50, 60 or 70 days.