U.S. patent application number 13/966117 was filed with the patent office on 2014-03-06 for compositions and methods for biological sample storage.
This patent application is currently assigned to Biomatrica, Inc.. The applicant listed for this patent is Sohela de Rozieres, Rolf Muller, Scott E. Whitney. Invention is credited to Sohela de Rozieres, Rolf Muller, Scott E. Whitney.
Application Number | 20140065627 13/966117 |
Document ID | / |
Family ID | 43085552 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140065627 |
Kind Code |
A1 |
Whitney; Scott E. ; et
al. |
March 6, 2014 |
COMPOSITIONS AND METHODS FOR BIOLOGICAL SAMPLE STORAGE
Abstract
Compositions and methods are disclosed for substantially dry
storage at ambient or elevated temperatures of biological samples
such as nucleic acids, proteins and cells in a form from which the
samples can be substantially recovered, using a dissolvable or
dissociable dry storage matrix comprising a borate composition and
a stabilizer as disclosed, such as any of a number of zwitterionic
stabilizers.
Inventors: |
Whitney; Scott E.; (San
Diego, CA) ; de Rozieres; Sohela; (San Diego, CA)
; Muller; Rolf; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Whitney; Scott E.
de Rozieres; Sohela
Muller; Rolf |
San Diego
San Diego
San Diego |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
Biomatrica, Inc.
San Diego
CA
|
Family ID: |
43085552 |
Appl. No.: |
13/966117 |
Filed: |
August 13, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12778100 |
May 11, 2010 |
8519125 |
|
|
13966117 |
|
|
|
|
61177161 |
May 11, 2009 |
|
|
|
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
A01N 1/00 20130101; A01N
1/0231 20130101; C12Q 1/6806 20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A matrix for substantially dry storage of a biological sample,
comprising: (a) a borate composition; and (b) at least one
stabilizer that is selected from the group consisting of: (i) a
compound of formula I: ##STR00070## wherein R.sub.1, R.sub.2,
R.sub.3 are independently selected from aryl, arylalkyl, --H,
--CH.sub.3 and --CH.sub.2--CH.sub.3, wherein when R.sub.1 and
R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3, R.sub.3 is either H or
absent, wherein X is selected from --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, ##STR00071## and wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-; (ii) a compound of
formula II: ##STR00072## wherein R.sub.1 is selected from CH.sub.3
and CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from
H and OH, and when X is CH.sub.2--CH, Y is H; (iii) a compound of
formula III: ##STR00073## wherein R.sub.1 and R.sub.2 are
independently selected from --H, --CH.sub.3, and
--CH.sub.2CH.sub.3, and wherein X is selected from H, OH and SH;
(iv) a compound of formula IV: ##STR00074## wherein R.sub.1 is
selected from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; (v) a compound of
formula V: ##STR00075## wherein R.sub.1 and R.sub.2 are each
independently selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH; and
CH.sub.2CH.sub.2CH.sub.2OH, (vi) a compound of formula VI:
##STR00076## wherein R.sub.1 and R.sub.2 are each independently
selected from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; (vii) a compound of formula VII:
##STR00077## wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from the group consisting
of trimethylammonium acetate, glycerol phosphate, diglycerol
phosphate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks.
2. The matrix of claim 1 wherein the time period is selected from
at least four weeks, at least eight weeks, at least 12 weeks, at
least 16 weeks, at least 20 weeks, at least 24 weeks, at least 30
weeks, at least 36 weeks, at least 40 weeks, at least 48 weeks, and
at least one year.
3. The matrix of claim 1 wherein the borate composition comprises
at least one compound selected from the group consisting of boric
acid, dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol, boric anhydride (B.sub.2O.sub.3) and boric-acid-1,3
propanediol.
4. The matrix of claim 1 wherein the stabilizer is selected from
the group consisting of hydroxyectoine, ectoine, homoectoine,
betaine, L-carnitine, sarcosine, N,N-dimethylglycine,
triethylammonium acetate, glycerol phosphate,
N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-Morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl
sulfonate, glycolic acid, lactic acid, malic acid, tartaric acid,
2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid, pyridine 2,5-dicarboxylic acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate,
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, and
4-[benzyl(2-hydroxyethyl)methylazaniumyl]butane-1-sulfonate.
5. The matrix of claim 1, further comprising a chelator.
6. The matrix of claim 5 wherein the chelator is selected from the
group consisting of ethylenediaminetetraacetic acid (EDTA),
ethylene glycol tetraacetic acid (EGTA),
diethylenetriaminepentaacetic acid (DTPA),
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).
7. The matrix of claim 1 which dissolves or dissociates in a
biocompatible solvent.
8. The matrix of claim 7 which dissolves in a biocompatible
solvent.
9. The matrix of claim 7 wherein the biocompatible solvent
comprises water.
10. The matrix of claim 9 wherein the biocompatible solvent
comprises a pH buffer.
11. The matrix of claim 10 wherein the pH buffer is selected from
the group consisting of Tris, citrate, acetate, phosphate, borate,
CAPS, CAPSO, HEPES, MES, MOPS, MOPSO, PIPES, carbonate and
bicarbonate.
12. The matrix of any one of claims 1 and 3-5, further comprising a
biological inhibitor or a biochemical inhibitor.
13. A matrix for substantially dry storage of a biological sample,
comprising: (a) a borate composition which comprises at least one
compound selected from the group consisting of boric acid, boric
anhydride, dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol and boric-acid-1,3 propanediol; (b) at least one
stabilizer that is selected from the group consisting of: (i)
##STR00078## wherein R.sub.1, R.sub.2, R.sub.3 are independently
selected from aryl, arylalkyl, --H, --CH.sub.3 or
--CH.sub.2--CH.sub.3, wherein when R.sub.1 and R.sub.2 are CH.sub.3
or CH.sub.2--CH.sub.3, R.sub.3 may be absent or H, wherein X is
selected from --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, ##STR00079## and wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-; (ii) ##STR00080##
wherein R.sub.1 is CH.sub.3 or CH.sub.2CH.sub.3, and wherein when X
is CH, Y is H or OH, and when X is CH.sub.2--CH, Y is H; (iii)
##STR00081## wherein R.sub.1 and R.sub.2 are independently selected
from --H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is H,
OH, or SH; (iv) a compound of formula IV: ##STR00082## wherein
R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; (v) a compound of
formula V: ##STR00083## wherein R.sub.1 and R.sub.2 are each
independently selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH; and
CH.sub.2CH.sub.2CH.sub.2OH, (vi) a compound of formula VI:
##STR00084## wherein R.sub.1 and R.sub.2 are each independently
selected from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; (vii) a compound of formula VII:
##STR00085## wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from the group consisting
of trimethylammonium acetate, glycerol phosphate, diglycerol
phosphate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks.
14. The matrix of either claim 1 or claim 13 wherein the biological
sample comprises at least one of (i) an isolated biomolecule that
is selected from the group consisting of a nucleic acid, a protein,
a polypeptide, a lipid, a glyconconjugate, an oligosaccharide, and
a polysaccharide, and (ii) a biological material that is selected
from the group consisting of a mammalian cell, a bacterium, a yeast
cell, a virus, a vaccine, blood, urine, a biological fluid, and a
buccal swab.
15. The matrix of either claim 1 or claim 13 wherein the biological
sample comprises at least one isolated nucleic acid that is
selected from DNA and RNA.
16. The matrix of claim 12 wherein the biological inhibitor or
biochemical inhibitor is selected from the group consisting of a
reducing agent, an alkylating agent, an antifungal agent and an
antimicrobial agent.
17. The matrix of either claim 1 or claim 13 which comprises at
least one detectable indicator.
18. The matrix of claim 17 wherein the detectable indicator
comprises a dye or a colorimetric indicator.
19. The matrix of claim 17 wherein the detectable indicator is
selected from the group consisting of phenol red, ethidium bromide,
a DNA polymerase, a restriction endonuclease, cobalt chloride,
Reichardt's dye and a fluorogenic protease substrate.
20. A method of storing a biological sample, comprising: contacting
a biological sample with a matrix for substantially dry storage of
a biological sample, the matrix comprising (a) a borate
composition; and (b) at least one stabilizer that is selected from
the group consisting of: (i) ##STR00086## wherein R.sub.1, R.sub.2,
R.sub.3 are independently selected from aryl, arylalkyl, --H,
--CH.sub.3 or --CH.sub.2--CH.sub.3, wherein when R.sub.1 and
R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3, R.sub.3 may be absent
or H, wherein X is selected from --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--, ##STR00087##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; (ii)
##STR00088## wherein R.sub.1 is CH.sub.3 or CH.sub.2CH.sub.3, and
wherein when X is CH, Y is H or OH, and when X is CH.sub.2--CH, Y
is H; (iii) ##STR00089## wherein R.sub.1 and R.sub.2 are
independently selected from --H, --CH.sub.3, and
--CH.sub.2CH.sub.3, and wherein X is H, OH, or SH; (iv) a compound
of formula IV: ##STR00090## wherein R.sub.1 is selected from aryl,
arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; (v) a compound of
formula V: ##STR00091## wherein R.sub.1 and R.sub.2 are each
independently selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH; and
CH.sub.2CH.sub.2CH.sub.2OH, (vi) a compound of formula VI:
##STR00092## wherein R.sub.1 and R.sub.2 are each independently
selected from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; (vii) a compound of formula VII:
##STR00093## wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from the group consisting
of trimethylammonium acetate, glycerol phosphate, diglycerol
phosphate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks.
21. The method of claim 20, comprising maintaining the matrix
without refrigeration subsequent to the step of contacting.
22. The method of claim 20, further comprising substantially drying
the matrix, and thereby storing said biological sample.
23. The method of claim 22, comprising maintaining the matrix
without refrigeration subsequent to the steps of contacting and
drying.
24. The method of claim 20 wherein the time period is selected from
at least four weeks, at least eight weeks, at least 12 weeks, at
least 16 weeks, at least 20 weeks, at least 24 weeks, at least 30
weeks, at least 36 weeks, at least 40 weeks, at least 48 weeks, and
at least one year.
25. The method of claim 20 wherein the borate composition comprises
at least one compound selected from the group consisting of boric
acid, dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol, boric anhydride (B.sub.2O.sub.3) and boric-acid-1,3
propanediol.
26. The method of claim 20 wherein the stabilizer is selected from
the group consisting of hydroxyectoine, ectoine, homoectoine,
betaine, L-carnitine, sarcosine, N,N-dimethylglycine,
triethylammonium acetate, glycerol phosphate,
N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-Morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl
sulfonate, glycolic acid, lactic acid, malic acid, tartaric acid,
2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid, pyridine 2,5-dicarboxylic acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate,
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, and
4-[benzyl(2-hydroxyethyl)methylazaniumyl]butane-1-sulfonate.
27. The method of claim 20 wherein the matrix further comprises a
chelator.
28. The method of claim 27 wherein the chelator is selected from
the group consisting of ethylenediaminetetraacetic acid (EDTA),
ethylene glycol tetraacetic acid (EGTA),
diethylenetriaminepentaacetic acid (DTPA),
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).
29. The method of claim 20 wherein the matrix dissolves or
dissociates in a biocompatible solvent.
30. The method of claim 20 wherein the matrix dissolves in a
biocompatible solvent.
31. The method of claim 29 wherein the biocompatible solvent
comprises water.
32. The matrix of claim 31 wherein the biocompatible solvent
comprises a pH buffer.
33. The method of claim 32 wherein the pH buffer is selected from
the group consisting of Tris, citrate, acetate, phosphate, borate,
CAPS, CAPSO, HEPES, MES, MOPS, MOPSO, PIPES, carbonate and
bicarbonate.
34. The method of any one of claims 20 and 25-27 wherein the matrix
further comprises a biological inhibitor or a biochemical
inhibitor.
35. The method of claim 20 wherein the matrix comprises: (a) a
borate composition which comprises at least one compound selected
from the group consisting of boric acid, boric anhydride,
dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol and boric-acid-1,3 propanediol; (b) at least one
stabilizer that is selected from the group consisting of: (i)
##STR00094## wherein R.sub.1, R.sub.2, R.sub.3 are independently
selected from aryl, arylalkyl, --H, --CH.sub.3 or
--CH.sub.2--CH.sub.3, wherein when R.sub.1 and R.sub.2 are CH.sub.3
or CH.sub.2--CH.sub.3, R.sub.3 may be absent or H, wherein X is
selected from --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, ##STR00095## and wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-; (ii) ##STR00096##
wherein R.sub.1 is CH.sub.3 or CH.sub.2CH.sub.3, and wherein when X
is CH, Y is H or OH, and when X is CH.sub.2--CH, Y is H; (iii)
##STR00097## wherein R.sub.1 and R.sub.2 are independently selected
from --H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is H,
OH, or SH; (iv) a compound of formula IV: ##STR00098## wherein
R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; (v) a compound of
formula V: ##STR00099## wherein R.sub.1 and R.sub.2 are each
independently selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH; and
CH.sub.2CH.sub.2CH.sub.2OH, (vi) a compound of formula VI:
##STR00100## wherein R.sub.1 and R.sub.2 are each independently
selected from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; (vii) a compound of formula VII:
##STR00101## wherein R1 is selected from aryl, arylalkyl, --H,
--CH3-CH2-CH3, --CH2CH2OH, CH.sub.2CHOHCH.sub.3,
CH.sub.2CHOHCH.sub.2OH, and CH.sub.2CH.sub.2CH.sub.2OH, wherein X
is selected from --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CHOH, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CHOHCH.sub.2--, and
--CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is selected from
CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z is selected from
CH.sub.2, CHOH, O and S; and (viii) an osmoprotectant compound that
is selected from the group consisting of trimethylammonium acetate,
glycerol phosphate, diglycerol phosphate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks.
36. The method of claim 35 wherein the biological sample comprises
at least one of (i) an isolated biomolecule that is selected from
the group consisting of a nucleic acid, a protein, a polypeptide, a
lipid, a glyconconjugate, an oligosaccharide, and a polysaccharide,
and (ii) a biological material that is selected from the group
consisting of a mammalian cell, a bacterium, a yeast cell, a virus,
a vaccine, blood, urine, a biological fluid, and a buccal swab.
37. The method of claim 35 wherein the biological sample comprises
at least one isolated nuclei.
38. The method of claim 34 wherein the biological inhibitor or
biochemical inhibitor is selected from the group consisting of a
reducing agent, an alkylating agent, an antifungal agent and an
antimicrobial agent.
39. The method of claim 35 wherein the matrix further comprises at
least one detectable indicator.
40. The method of claim 39 wherein the detectable indicator
comprises a dye or a colorimetric indicator.
41. The method of claim 39 wherein the detectable indicator is
selected from the group consisting of phenol red, ethidium bromide,
a DNA polymerase, a restriction endonuclease, cobalt chloride,
Reichardt's dye and a fluorogenic protease substrate.
42. The method of claim 23 wherein biological activity of the
sample subsequent to the step of maintaining is substantially the
same as biological activity of the sample prior to the step of
contacting.
43. The method of claim 23 wherein degradation of the biological
sample is decreased relative to degradation of a control biological
sample maintained without refrigeration in the absence of the
matrix material.
44. The method of claim 22 which is selected from (i) the method
wherein the step of contacting comprises simultaneously dissolving
or dissociating the matrix material in a solvent, (ii) the method
wherein the step of contacting is preceded by dissolving or
dissociating the matrix material in a solvent, and (iii) the method
wherein the step of contacting is followed by dissolving or
dissociating the matrix material in a solvent.
45. A method of preparing a biological sample storage device for
one or a plurality of biological samples, comprising: (a)
administering a matrix to one or a plurality of sample wells of a
biological sample storage device, wherein (1) said biological
sample storage device comprises a sample plate comprising one or a
plurality of sample wells that are capable of containing a
biological sample, and wherein (2) the matrix comprises (a) a
borate composition; and (b) at least one stabilizer that is
selected from the group consisting of: (i) ##STR00102## wherein
R.sub.1, R.sub.2, R.sub.3 are independently selected from aryl,
arylalkyl, --H, --CH.sub.3 or --CH.sub.2--CH.sub.3, wherein when
R.sub.1 and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3, R.sub.3 may
be absent or H, wherein X is selected from --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--, ##STR00103##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; (ii)
##STR00104## wherein R.sub.1 is CH.sub.3 or CH.sub.2CH.sub.3, and
wherein when X is CH, Y is H or OH, and when X is CH.sub.2--CH, Y
is H; (iii) ##STR00105## wherein R.sub.1 and R.sub.2 are
independently selected from --H, --CH.sub.3, and
--CH.sub.2CH.sub.3, and wherein X is H, OH, or SH; (iv) a compound
of formula IV: ##STR00106## wherein R.sub.1 is selected from aryl,
arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; (v) a compound of
formula V: ##STR00107## wherein R.sub.1 and R.sub.2 are each
independently selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH; and
CH.sub.2CH.sub.2CH.sub.2OH, (vi) a compound of formula VI:
##STR00108## wherein R.sub.1 and R.sub.2 are each independently
selected from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; (vii) a compound of formula VII:
##STR00109## wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from the group consisting
of trimethylammonium acetate, glycerol phosphate, diglycerol
phosphate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks; and (b) substantially drying one or more of the
sample wells, and thereby preparing the biological sample storage
device.
46. The method of claim 45 wherein the step of administering
comprises administering a liquid solution or a liquid suspension
that contains the matrix and a solvent.
47. The method of claim 45 wherein at least one well comprises at
least one detectable indicator.
48. The method of claim 47 wherein the detectable indicator
comprises a dye or colorimetric indicator.
49. The method of claim 47 wherein the detectable indicator is
selected from the group consisting of phenol red, a food dye,
ethidium bromide, a dye compatible with qPCR, a DNA polymerase, a
restriction endonuclease, cobalt chloride, Reichardt's dye and a
fluorogenic protease substrate.
50. A method of recovering a stored biological sample, comprising:
(a) contacting, simultaneously or sequentially and in either order
in a biological sample storage device, one or a plurality of
biological samples with a matrix for substantially dry storage of a
biological sample, wherein (1) said biological sample storage
device comprises a sample plate comprising one or a plurality of
sample wells that are capable of containing the biological sample,
wherein one or more of said wells comprises the matrix, and wherein
(2) the matrix comprises (a) a borate composition; and (b) at least
one stabilizer that is selected from the group consisting of: (i)
##STR00110## wherein R.sub.1, R.sub.2, R.sub.3 are independently
selected from aryl, arylalkyl, --H, --CH.sub.3 or
--CH.sub.2--CH.sub.3, wherein when R.sub.1 and R.sub.2 are CH.sub.3
or CH.sub.2--CH.sub.3, R.sub.3 may be absent or H, wherein X is
selected from --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, ##STR00111## and wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-; (ii) ##STR00112##
wherein R.sub.1 is CH.sub.3 or CH.sub.2CH.sub.3, and wherein when X
is CH, Y is H or OH, and when X is CH.sub.2--CH, Y is H; (iii)
##STR00113## wherein R.sub.1 and R.sub.2 are independently selected
from --H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is H,
OH, or SH; (iv) a compound of formula IV: ##STR00114## wherein
R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; (v) a compound of
formula V: ##STR00115## wherein R.sub.1 and R.sub.2 are each
independently selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH; and
CH.sub.2CH.sub.2CH.sub.2OH, (vi) a compound of formula VI:
##STR00116## wherein R.sub.1 and R.sub.2 are each independently
selected from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; (vii) a compound of formula VII:
##STR00117## wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from the group consisting
of trimethylammonium acetate, glycerol phosphate, diglycerol
phosphate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks; (b) substantially drying one or more of the sample
wells; (c) maintaining the biological sample storage device without
refrigeration subsequent to the steps of contacting and drying; and
(d) resuspending or redissolving the biological sample in a
biocompatible solvent, and therefrom recovering the stored
biological sample.
51. The method of claim 50 wherein biological activity of the
sample subsequent to the step of maintaining is substantially the
same as biological activity of the sample prior to the step of
contacting.
52. A matrix for substantially dry storage of a biological sample,
comprising: (a) a borate composition which comprises at least one
compound selected from the group consisting of boric acid,
dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol, boric anhydride (B2O.sub.3) and boric-acid-1,3
propanediol; (b) at least one stabilizer selected from the group
consisting of hydroxyectoine, ectoine, homoectoine, betaine,
L-carnitine, sarcosine, N,N-dimethylglycine, triethylammonium
acetate, glycerol phosphate, tricine, MOPSO, pentaerythritol and
N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl sulfonate,
glycolic acid, lactic acid, malic acid and tartaric acid; and (c) a
sample treatment composition, wherein the borate composition and
the stabilizer are present at a molar ratio of from about 10:1 to
about 1:10, and wherein the matrix is capable of preventing
degradation of an isolated DNA fragment of at least 10 kilobases
during substantially dry storage of the DNA fragment in the matrix
at 85.degree. C. for a time period of at least two weeks.
53. The matrix of claim 52 wherein the sample treatment composition
comprises a composition that is selected from the group consisting
of an activity buffer, a cell lysis buffer, a free radical trapping
agent, a sample denaturant and a pathogen-neutralizing agent.
Description
CROSS-REFERENCE
[0001] This application is a divisional of U.S. application Ser.
No. 12/778,100, filed on May 11, 2010, which claims priority to
U.S. Provisional Application No. 61/177,161, filed on May 11, 2009,
each of which is incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to improved
compositions and methods for biological sample protection, storage,
and retrieval. The invention also relates to the use, storage,
retrieval and analysis of such biological materials and
samples.
BACKGROUND OF THE INVENTION
[0003] Research in the life sciences field is based upon the
analysis of biological materials and samples, such as DNA, RNA,
blood, urine, feces, buccal swabs or samples, bacteria,
archaebacteria, viruses, phage, plants, algae, yeast,
microorganisms, PCR products, cloned DNA, proteins, enzymes,
peptides, prions, eukaryotes (e.g. protoctisca, fungi, plantae and
animalia), prokaryotes, cells and tissues, germ cells (e.g. sperm
and oocytes), stem cells, vaccines, and of minerals or chemicals.
Such samples are typically collected or obtained from appropriate
sources and placed into storage and inventory for further
processing and analysis. Oftentimes, transportation of samples is
required, and attention is given to preserve their integrity,
sterility and stability. Biological samples can be transported in a
refrigerated environment using ice, dry ice or other freezing
facility. However, adequate low temperatures often cannot
conveniently be maintained for extended time periods such as those
required for transportation within or between countries or
continents, particularly where an energy source for the
refrigeration device is lacking.
[0004] Storage containers or storage vessels for such samples
include bottles, tubes, vials, bags, boxes, racks, multi-well
dishes and multi-well plates, which are typically sealed by
individual screw caps or snap caps, snap or seal closures, lids,
adhesive strips or tape, multi-cap strips, or other means for
containing such samples. The standard container format for medium
to high throughput of sample storage, processing and automation of
biological processes is a 96-, 384-, or 1536-well plate or array.
The containers and the samples contained therein are stored at
various temperatures, for example at ambient temperature or at
4.degree. C. or at temperatures below 0.degree. C., typically at
about -20.degree. C. or at -70.degree. C. to -80.degree. C. The
samples that are placed and stored in the devices are most
frequently contained in liquid medium or a buffer solution, and
they require storage at such subzero temperatures (e.g.,
-20.degree. C. or -70 to -80.degree. C.). In some cases, samples
are first dried and then stored at ambient temperature, or at
4.degree. C., at -20.degree. C. or at -70 to -80.degree. C.
[0005] For example, presently, nucleic acids are stored in liquid
form at low temperatures. For short term storage, nucleic acids can
be stored at 4.degree. C. For long term storage the temperature is
generally lowered to -20.degree. C. to -70.degree. C. to prevent
degradation of the genetic material, particularly in the case of
genomic DNA and RNA. Nucleic acids are also stored at room
temperature on solid matrices such as cellulose membranes. Both
storage systems are associated with disadvantages. Storage under
low temperature requires costly equipment such as cold rooms,
freezers, and/or electric generator back-up systems; such equipment
can be unreliable in cases of unexpected power outage or may be
difficult to use in areas without a ready source of electricity or
having unreliable electric systems. The storage of nucleic acids on
cellulose fibers also results in a substantial loss of material
during the rehydration process, since the nucleic acid stays
trapped by, and hence associated with, the cellulose fibers instead
of being quantitatively recoverable. Nucleic acid dry storage on
cellulose also requires the separation of the cellulose from the
biological material, since the cellulose fibers otherwise
contaminate the biological samples. The separation of the nucleic
acids from cellulose filters requires additional handling,
including steps of pipetting, transferring of the samples into new
tubes or containers, and centrifugation, all of which can result in
reduced recovery yields and increased opportunity for the
introduction of unwanted contaminants or exposure to conditions
that promote sample degradation, and which are also cost- and
labor-intensive.
[0006] Proteins are presently handled primarily in liquid stages,
in cooled or frozen environments typically ranging from -20.degree.
C. to storage in liquid nitrogen. In some exceptions proteins may
be freeze-dried, or dried at room temperature, for example, in the
presence of trehalose and applied directly to an untreated surface.
(Garcia de Castro et al., 2000 Appl. Environ. Microbiol. 66:4142;
Manzanera et al., 2002 Appl. Environ. Microbiol. 68:4328) Proteins
often degrade and/or lose activity even when stored cooled
(4.degree. C.), or frozen (-20.degree. C. or -80.degree. C.). The
freeze-thaw stress on proteins reduces bioactivity (e.g., enzymatic
activity, specific binding to a cognate ligand, etc.) especially if
repeated freeze-thawing of aliquots of a protein sample is
required. The consequent loss of protein activity that may be
needed for biological assays typically requires the readjustment of
the protein concentration in order to obtain comparable assay
results, or costly rejection of compromised protein reagents in
favor of procuring new lots. The common practice of having multiple
users of enzyme reagents stored in a laboratory, especially by
different users at different times and employing non-standardized
handling procedures, further reduces the reliability of
experimental data generated with such reagents. As a result, the
half-life of proteins is reduced and expensive reagents have to be
replaced frequently, amounting to enormous financial costs to the
user. For the supplier of the proteins, high costs are required to
maintain an undisrupted frozen supply chain starting with initial
cold room work-ups, for shipment, frozen storage of the sample, and
frozen transport of the protein from production to the site of use.
For example, delays during shipment can result in inactivation of
proteins, which then have to be replaced at great cost to the
supplier; receipt of inactive product can also result in
dissatisfied customers.
[0007] Drying of proteins and nucleic acids has yet to be
universally adopted by the research scientific, biomedical,
biotechnology and other industrial business communities because of
the lack of standard established and reliable processes,
difficulties with recoveries of functional properties and with
quantitative recoveries of biological sample material, variable
buffer and solvent compatibilities and tolerances, and other
difficulties arising from the demands of handling nucleic acids and
proteins. The same problems apply to the handling, storage, and use
of other biological materials, such as viruses, phage, bacteria,
cells and multicellular organisms. See, e.g., Roberts, 2005 Saline
Systems 1:5; Galinski et al., 1985 Eur. J. Biochem. 149:135; Malin
et al., 1999 J Biol. Chem. 274:6920; Mascellani et al., 2007 BMC
Biotechnol. 7:82. Dissacharides such as trehalose or lactitol, for
example, have been described as additives for dry storage of
protein-containing samples (e.g., U.S. Pat. No. 4,891,319; U.S.
Pat. No. 5,834,254; U.S. Pat. No. 6,896,894; U.S. Pat. No.
5,876,992; U.S. Pat. No. 5,240,843; WO 90/05182; WO 91/14773) but
usefulness of such compounds in the described contexts has been
compromised by their serving as energy sources for undesirable
microbial contaminants, by their limited stabilizing effects when
used as described, by their lack of general applicability across a
wide array of biological samples, and by other factors.
[0008] The genomic age and the recent deciphering of the human and
many other genomes, proteomes, transcriptomes, etc. have led to the
industrialization of life sciences research. Millions of biological
samples including genes and/or gene products from a multitude of
organisms are being analyzed in order to advance scientific
knowledge and develop commercial products. The development of high
throughput technologies has resulted in a vast pool of information
and samples, such that there is an increasing need to store these
samples for analysis at a later timepoint. Typically samples that
may be tested at later times are stored frozen in freezers at
-20.degree. C. to -80.degree. C. However with the rapid expansion
of demand and capability for analyzing samples by techniques such
as polymerase chain reaction (PCR), nucleic acid sequencing, single
nucleotide polymorphism (SNP) analyses and other biochemical and/or
molecular biology techniques, the available space for storing these
samples is rapidly diminishing. Also, universities and other
research institutions, reference and diagnostic laboratories and
the like are beginning to recognize that the electricity demands
for such frozen storage capabilities are constantly growing. Hence,
and as the energy pricing rates rise concomitantly, the long term
sustainability of this approach is being questioned. It is apparent
that a long term sustainable solution to sample storage is vital to
the research and diagnostic communities. Clearly there is a need in
the industry for convenient, low-cost, energy efficient and
accessible life sciences sample storage and retrieval systems. The
present disclosure addresses such needs by providing compositions
and methods for stably and recoverably storing biological samples
such as DNA, RNA and proteins obtained from various biological
sources, under anhydrobiotic conditions at room temperature, and
offers other related advantages
SUMMARY OF THE INVENTION
[0009] According to certain embodiments of the present invention
there is provided a matrix for substantially dry storage of a
biological sample, comprising (a) a borate composition; and (b) at
least one stabilizer that is selected from (i) a compound of
formula I:
##STR00001##
wherein R.sub.1, R.sub.2, R.sub.3 are independently selected from
aryl, arylalkyl, --H, --CH.sub.3 and --CH.sub.2--CH.sub.3, wherein
when R.sub.1 and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3,
R.sub.3 is either H or absent, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
##STR00002##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; [0010]
(ii) a compound of formula II:
##STR00003##
[0010] wherein R.sub.1 is selected from CH.sub.3 and
CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from H
and OH, and when X is CH.sub.2--CH, Y is H; [0011] (iii) a compound
of formula III:
##STR00004##
[0011] wherein R.sub.1 and R.sub.2 are independently selected from
--H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is selected
from H, OH and SH; [0012] (iv) a compound of formula IV:
##STR00005##
[0012] wherein R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; [0013] (v) a
compound of formula V:
##STR00006##
[0013] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH;
and CH.sub.2CH.sub.2CH.sub.2OH, [0014] (vi) a compound of formula
VI:
##STR00007##
[0014] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; [0015] (vii) a compound of formula
VII:
##STR00008##
[0015] wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and [0016] (viii) an
osmoprotectant compound that is selected from trimethylammonium
acetate, glycerol phosphate, diglycerol phosphate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks.
[0017] In certain embodiments the time period is selected from at
least four weeks, at least eight weeks, at least 12 weeks, at least
16 weeks, at least 20 weeks, at least 24 weeks, at least 30 weeks,
at least 36 weeks, at least 40 weeks, at least 48 weeks, and at
least one year. In certain embodiments the borate composition
comprises at least one compound selected from boric acid,
dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol, boric anhydride (B.sub.2O.sub.3) and boric-acid-1,3
propanediol. In certain embodiments the stabilizer is selected from
hydroxyectoine, ectoine, homoectoine, betaine, L-carnitine,
sarcosine, N,N-dimethylglycine, triethylammonium acetate, glycerol
phosphate, N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-Morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl
sulfonate, glycolic acid, lactic acid, malic acid, tartaric acid,
2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid, pyridine 2,5-dicarboxylic acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate,
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, and
4-[benzyl(2-hydroxyethyl)methylazaniumyl]butane-1-sulfonate.
[0018] In certain embodiments the matrix further comprises a
chelator, which in certain embodiments is selected from
ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic
acid (EGTA), diethylenetriaminepentaacetic acid (DTPA),
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). In certain embodiments the matrix
dissolves or dissociates in a biocompatible solvent. In certain
embodiments the matrix dissolves in a biocompatible solvent. In
certain embodiments the biocompatible solvent comprises water. In
certain embodiments the biocompatible solvent comprises a pH
buffer. In certain embodiments the pH buffer is selected from Tris,
citrate, acetate, phosphate, borate, CAPS, CAPSO, HEPES, MES, MOPS,
MOPSO, PIPES, carbonate and bicarbonate. In certain embodiments the
matrix further comprises a biological inhibitor or a biochemical
inhibitor.
[0019] In another embodiment there is provided a matrix for
substantially dry storage of a biological sample, comprising (a) a
borate composition which comprises at least one compound selected
from boric acid, boric anhydride, dihydrogen borate, hydrogen
borate, diborate, triborate, tetraborate, metaborate, hydroxoborate
(borax), borate salt, boric acid-glycerol and boric-acid-1,3
propanediol; (b) at least one stabilizer that is selected from: (i)
a compound of formula I:
##STR00009##
wherein R.sub.1, R.sub.2, R.sub.3 are independently selected from
aryl, arylalkyl, --H, --CH.sub.3 and --CH.sub.2--CH.sub.3, wherein
when R.sub.1 and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3,
R.sub.3 is either H or absent, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
##STR00010##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; [0020]
(ii) a compound of formula II:
##STR00011##
[0020] wherein R.sub.1 is selected from CH.sub.3 and
CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from H
and OH, and when X is CH.sub.2--CH, Y is H; [0021] (iii) a compound
of formula III:
##STR00012##
[0021] wherein R.sub.1 and R.sub.2 are independently selected from
--H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is selected
from H, OH and SH; [0022] (iv) a compound of formula IV:
##STR00013##
[0022] wherein R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; [0023] (v) a
compound of formula V:
##STR00014##
[0023] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH;
and CH.sub.2CH.sub.2CH.sub.2OH, [0024] (vi) a compound of formula
VI:
##STR00015##
[0024] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; [0025] (vii) a compound of formula
VII:
##STR00016##
[0025] wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from trimethylammonium
acetate, glycerol phosphate, diglycerol phosphate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks.
[0026] According to certain further embodiments the biological
sample comprises at least one of (i) an isolated biomolecule that
is selected from the group consisting of a nucleic acid, a protein,
a polypeptide, a lipid, a glyconconjugate, an oligosaccharide, and
a polysaccharide, and (ii) a biological material that is selected
from the group consisting of a mammalian cell, a bacterium, a yeast
cell, a virus, a vaccine, blood, urine, a biological fluid, and a
buccal swab. In certain embodiments the biological sample comprises
at least one isolated nucleic acid that is selected from DNA and
RNA. In certain embodiments the biological inhibitor or biochemical
inhibitor is selected from the group consisting of a reducing
agent, an alkylating agent, an antifungal agent and an
antimicrobial agent. In certain embodiments the matrix comprises at
least one detectable indicator, which in certain further
embodiments comprises a dye or a colorimetric indicator, and in
certain other further embodiments is selected from phenol red,
ethidium bromide, a DNA polymerase, a restriction endonuclease,
cobalt chloride, Reichardt's dye and a fluorogenic protease
substrate.
[0027] In another embodiment the present invention provides a
method of storing a biological sample, comprising contacting a
biological sample with a matrix for substantially dry storage of a
biological sample, the matrix comprising (a) a borate composition;
and (b) at least one stabilizer that is selected from: (i) a
compound of formula I:
##STR00017##
wherein R.sub.1, R.sub.2, R.sub.3 are independently selected from
aryl, arylalkyl, --H, --CH.sub.3 and --CH.sub.2--CH.sub.3, wherein
when R.sub.1 and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3,
R.sub.3 is either H or absent, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
##STR00018##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; [0028]
(ii) a compound of formula II:
##STR00019##
[0028] wherein R.sub.1 is selected from CH.sub.3 and
CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from H
and OH, and when X is CH.sub.2--CH, Y is H; [0029] (iii) a compound
of formula III:
##STR00020##
[0029] wherein R.sub.1 and R.sub.2 are independently selected from
--H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is selected
from H, OH and SH; [0030] (iv) a compound of formula IV:
##STR00021##
[0030] wherein R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; [0031] (v) a
compound of formula V:
##STR00022##
[0031] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH;
and CH.sub.2CH.sub.2CH.sub.2OH, [0032] (vi) a compound of formula
VI:
##STR00023##
[0032] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; [0033] (vii) a compound of formula
VII:
##STR00024##
[0033] wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from trimethylammonium
acetate, glycerol phosphate, diglycerol phosphate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks
[0034] In certain embodiments the method comprises maintaining the
matrix without refrigeration subsequent to the step of contacting.
In certain embodiments the method comprises substantially drying
the matrix, and thereby storing the biological sample. In certain
further embodiments the method comprises maintaining the matrix
without refrigeration subsequent to the steps of contacting and
drying. In certain embodiments the time period is selected from at
least four weeks, at least eight weeks, at least 12 weeks, at least
16 weeks, at least 20 weeks, at least 24 weeks, at least 30 weeks,
at least 36 weeks, at least 40 weeks, at least 48 weeks, and at
least one year. In certain embodiments the borate composition
comprises at least one compound selected from boric acid,
dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol, boric anhydride (B.sub.2O.sub.3) and boric-acid-1,3
propanediol. In certain embodiments the stabilizer is selected from
hydroxyectoine, ectoine, homoectoine, betaine, L-carnitine,
sarcosine, N,N-dimethylglycine, triethylammonium acetate, glycerol
phosphate, N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-Morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl
sulfonate, glycolic acid, lactic acid, malic acid, tartaric acid,
2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid, pyridine 2,5-dicarboxylic acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate,
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, and
4-[benzyl(2-hydroxyethyl)methylazaniumyl]butane-1-sulfonate.
[0035] In certain embodiments the matrix further comprises a
chelator, which in certain further embodiments is selected from
EDTA, EGTA, DTPA, CDTA, BAPTA, DOTA,
N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid, and NTA.
In certain embodiments the matrix dissolves or dissociates in a
biocompatible solvent. In certain embodiments the matrix dissolves
in a biocompatible solvent. In certain embodiments the
biocompatible solvent comprises water, and in certain further
embodiments the biocompatible solvent comprises a pH buffer, which
in certain further embodiments is selected from the Tris, citrate,
acetate, phosphate, borate, CAPS, CAPSO, HEPES, MES, MOPS, MOPSO,
PIPES, carbonate and bicarbonate. In certain embodiments of the
herein described method, the matrix further comprises a biological
inhibitor or a biochemical inhibitor.
[0036] In certain embodiments of the above described method, the
matrix comprises (a) a borate composition which comprises at least
one compound selected from boric acid, boric anhydride, dihydrogen
borate, hydrogen borate, diborate, triborate, tetraborate,
metaborate, hydroxoborate (borax), borate salt, boric acid-glycerol
and boric-acid-1,3 propanediol; (b) at least one stabilizer that is
selected from: (i) a compound of formula I:
##STR00025##
wherein R.sub.1, R.sub.2, R.sub.3 are independently selected from
aryl, arylalkyl, --H, --CH.sub.3 and --CH.sub.2--CH.sub.3, wherein
when R.sub.1 and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3,
R.sub.3 is either H or absent, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
##STR00026##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; [0037]
(ii) a compound of formula II:
##STR00027##
[0037] wherein R.sub.1 is selected from CH.sub.3 and
CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from H
and OH, and when X is CH.sub.2--CH, Y is H; [0038] (iii) a compound
of formula III:
##STR00028##
[0038] wherein R.sub.1 and R.sub.2 are independently selected from
--H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is selected
from H, OH and SH; [0039] (iv) a compound of formula IV:
##STR00029##
[0039] wherein R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; [0040] (v) a
compound of formula V:
##STR00030##
[0040] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH;
and CH.sub.2CH.sub.2CH.sub.2OH, [0041] (vi) a compound of formula
VI:
##STR00031##
[0041] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; [0042] (vii) a compound of formula
VII:
##STR00032##
[0042] wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected trimethylammonium acetate,
glycerol phosphate, diglycerol phosphate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks. In certain embodiments the biological sample
comprises at least one of (i) an isolated biomolecule that is
selected from a nucleic acid, a protein, a polypeptide, a lipid, a
glyconconjugate, an oligosaccharide, and a polysaccharide, and (ii)
a biological material that is selected from a mammalian cell, a
bacterium, a yeast cell, a virus, a vaccine, blood, urine, a
biological fluid, and a buccal swab. In certain embodiments the
biological sample comprises at least one isolated nucleic acid that
is selected from DNA and RNA. In certain embodiments the biological
inhibitor or biochemical inhibitor is selected from a reducing
agent, an alkylating agent, an antifungal agent and an
antimicrobial agent. In certain embodiments the matrix further
comprises at least one detectable indicator, which in certain
further embodiments comprises a dye or a colorimetric indicator. In
certain embodiments the detectable indicator is selected from
phenol red, ethidium bromide, a DNA polymerase, a restriction
endonuclease, cobalt chloride, Reichardt's dye and a fluorogenic
protease substrate.
[0043] In certain embodiments of the above described methods,
biological activity of the sample subsequent to the step of
maintaining is substantially the same as biological activity of the
sample prior to the step of contacting. In certain embodiments of
the above described methods, degradation of the biological sample
is decreased relative to degradation of a control biological sample
maintained without refrigeration in the absence of the matrix
material. In certain embodiments of the above described methods,
the method is selected from (i) the method wherein the step of
contacting comprises simultaneously dissolving or dissociating the
matrix material in a solvent, (ii) the method wherein the step of
contacting is preceded by dissolving or dissociating the matrix
material in a solvent, and (iii) the method wherein the step of
contacting is followed by dissolving or dissociating the matrix
material in a solvent.
[0044] In another embodiment of the present invention, there is
provided a method of preparing a biological sample storage device
for one or a plurality of biological samples, comprising (a)
administering a matrix to one or a plurality of sample wells of a
biological sample storage device, wherein (1) said biological
sample storage device comprises a sample plate comprising one or a
plurality of sample wells that are capable of containing a
biological sample, and wherein (2) the matrix comprises (a) a
borate composition; and (b) at least one stabilizer that is
selected from: (i) a compound of formula I:
##STR00033##
wherein R.sub.1, R.sub.2, R.sub.3 are independently selected from
aryl, arylalkyl, --H, --CH.sub.3 and --CH.sub.2--CH.sub.3, wherein
when R.sub.1 and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3,
R.sub.3 is either H or absent, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
##STR00034##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; [0045]
(ii) a compound of formula II:
##STR00035##
[0045] wherein R.sub.1 is selected from CH.sub.3 and
CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from H
and OH, and when X is CH.sub.2--CH, Y is H; [0046] (iii) a compound
of formula III:
##STR00036##
[0046] wherein R.sub.1 and R.sub.2 are independently selected from
--H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is selected
from H, OH and SH; [0047] (iv) a compound of formula IV:
##STR00037##
[0047] wherein R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; [0048] (v) a
compound of formula V:
##STR00038##
[0048] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH;
and CH.sub.2CH.sub.2CH.sub.2OH, [0049] (vi) a compound of formula
VI:
##STR00039##
[0049] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; [0050] (vii) a compound of formula
VII:
##STR00040##
[0050] wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from trimethylammonium
acetate, glycerol phosphate, diglycerol phosphate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
142-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks; and (b) substantially drying one or more of the
sample wells, and thereby preparing the biological sample storage
device. In a further embodiment the step of administering comprises
administering a liquid solution or a liquid suspension that
contains the matrix and a solvent. In another further embodiment at
least one well comprises at least one detectable indicator, which
in a still further embodiment comprises a dye or colorimetric
indicator. In another further embodiment the detectable indicator
is selected from phenol red, a food dye, ethidium bromide, a dye
compatible with qPCR, a DNA polymerase, a restriction endonuclease,
cobalt chloride, Reichardt's dye and a fluorogenic protease
substrate.
[0051] In another embodiment of the present invention there is
provided a method of recovering a stored biological sample,
comprising (a) contacting, simultaneously or sequentially and in
either order in a biological sample storage device, one or a
plurality of biological samples with a matrix for substantially dry
storage of a biological sample, wherein (1) said biological sample
storage device comprises a sample plate comprising one or a
plurality of sample wells that are capable of containing the
biological sample, wherein one or more of said wells comprises the
matrix, and wherein (2) the matrix comprises (a) a borate
composition; and (b) at least one stabilizer that is selected from:
(i) a compound of formula I:
##STR00041##
wherein R.sub.1, R.sub.2, R.sub.3 are independently selected from
aryl, arylalkyl, --H, --CH.sub.3 and --CH.sub.2--CH.sub.3, wherein
when R.sub.1 and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3,
R.sub.3 is either H or absent, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
##STR00042##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; [0052]
(ii) a compound of formula II:
##STR00043##
[0052] wherein R.sub.1 is selected from CH.sub.3 and
CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from H
and OH, and when X is CH.sub.2--CH, Y is H; [0053] (iii) a compound
of formula III:
##STR00044##
[0053] wherein R.sub.1 and R.sub.2 are independently selected from
--H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is selected
from H, OH and SH; [0054] (iv) a compound of formula IV:
##STR00045##
[0054] wherein R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; [0055] (v) a
compound of formula V:
##STR00046##
[0055] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH;
and CH.sub.2CH.sub.2CH.sub.2OH, [0056] (vi) a compound of formula
VI:
##STR00047##
[0056] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; [0057] (vii) a compound of formula
VII:
##STR00048##
[0057] wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S; and (viii) an
osmoprotectant compound that is selected from trimethylammonium
acetate, glycerol phosphate, diglycerol phosphate,
N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, wherein the
borate composition and the stabilizer are present at a molar ratio
that is selected from a molar ratio of from about 10:1 to about
1:10, a molar ratio of from about 5:1 to about 1:5, and a molar
ratio of from about 20:1 to about 1:20, and wherein the matrix is
capable of preventing degradation of an isolated DNA fragment of at
least 10 kilobases during substantially dry storage of the DNA
fragment in the matrix at 85.degree. C. for a time period of at
least two weeks; (b) substantially drying one or more of the sample
wells; (c) maintaining the biological sample storage device without
refrigeration subsequent to the steps of contacting and drying; and
(d) resuspending or redissolving the biological sample in a
biocompatible solvent, and therefrom recovering the stored
biological sample. In certain further embodiments the biological
activity of the sample subsequent to the step of maintaining is
substantially the same as biological activity of the sample prior
to the step of contacting.
[0058] In another embodiment of the present invention there is
provided a matrix for substantially dry storage of a biological
sample, comprising (a) a borate composition which comprises at
least one compound selected from the group consisting of boric
acid, dihydrogen borate, hydrogen borate, diborate, triborate,
tetraborate, metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol, boric anhydride (B.sub.2O.sub.3) and boric-acid-1,3
propanediol; (b) at least one stabilizer selected from the group
consisting of hydroxyectoine, ectoine, homoectoine, betaine,
L-carnitine, sarcosine, N,N-dimethylglycine, triethylammonium
acetate, glycerol phosphate, tricine, MOPSO, pentaerythritol and
N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl sulfonate,
glycolic acid, lactic acid, malic acid and tartaric acid; and (c) a
sample treatment composition, wherein the borate composition and
the stabilizer are present at a molar ratio of from about 10:1 to
about 1:10, and wherein the matrix is capable of preventing
degradation of an isolated DNA fragment of at least 10 kilobases
during substantially dry storage of the DNA fragment in the matrix
at 85.degree. C. for a time period of at least two weeks. In
certain further embodiments the sample treatment composition
comprises a composition that is selected from an activity buffer, a
cell lysis buffer, a free radical trapping agent, a sample
denaturant and a pathogen-neutralizing agent.
[0059] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entirety as if each was
incorporated individually. Background information pertaining to
storage and stabilization of biological samples, including
compositions and methods for unrefrigerated dry storage, may be
found, for example, in US 2005/0276728, WO 2005/113147, US
2006/0099567, WO 2007/075253, US 2008/0176209, US 2008/0268514, US
2008/0307117, US 2009/0291427, US 2009/0298132, WO 2009/009210, and
WO 2009/038853.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIG. 1 summarizes DNA recoveries determined by quantitative
PCR. Data were obtained using as PCR templates for each PCR
reaction the DNA recovered following dry storage on the indicated
dry storage matrix of 10 nanograms of DNA from HEK-293T cells at
ambient temperature (25.degree. C.) for 10 days, or for seven days
at elevated temperature (85.degree. C.). Set #1, dry storage matrix
was prepared from 50 mM hydroxyectoine, 10 mM boric acid, 0.4 mM
DTPA, pH 8.3; Set #2, 50 mM hydroxyectoine, 10 mM boric acid, 1 mM
sodium tetraborate, 0.4 mM DTPA, pH 8.3; Set #3, 25 mM
hydroxyectoine, 10 mM boric acid, 0.4 mM DTPA, pH 8.3; Set #4, 25
mM hydroxyectoine, 10 mM boric acid, 1 mM sodium tetraborate, 0.4
mM DTPA, pH 8.3; NP, dry storage with no matrix material present;
-20.degree. C., data from control DNA samples analyzed after
-20.degree. C. storage for comparable time periods.
[0061] FIG. 2 shows an electrophoretogram of human genomic (293T
cell) DNA samples following dry storage for one year at 85.degree.
C. on a borate/hydroxyectoine matrix of indicated composition, or
in Gentegra.TM. tubes (Genvault, San Diego, Calif.) according to
the manufacturer's instructions. Control lanes show DNA samples
analyzed after -20.degree. C. storage for a comparable time period
(20.degree. C.), and after one year at 85.degree. C. in the absence
of any protective dry storage matrix (NP). Outer lanes (unmarked)
contain 1 kb ladder reference standard from New England Biolabs
(Beverly, Mass.), in kb (from the top), 10, 8, 6, 5, 4, 3, 2, 1.5,
1.0, 0.5 kb.
[0062] FIG. 3 summarizes DNA recoveries determined by quantitative
PCR. Data were obtained using as PCR templates for each PCR
reaction the DNA recovered following dry storage on the indicated
dry storage matrix of 10 nanograms of DNA from HEK-293T cells for
seven days at elevated temperature (85.degree. C.). Set #1, dry
storage matrix was prepared from 50 mM hydroxyectoine, 10 mM boric
acid, 0.4 mM DTPA, pH 8.3; Set #2, 50 mM hydroxyectoine, 10 mM
boric acid, 1 mM sodium tetraborate, 0.4 mM DTPA, pH 8.3; Set #3,
25 mM hydroxyectoine, 10 mM boric acid, 0.4 mM DTPA, pH 8.3; Set
#4, 25 mM hydroxyectoine, 10 mM boric acid, 1 mM sodium
tetraborate, 0.4 mM DTPA, pH 8.3; NP, dry storage with no matrix
material present; -20.degree. C., data from control DNA samples
analyzed after -20.degree. C. storage for seven days. summarizes
DNA recoveries determined by quantitative PCR. Data were obtained
using as PCR templates for each PCR reaction the DNA recovered
following dry storage on the indicated dry storage matrix of 10
nanograms of DNA from HEK-293T cells at ambient temperature
(25.degree. C.) for 10 days. Set #1, dry storage matrix was
prepared from 50 mM hydroxyectoine, 10 mM boric acid, 0.4 mM DTPA,
pH 8.3; Set #2, 50 mM hydroxyectoine, 10 mM boric acid, 1 mM sodium
tetraborate, 0.4 mM DTPA, pH 8.3; Set #3, 25 mM hydroxyectoine, 10
mM boric acid, 0.4 mM DTPA, pH 8.3; Set #4, 25 mM hydroxyectoine,
10 mM boric acid, 1 mM sodium tetraborate, 0.4 mM DTPA, pH 8.3; NP,
dry storage with no matrix material present; -20.degree. C., data
from control DNA samples analyzed after -20.degree. C. storage for
10 days.
[0063] FIG. 4 summarizes DNA recoveries determined by quantitative
PCR. Data were obtained using as PCR templates for each PCR
reaction the DNA recovered following dry storage on the indicated
dry storage matrix of 10 nanograms of DNA from HEK-293T cells at
ambient temperature (25.degree. C.) for 10 days. Set #1, dry
storage matrix was prepared from 50 mM hydroxyectoine, 10 mM boric
acid, 0.4 mM DTPA, pH 8.3; Set #2, 50 mM hydroxyectoine, 10 mM
boric acid, 1 mM sodium tetraborate, 0.4 mM DTPA, pH 8.3; Set #3,
25 mM hydroxyectoine, 10 mM boric acid, 0.4 mM DTPA, pH 8.3; Set
#4, 25 mM hydroxyectoine, 10 mM boric acid, 1 mM sodium
tetraborate, 0.4 mM DTPA, pH 8.3; NP, dry storage with no matrix
material present; -20.degree. C., data from control DNA samples
analyzed after -20.degree. C. storage for 10 days.
[0064] FIG. 5 shows an electrophoretogram of purified RNA samples
recovered from borate-stabilizer storage matrices (lanes 1-6)
following dry storage for 72 hours at 60.degree. C. Control samples
were dry-stored in the absence of borate-stabilizer matrix (NP) or
at -80.degree. C.
[0065] FIG. 6 shows an electrophoretogram of purified RNA samples
recovered from borate-stabilizer storage matrices (lanes 7-11)
following dry storage for 72 hours at 60.degree. C. Control samples
were dry-stored in the absence of borate-stabilizer matrix (NP) or
at -80.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0066] The present invention is directed in certain embodiments as
described herein to compositions and methods for substantially dry
storage of a biological sample, based on the surprising discovery
that in the presence of certain matrix compositions that comprise a
borate composition and a stabilizer as provided herein, a
biological sample can be dried and stored at ambient or elevated
temperatures for extended periods of time, such that upon
subsequent restoration of solvent conditions substantially all of
the biological activity of the sample can be recovered.
[0067] As described herein, certain invention embodiments relate in
part to unexpected advantages provided by selection of a matrix
that dissolves or dissociates in a biocompatible solvent (e.g., a
solvent which is compatible with preserving structure and/or
activity of a biological sample), and in part to unexpected
advantages provided by the selection of the combination of a borate
composition with a stabilizer such as a stabilizer of at least one
of formulae (I)-(VII) or another stabilizer disclosed herein, from
which combination certain particularly useful dry storage matrices
may be comprised.
[0068] Certain embodiments described herein advantageously combine
a borate composition, such as one or more of boric acid, dihydrogen
borate, hydrogen borate, diborate, triborate, tetraborate,
metaborate, hydroxoborate (borax), borate salt, boric
acid-glycerol, boric anhydride (B.sub.2O.sub.3) and boric-acid-1,3
propanediol, with a stabilizer as provided herein such as a
stabilizer of formula (I)-(VII), including in certain preferred
embodiments a stabilizer that comprises a zwitterion (e.g., a
typically water-soluble compound having charged and typically
non-adjacent atoms but a net neutral, i.e., zero, charge) wherein
the borate composition and the stabilizer are present at a molar
ratio that is selected from a molar ratio of from about 10:1 to
about 1:10, a molar ratio of from about 5:1 to about 1:5, and a
molar ratio of from about 20:1 to about 1:20, including any
intermediate molar ratios therein.
[0069] These and related embodiments permit efficient, convenient
and economical storage of a wide variety of biological samples
including polynucleotides (e.g., nucleic acids such as DNA, RNA,
oligonucleotides and other naturally or artificially produced
nucleic acids), enzymes and other proteins, and cells, without
refrigeration or frozen storage. Samples may be dried without
lyophilization (although lyophilization may be employed if
desired), and following dry storage the samples may be used
immediately upon solvent reconstitution without a need for
separating the sample from the matrix material, which dissolves or
dissociates in the solvent and does not interfere with biological
activity of the sample.
[0070] Certain invention embodiments offer advantageously superior
recoveries of stored biological samples, including enhanced
detection sensitivity for interrogating samples containing minute
quantities of biomolecules of interest, and may find uses in
clinical, healthcare and diagnostic contexts, in biomedical
research, biological research and forensic science, and in
biological products and other settings where sample storage for
life sciences may be desired. Of particular note, the compositions
and methods described herein afford preservation and protection of
biological samples under conditions typically regarded as
inhospitable to biological sample storage, such as elevated
temperatures (e.g., increased in a statistically significant manner
over common ambient or room temperature ranges, such as sustained
temperatures in excess of 25.degree. C., 30.degree. C., 35.degree.
C., 40.degree. C., 45.degree. C., 50.degree. C., 55.degree. C.,
60.degree. C., 65.degree. C., 70.degree. C. and significantly
higher), without any need for expensive, cumbersome and
energy-demanding refrigeration or freezing equipment. For example,
these and related embodiments may be particularly attractive for
collection, shipping, storage and retrieval of biological samples
in undeveloped or underdeveloped regions of the world, as well as
in highly developed areas.
[0071] Certain embodiments of the present invention thus relate to
storage of dry samples including storage at ambient temperature,
and also may have use for the storage of diverse biological
materials and biological samples, such as but not limited to DNA,
RNA, blood, urine, feces, other biological fluids (e.g., serum,
serosal fluids, plasma, lymph, cerebrospinal fluid, saliva, mucosal
secretions of the secretory tissues and organs, vaginal secretions,
ascites fluids, fluids of the pleural, pericardial, peritoneal,
abdominal and other body cavities, cell and organ culture medium
including cell or organ conditioned medium, lavage fluids and the
like, etc.), buccal cells from the inner lining of the cheek
present in a buccal swab or sample, bacteria, viruses, yeast cells,
PCR products, cloned DNA, genomic DNA, oligonucleotides, plasmid
DNA, mRNA, tRNA, rRNA, siRNA, micro RNA, hnRNA, cDNA, proteins,
polypeptides, lipids, glycoconjugates (e.g., glycolipids,
glycoproteins), oligosaccharides, polysaccharides, vaccines (e.g.,
natural or synthetic, live or attenuated in the case of intact
biological particles such as viral or other microbial vaccines, or
extracts of natural, synthetic or artificial materials including
products of genetic engineering), cells and tissues, cell or tissue
lysates, cell or tissue homogenates or extracts, and the like, or
other biological samples.
[0072] Biological samples may therefore also include a blood
sample, biopsy specimen, tissue explant, organ culture, biological
fluid or any other tissue or cell preparation, or fraction or
derivative thereof or isolated therefrom, from a subject or a
biological source. The subject or biological source may be a human
or non-human animal, including mammals and non-mammals, vertebrates
and invertebrates, and may also be any other multicellular organism
or single-celled organism such as a eukaryotic (including plants
and algae) or prokaryotic organism archaeon, microorganisms (e.g.
bacteria, archaea, fungi, protists, viruses), aquatic plankton, a
primary cell culture or culture adapted cell line including but not
limited to genetically engineered cell lines that may contain
chromosomally integrated or episomal recombinant nucleic acid
sequences, immortalized or immortalizable cell lines, somatic cell
hybrid cell lines, differentiated or differentiable cell lines,
transformed cell lines, stem cells, germ cells (e.g. sperm,
oocytes), transformed cell lines and the like.
[0073] According to certain embodiments described herein there are
provided methods and compositions related to isolating nucleic
acids from a biological sample such as, but not limited to, cells
(e.g. eukaryotic, prokaryotic, bacteria, yeast) or viruses after
dry storage in a dry storage matrix and subsequent rehydration of
the sample. An unexpected advantage of the presently disclosed
embodiments is the ability to isolate and extract nucleic acids
from intact cells or viruses upon rehydration following dry storage
without refrigeration in a storage matrix. The simple one-step
addition of solvent, which in certain preferred embodiments may
comprise water, to rehydrate samples stored dry in the matrix
results, surprisingly, in isolation of nucleic acids that are ready
for use in downstream applications; further purification of such
extracted nucleic acids is unnecessary, but may be optionally
performed.
[0074] As disclosed herein, the steps for sample preparation, dry
storage and subsequent nucleic acid isolation by simple rehydration
can all be performed under ambient conditions (e.g., at room
temperature), thus eliminating the need for cold-storage and also
eliminating the need for the use of any heating sources as part of
the nucleic acid extraction procedure. A further advantage afforded
by certain embodiments based on the present disclosure that will be
appreciated by those skilled in the art is that the conditions
optimized for the isolation of nucleic acids after dry storage in
the matrix (e.g., the dry-storage matrix) render cells and viruses
non-viable, thus significantly increasing biosafety levels, and
further offering added convenience to many operations that may be
involved in the handling of potentially pathogenic biological
samples.
[0075] According to non-limiting theory, cells or viruses stored
dry as described herein, in a dry-storage matrix for appropriate
time periods at room temperature, are no longer viable due to
breakdown of cell membranes and viral envelopes. Presumably (and
further according to non-limiting theory) storage in the matrix
renders the cell membranes or viral envelope remnants passive and
completely penetrable to the matrix materials. Consequently, the
nucleic acids contained within the cell or virus are protected from
degradation by the storage matrix. Simple rehydration of the sample
results in isolation and recovery of nucleic acid, thus eliminating
the need for time-consuming and labor intensive purification
methods, as well as reducing or eliminating dangers associated with
handling suspected pathogens.
[0076] A further advantage that will be appreciated by one skilled
in the art is the usefulness of the herein disclosed methods and
compositions for replacing or augmenting costly freezer stocks of
precious, and oftentimes numerous, biological samples. For example,
bacterial cultures (from as little as a few microliters) can be
applied directly into the storage matrix for long-term dry, room
temperature storage and subsequent isolation of bacterial nucleic
acids (e.g. plasmid or genomic DNA). The presently described
compositions and methods thus provide an attractive and convenient
alternative to maintaining glycerol stocks that are extremely
labile to temperature fluctuations and that rely on costly and
potentially vulnerable freezer equipment, particularly if numerous
samples are involved. Hence, from as little as a few microliters of
a typical suspension of cells or viruses, rapid and safe collection
and processing of a large number of samples is possible. As
disclosed herein, cell-based isolation of nucleic acids from
samples stored dry in a dry-storage matrix as described below has
the additional utility in that long-term cataloging, storage and
processing of samples is possible via the simple addition of water
(or another solvent such as a biocompatible solvent that comprises
water) to isolate and recover nucleic acids. Sample processing
(e.g., nucleic acid isolation) can be performed at the user's
convenience, after collection of the biological sample, and can be
delayed indefinitely.
[0077] As disclosed herein, the duration of the period for
unrefrigerated dry storage of biological samples such as nucleic
acids, proteins, cells or viruses on a dry-storage matrix, the
particular biological source material such as the cells or viruses
used (e.g., strains, substrains, variants, types, subtypes,
isolates, quasi-species, and the like), and other factors may be
varied to affect the nucleic acid storage, isolation and recovery
methods. As will be appreciated by those skilled in the art and
based on the present disclosure, preliminary studies may be done
routinely to determine the optimal length of time for dry storage
of, e.g., isolated nucleic acids or of intact cells or viruses in
the matrix for protection and subsequent recovery of isolated
nucleic acids. Conditions for substantially dry storage of a cell
sample for purposes of recovering cellular nucleic acid from the
sample are distinct from conditions that may permit recovery of
viable cells (or of infective viral particles) following
substantially dry storage on a matrix such as those described in US
2006/0099567, according to which viable cell recovery typically
will involve storage periods of shorter duration than may be
employed for recovering cellular nucleic acid. Thus, for example,
in a preliminary study to determine a storage period beyond which
few or no detectable viable cells may be recovered, the viability
of a given preparation of bacterial cells, after rehydration
following dry storage at room temperature in the storage matrix,
can be determined by inoculating growth media directly with an
aliquot of the rehydrated sample and growing or attempting to grow
the culture under appropriate conditions (e.g. overnight at
37.degree. C.).
[0078] Isolation and recovery of nucleic acids following dry
storage of previously isolated nucleic acids, or of cells or
viruses on a dry-storage matrix as described herein, can be
determined using any of a number of assays practiced by those
skilled in the relevant art, including those described herein (see
for example, Maniatis, T. et al. 1982. Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor University Press, Cold Spring
Harbor, N.Y.; Ausubel et al., 1993 Current Protocols in Molecular
Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons,
Inc., Boston, Mass.). For example, to determine if plasmid DNA has
been successfully isolated from bacterial cells after dry storage
in the matrix at room temperature, rehydrated samples can be
directly transformed into competent bacteria. Growth of bacterial
colonies in the appropriate selection medium indicates successful
incorporation of plasmid DNA, and colony counts provide an easy
assay to determine transfection efficiency. Restriction enzyme
analysis can also be performed to verify successful isolation of
the appropriate plasmid DNA as recovered according to the presently
described methods from bacterial cells that have been stored dry
without refrigeration in the storage matrix.
[0079] Isolation and recovery of genomic DNA (or RNA) following dry
storage without refrigeration on a dry-storage matrix as herein
described can be determined using nucleic acid hybridization
analysis (such as PCR, real-time PCR, reverse transcription PCR,
quantitative PCR, etc.) with oligonucleotide primers that are
specific for target genomic nucleic acid sequences that may be
present in a dry-stored cell or virus. For example, PCR ribotyping
can be used to identify bacterial strains (Kostman et al. 1995. J.
Infect. Dis. 171:204-208). Other assays used for genomic
phenotyping analysis include, for example, but are not intended to
be limited to, restriction fragment length polymorphism analysis of
PCR products, randomly amplified polymorphic DNA, repetitive
element-based PCR, pulse-field gel electrophoresis, sequencing of
individual genes that may be related to virulence, and multi-locus
enzyme electrophoresis, (see for example, Baumforth, K. R. N. et
al. 1999. J Clin Pathol: Mol. Pathol. 52:112-10; Becker Y, Darai G.
1995. PCR: protocols for diagnosis of human and animal virus
diseases, Springer Lab Manual. Berlin: Springer-Verlag; Read, S. J.
2000. J. Clinical Path. 53(7):502-506; Shaw, K. J. (ed). 2002.
Pathogen Genomics: Impact on Human Health, Humana Press, Inc.,
Totowa, N.J.; Maiden, M. C. et al. 1998. Proc. Natl. Acad. Sci. USA
95:3140-3145; Lindstedt, B. A. et al. 2003. J. Clin. Microbiol.
41:1469-1479; Klevytska, A. M. et al. 2001. J. Clin. Microbiol.
39:3179-3185; and Yazdankhah, S. P. et al. 2005. J. Clin.
Microbiol. 43(4):1699-1705).
[0080] As described herein, a nucleic acid refers to a polymer of
two or more modified and/or unmodified deoxyribonucleotides or
ribonucleotides, either in the form of a separate fragment or as a
component of a larger construction. Examples of polynucleotides
include, but are not limited to, DNA, RNA, or DNA analogs such as
PNA (peptide nucleic acid), and any chemical modifications thereof.
The DNA may be a single- or double-stranded DNA, cDNA, or a DNA
amplified by any amplification technique, or any DNA polymer. The
RNA may be mRNA, rRNA, tRNA, siRNA, total RNA, small nuclear RNA
(snRNA), RNAi, micro RNA, genomic RNA, RNA isolated from cells or
tissues, a ribozyme, or any RNA polymer. Encompassed are not only
native nucleic acid molecules, such as those that can be isolated
from natural sources, but also forms, fragments and derivatives
derived therefrom, as well as recombinant forms and artificial
molecules, as long as at least one property of the native molecules
is present. Preferred biological samples are those that can be
applied to analytical, diagnostic and/or pharmaceutical purposes,
such as, but not limited to, nucleic acids and their derivatives
(e.g. oligonucleotides, DNA, cDNA, PCR products, genomic DNA,
plasmids, chromosomes, artificial chromosomes, gene transfer
vectors, RNA, mRNA, tRNA, siRNA, miRNA, hnRNA, ribozymes, genomic
RNA, peptide nucleic acid (PNA), and bacterial artificial
chromosomes (BACs)).
[0081] Nucleic acid molecule(s), oligonucleotide(s), and
polynucleotide(s), include RNA or DNA (either single or double
stranded, coding, complementary or antisense), or RNA/DNA hybrid
sequences of more than one nucleotide in either single chain or
duplex form (although each of the above species may be particularly
specified). The term "nucleotide" may be used herein as an
adjective to describe molecules comprising RNA, DNA, or RNA/DNA
hybrid sequences of any length in single-stranded or duplex form.
More precisely, the expression "nucleotide sequence" encompasses
the nucleic material itself and is thus not restricted to the
sequence information (i.e., the succession of letters chosen among
the four base letters) that biochemically characterizes a specific
DNA or RNA molecule. The term "nucleotide" is also used herein as a
noun to refer to individual nucleotides or varieties of
nucleotides, meaning, e.g., a molecule, or individual subunit in a
larger nucleic acid molecule, comprising a purine or pyrimidine, a
ribose or deoxyribose sugar moiety, and a phosphate group, or
phosphodiester linkage in the case of nucleotides within an
oligonucleotide or polynucleotide. The term "nucleotide" is also
used herein to encompass "modified nucleotides" which comprise at
least one modification such as (a) an alternative linking group,
(b) an analogous form of purine, (c) an analogous form of
pyrimidine, or (d) an analogous sugar.
[0082] Certain embodiments of the present invention relate to the
preservation, storage, retrieval, and/or analysis of nucleic acids
isolated from intact cells or viruses. An intact cell preferably
has an intact plasma membrane that is capable of selectively
excluding solutes and/or of retaining cellular cytoplasmic
components such as organelles (e.g., nuclei, ribosomes,
mitochondria, endoplasmic reticulum, vacuoles) vesicles and other
membrane-bound compartments, intracellular biomolecules
(polynucleotides, polypeptides, lipids, carbohydrates,
intracellular mediators, co-factors and the like), macromolecular
structures and/or assemblies (e.g., cytoskeletal elements,
centrioles, chromatin), cytosol, etc. Preferably and in certain
non-limiting embodiments, an intact cell is viable, but the
invention need not be so limited. Certain embodiments are provided
for the isolation and/or extraction from cells and/or viruses, and
storage of cellular nucleic acids at ambient temperature, that are
obtained or derived from biological samples that may include but
are not limited to blood and cells contained therein (e.g.,
lymphocytes, polymorphonuclear leukocytes, monocytes, granulocytes,
platelets, erythrocytes and other circulating cells including cells
of hematopoietic origin), urine, other biological fluids (e.g.,
serum, serosal fluids, plasma, lymph, cerebrospinal fluid, saliva,
mucosal secretions of the secretory tissues and organs, vaginal
secretions, ascites fluids, fluids of the pleural, pericardial,
peritoneal, abdominal and other body cavities, cell and organ
culture medium including cell or organ conditioned medium, lavage
fluids and the like, etc.), cells from the inner lining of the
cheek present in a buccal swab or sample, bacteria, biofilms,
viruses, yeast cells, cells and tissues, cell or tissue lysates,
cell or tissue homogenates or extracts, and the like, or other
biological samples.
[0083] Other sources of intact cells for isolation or extraction of
nucleic acids that are contemplated herein may also include a blood
sample, biopsy specimen (including tumor specimens), tissue
explant, organ culture, cancer cell, biological fluid or any other
tissue or cell preparation, or fraction or derivative thereof or
isolated therefrom, from a subject or a biological source. The
subject or biological source may be a human or non-human animal,
including mammals and non-mammals, vertebrates and invertebrates,
and may also be any other multicellular organism or single-celled
organism or biofilm such as a eukaryotic (including plants) or
prokaryotic organism or archaea, a primary cell culture or culture
adapted cell line including but not limited to genetically
engineered cell lines that may contain chromosomally integrated or
episomal recombinant nucleic acid sequences, immortalized or
immortalizable cell lines, somatic cell hybrid cell lines,
differentiated or differentiable cell lines, transformed cell lines
and the like.
[0084] Bacterial cells according to certain embodiments described
herein may include bacteria that belong to a genus selected from
Caulobacter, Staphylococcus, Bacillus, Salmonella, Campylobacter,
Aerobacter, Rhizobium, Agrobacterium, Clostridium, Nostoc,
Tricodesium, Pseudomonas, Xanthomonas, Nitrobacteriaceae,
Nitrobacter, Nitrosomonas, Thiobacillus, Spririllum, Vibrio,
Baceroides, Kelbsilla, Escherichia, Klebsiella, Shigella, Erwinia,
Rickettsia, Chlamydia, Mycobacterium, Polyangium, Micrococcus,
Lactobacillus, Diplococcus, Streptococcus, Spirochaeta, Treponema,
Borrelia, Leptospira, or Streptomyces.
[0085] Certain embodiments relate to a biological sample that may
comprise an isolated biomolecule, where the term "isolated" means
that the material is removed from its original environment (e.g.,
the natural environment if it is naturally occurring). For example,
a naturally occurring nucleic acid or polypeptide present in an
intact cell or in a living animal is not isolated, but the same
nucleic acid or polypeptide, separated from some or all of the
co-existing materials in the natural system, is isolated. Such
nucleic acids could be part of a vector and/or such nucleic acids
or polypeptides could be part of a composition, and still be
isolated in that such vector or composition is not part of its
natural environment.
[0086] Certain other embodiments relate to a biological sample that
may comprise an intact cell or a living animal or organism that has
not been depleted of, or from which has not been removed, a
cell-derived molecular component such as a protein or peptide,
lipid (including phospholipids, glycolipids and other lipids),
nucleic acid (including DNA and RNA), carbohydrate (including
oligosaccharides and polysaccharides and their derivatives),
metabolite, intermediate, cofactor or the like, or any covalently
or non-covalently complexed combination of these components and any
other biological molecule that is a stable or transient constituent
of a viable cell.
[0087] Techniques for isolating and/or purifying a cellular
molecular component may include any biological and/or biochemical
methods useful for separating the component from its biological
source, and subsequent characterization may be performed according
to standard biochemical and molecular biology procedures. Those
familiar with the art will be able to select an appropriate method
depending on the biological starting material and other factors.
Such methods may include, but need not be limited to, radiolabeling
or otherwise detectably labeling cellular and subcellular
components in a biological sample, cell fractionation, density
sedimentation, differential extraction, salt precipitation,
ultrafiltration, gel filtration, ion-exchange chromatography,
partition chromatography, hydrophobic chromatography,
electrophoresis, affinity techniques or any other suitable
separation method that can be adapted for use with the agent with
which the cellular molecular component interacts. Antibodies to
partially purified components may be developed according to methods
known in the art and may be used to detect and/or to isolate such
components.
[0088] Certain other embodiments relate to a biological sample that
may comprise a purified biomolecule, such as but not limited to a
nucleic acid, where the terms "purified" or "substantially
purified" refer to recovery of a biomolecule (such as a nucleic
acid) which is at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%,
75-80%, 80-85%, 85-90%, 90-95%, 92%, 94%, 96%, 98%, 95-100% or
98-100% purified with respect to removal of a contaminant, e.g.,
cellular components such as protein, lipid or salt; thus, the term
"substantially purified" generally refers to separation of a
majority of cellular proteins or reaction contaminants from the
biological sample, so that compounds capable of interfering with
the subsequent use of the isolated biomolecule (such as a nucleic
acid) are removed.
[0089] Certain herein described embodiments relate to stabilization
and/or preservation of a biological sample, which involves
maintenance, retention or reconstitution of the structural and/or
functional integrity of biological samples (including of molecular,
multimolecular or oligomeric, organellar, subcellular, cellular,
multicellular, or higher organizational levels of biological
structure and/or function) and of the biological properties based
thereupon. The biological activity of a biological sample that
comprises, in a particular embodiment, a macromolecule or
biopolymer or the like such as a polypeptide or polynucleotide, may
involve, for example, the extensive maintenance of its primary,
secondary and/or tertiary structure. The biological activity of a
nucleic acid probe comprises, for example, its property of forming
in a sequence-specific manner a hybridization complex (e.g., a
duplex) with a nucleic acid target which is complementary to the
probe. The biological activity of a nucleic acid, for example, may
comprise a DNA encoding a cytocide, a prodrug, a therapeutic
molecule, or another nucleic acid molecule or encoded product that
has a discernible or detectable effect upon or within cells. Such
biological activity may be assayed by any method known to those of
skill in the art, including, but not limited to, in vitro and/or in
vivo assays that assess efficacy by measuring the effect on cell
proliferation or on protein synthesis (see for example, Sambrook et
al., 1989; Current Protocols, Nucleic Acid Chemistry, Molecular
Biology, Wiley and Sons, 2003; and Asubel, F M et al. (Eds.). 2007.
Current Protocols in Molecular Biology, Wiley and Sons, Inc.
Hoboken, N.J.). Additional non-limiting examples of the biological
activity of nucleic acids and polynucleotides include transfection,
transformation, amplification, enzymatic reaction, gene expression,
translation, transcription, and hybridization. The biological
activity of an antibody comprises, for example, a specific binding
interaction with its cognate antigen.
[0090] As described herein, the biological activity of a substance
means any activity which can affect any physical or biochemical
properties of a biological system, pathway, molecule, or
interaction relating to an organism, including for example but not
limited to, viruses, bacteria, bacteriophage, prions, insects,
fungi, plants, animals, and humans. Examples of substances with
biological activity include, but are not limited to,
polynucleotides, peptides, proteins, enzymes, antibodies, small
molecules (e.g. a bioactive small molecule, whether naturally
occurring or artificial, preferably of less than 10.sup.5 daltons
molecular mass, more preferably less than 10.sup.4 daltons, and
more preferably less than 10.sup.3 daltons, as provided herein),
pharmaceutical compositions (e.g., drugs), vaccines, carbohydrates,
lipids, steroids, hormones, chemokines, growth factors, cytokines,
liposomes, and toxins, liposomes. Persons familiar with the
relevant art will recognize appropriate assays and methods for
determining the biological activity of substances that affect the
physical or biochemical properties of a biological system,
including for example but not limited to, gene expression (see for
example, Asubel, F M et al. (Eds.). 2007. Current Protocols in
Molecular Biology, Wiley and Sons, Inc. Hoboken, N.J.),
receptor-ligand interactions (see for example, Coligan et al.
(Eds.). 2007. Current Protocols in Immunology, Wiley and Sons, Inc.
Hoboken, N.J.), enzymatic activity (see for example, Eisenthal and
Hanson (Eds.), 2002 Enzyme Assays. Second Edition. Practical
Approaches series, no 257. Oxford University Press, Oxford, UK;
Kaplan and Colowick (Eds.), 1955 and 1961 Preparation and Assay of
Enzymes, Methods in Enzymology, (vols. 1, 2 and 6). Academic Press,
Ltd., Oxford, UK), cytokine and cell proliferation and/or
differentiation activities (see for example, Coligan et al. (Eds.).
2007. Current Protocols in Immunology, Wiley and Sons, Inc.
Hoboken, N.J.), signal transduction (see for example, Bonifacino et
al. (Eds.). 2007. Current Protocols in Cell Biology, Wiley and
Sons, Inc. Hoboken, N.J.) and cell toxicity (see for example, Bus J
S et al. (Eds). 2007. Current Protocols in Toxicology, Wiley and
Sons, Inc. Hoboken, N.J.), apoptosis and necrosis (Green, D R and
Reed, J C. 1998 Science August 28; 281(5381):1309-12; Green, D R.
1998. Nature December 17: 629; Green D R. 1998 Cell 94(6):695-69;
Reed, J C (Ed.), 2000 Apoptosis, Methods in Enzymology (vol. 322).
Academic Press Ltd., Oxford, UK).
[0091] As described herein, recovery, following storage, of
substantially all biological activity refers to recovery of at
least 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 92%, 94%, 96%, 98%,
99%, 95-100% or 98-100% of the biological activity of a sample as
compared to the biological activity of the sample as determined
prior to storage according to the methods and compositions as
provided herein. In other embodiments as described herein,
substantial loss of the biological activity of a sample may be
apparent when, for instance, following unrefrigerated substantially
dry storage of an isolated nucleic acid sample or of a dry-storable
cell sample, the biological activity after storage decreases in a
statistically significant manner compared to the biological
activity present in the sample prior to storage, which decrease may
in some embodiments refer to any decrease in activity having
statistical significance relative to an appropriate control sample
as will be familiar to those skilled in the art, but which may in
some other embodiments refer to a decrease having statistical
significance that is more than a decrease of 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 10-12%, 12-15%, 15-20%, 20-25% or 25-30% of
the biological activity present in the sample prior to storage.
[0092] For example, and remarkably, according to certain herein
disclosed embodiments, dry storage of isolated intact cells
comprising, as provided herein, contacting one or a plurality of
isolated intact cells that contain nucleic acid with the presently
disclosed dry-storage matrix that dissolves in a biocompatible
solvent, drying the matrix to substantially remove the solvent,
maintaining without refrigeration for one or more days the
dry-storable cell sample so obtained, and resuspending or
redissolving the sample in a biocompatible solvent, permits simple
and efficient recovery of substantially purified cellular nucleic
acid having substantially all of the biological activity present in
the cellular nucleic acid prior to dry storage. Preferably in such
embodiments the cell is a bacterial cell. For example, and
according to non-limiting theory, substantially dry storage of a
bacterial cell sample on a dry-storage matrix followed by solvent
reconstitution (e.g., rehydration) under conditions and for a time
sufficient as described herein, is believed to release gently and
efficiently the cellular nucleic acid from the bacterial cell, such
that the simple resuspension or resolubilization of the dried cell
sample in a biocompatible solvent permits ready recovery of
isolated cellular nucleic acid. In other related embodiments
wherein the cell is a non-bacterial cell, the step of recovering
isolated nucleic acid from a dry-stored cell sample preferably
includes purifying the nucleic acid according to any of a number of
methodologies for nucleic acid extraction, separation, differential
solubilization, isolation, etc. such as those described herein and
known to the art.
[0093] In certain embodiments, the invention thus relates to the
long-term storage of biological, chemical and biochemical material
under dry conditions, and in a manner ready for immediate use after
hydration (e.g., upon rehydration). As described herein, there are
provided embodiments which include a) the specific dissolvable (or
dissociatable) storage matrix comprising a borate composition and a
stabilizer (which in certain embodiments may be a zwitterionic
compound) as provided herein, and optionally comprising additional
components as described herein, such as one or more of a chelator,
a pH buffer, a biological or biochemical inhibitor and/or a
detectable indicator, b) preparation and optimization of the
storage matrix with chemicals that increase the durability of the
long term storage conditions, including in certain embodiments,
e.g., the use of a stabilizer which may comprise a compound of
formula (I)-(VII) and/or another osmoprotectant compound, c)
preparation of different biological materials prior to the drying
process that allow immediate activity and usability of the
materials after rehydration, and d) the process of simplifying
complex biochemical processes through the use of dry stored
biologically active materials.
[0094] These and related embodiments thus provide surprising
advantages associated with unrefrigerated dry storage of
biologicals, including improved stabilization and preservation of
biological activity in biological samples, reduced degradation of
biological samples during storage at room temperature in dried form
(and in particular through the use of a protective matrix), and
simplification of the processes for preparing biological samples
for further use by reducing or eliminating the need for
time-consuming re-calibration and aliquoting of such samples, and
by eliminating the need for physically separating a sample from the
storage medium. Invention embodiments as described herein
additionally provide unexpectedly superior biological sample
recoveries by reducing or eliminating factors that can otherwise
reduce sample recovery yields, such as undesirable sample
denaturation and/or sample loss due to adsorption of the sample on
sample container surfaces.
[0095] According to certain embodiments the invention allows for
purification and optionally size fractionation of DNA, RNA,
proteins and other biomolecules, cells, cellular components and
other biological materials, minerals, chemicals, or compositions
derived from a biological sample or other life sciences related
sample. In certain embodiments the invention thus readily permits,
for example, the use of one or a plurality of biological materials
and/or biological samples in the performance of molecular biology
procedures, including but not limited to polymerase chain reaction
or PCR (including RT-PCR), biopolymer (e.g., polynucleotide,
polypeptide, oligosaccharide or other biopolymer) sequencing,
oligonucleotide primer extension, haplotyping (e.g., DNA
haplotyping) and restriction mapping in one unified, integrated and
easy-to-use platform. The invention also readily permits, for
example and in certain embodiments, the use of one or a plurality
of biological samples and/or biological materials for the
performance of protein crystallography. In other embodiments there
is provided a platform for use, testing or detection (including
diagnostic applications) of an antibody or small molecule (whether
naturally occurring or artificial) or other biological molecule
(e.g., a "biomolecule"), for example, a protein, polypeptide,
peptide, amino acid, or derivative thereof; a lipid, fatty acid or
the like, or derivative thereof; a carbohydrate, saccharide or the
like or derivative thereof, a nucleic acid, nucleotide, nucleoside,
purine, pyrimidine or related molecule, or derivative thereof, or
the like; or another biological molecule that is a constituent of a
biological sample.
Dry Storage of a Biological Sample
[0096] Compositions and methods described herein relate to dry
and/or substantially dry storage of a biological sample, and may
include the use of any suitable container, including, for example,
a dry storage device. The dry storage device is an application of
the biological sample storage device as herein disclosed, which
contains a matrix material for use as a dry storage matrix,
including in certain preferred embodiments a matrix material that
dissolves or dissociates in a solvent comprising borate and at
least one stabilizer that comprises a compound of formula (I)-(VII)
and/or an osmoprotectant compound as disclosed herein, preferably
selected according to certain embodiments from the group consisting
of hydroxyectoine, ectoine, homoectoine, betaine, L-carnitine,
sarcosine, N,N-Dimethylglycine, triethylammonium acetate, glycerol
phosphate, tricine, MOPSO, pentaerythritol and
N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl sulfonate as
described herein, for long-term storage of a biological sample or a
biological material, such as but not limited to blood, bacteria,
cells, viruses, chemical compounds (whether naturally occurring or
artificially produced), plasmid DNA, DNA fragments,
oligonucleotides, peptides, fluorogenic substrates, genomic DNA,
PCR products, cloned DNA, proteins, RNA, vaccines, minerals and
chemicals, and other biological samples as disclosed herein.
[0097] These and related embodiments derive from the surprising
observation that stable, long-term dry storage of biological
samples or biological materials may be effected without
refrigeration when such samples or materials are loaded onto a
suitable matrix material such as those described herein, including
a dissolvable (or dissociable) matrix material that comprises a
borate composition and a stabilizer. According to non-limiting
theory, biological materials present in a biological sample may
interact with the matrix material by absorption, adsorption,
specific or non-specific binding or other mechanism of attachment,
including those involving formation of non-covalent and/or covalent
chemical bonds and or intermolecular associative interactions such
as hydrophobic and/or hydrophilic interactions, hydrogen bond
formation, electrostatic interactions, and the like. Accordingly,
the present invention provides devices for stable, long-term dry
storage of biological samples at common indoor ambient room
temperatures (e.g., typically 20-27.degree. C. but varying as a
function of geography, season and physical plant from about
15-19.degree. C. or about 18-23.degree. C. to about 22-29.degree.
C. or about 28-32.degree. C.) for use in the sample storage and
processing methods and systems described herein.
[0098] Preferred embodiments employ the dissolvable matrix material
(e.g., borate composition/stabilizer matrix) or a dissociable
matrix material that may be dried before, during, or after being
contacted with the sample to provide dry storage, wherein in some
preferred embodiments such contact involves contacting the matrix
material and the sample in a fluid or liquid (e.g., fluidly
contacting), to provide substantially dry storage. Related
preferred embodiments thus involve the use of sample storage
devices as described herein that comprise a matrix material which
is capable of dry storage of a biological sample or a biological
material without refrigeration, for example, at ambient room
temperature. In certain related embodiments a drying step may be
performed to effect loading of the sample onto the matrix material
for dry storage, for example by air drying, drying at elevated
temperature or by the volatilization of solvent through exposure of
the sample loaded matrix material to reduced atmospheric pressure
(e.g., lyophilization or other vacuum drying method) or to a gentle
flowstream of a compatible gas such as nitrogen. The samples are
preferably stored dry under conditions that stabilize the sample,
i.e., little or no detectable (e.g., with statistical significance)
degradation or undesirable chemical or physical modification of the
sample occurs, according to criteria that will vary as a factor of
the nature of the sample being stored and that will in any event be
familiar to those having skill in the relevant art. In other
embodiments using the dry storage device, sample loading results in
dry storage, for example, whereby a liquid sample is absorbed by,
adsorbed to or otherwise entrapped by the matrix material such that
after loading no free liquid is readily discernible in or on, or
easily dislodged from, the matrix material, which may be dried as
just described.
[0099] Certain preferred embodiments provide compositions and
methods for storing biological material (e.g., polynucleotides,
genomic DNA, plasmid DNA, DNA fragments, RNA, oligonucleotides,
proteins, peptides, fluorogenic substances, cells, viruses,
chemical compounds, vaccines, etc.) or other biological samples as
provided herein on a matrix comprised of a material that dissolves
or dissociates, the matrix comprising a borate composition and at
least one stabilizer comprising a compound of formula (I)-(VII)
and/or an osmoprotectant compound as provided herein, in a solvent
that allows complete recovery or substantial recovery (e.g.,
recovery of at least 50 percent, preferably at least 60 percent,
more preferably at least 70 percent, more preferably at least 80
percent, and typically in more preferred embodiments at least 85
percent, more preferably at least 90, 91, 92, 93 or 94 percent,
more preferably at least 95 percent, still more preferably greater
than 96, 97, 98 or 99 percent) of the dried sample material after
hydration, rehydration or other solvent reconstitution of the
sample.
[0100] For example, a dissolvable matrix comprising a borate
composition and at least one stabilizer of formula (I)-(VII) and/or
an osmoprotectant may be capable of being solubilized in a suitable
solvent that can be selected based on the properties of the matrix
material and/or of the sample depending on the particular
methodology being employed and in a manner that permits recovery of
one or more desired structural or functional properties of the
sample (e.g., biological activity). Similarly, as another example,
the matrix comprising a borate composition and at least one
stabilizer of formula (I)-(VII) and/or an osmoprotectant may
dissociate in a solvent and may, but need not, become fully
solubilized, such that a dispersion, suspension, colloid, gel, sap,
slurry, syrup, or the like may be obtained. In other embodiments a
matrix comprising borate composition and at least one stabilizer of
formula (I)-(VII) and/or an osmoprotectant may further include, in
addition to the dissolvable/dissociable borate/stabilizer matrix
components, one or more additional components such as, but not
limited to, a sponge-like material, silica, silica powder, silica
filter paper, absorbent powder, cotton, wool, linen, polyvinyl
alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylamide,
poly(N-vinylacetamide), polyester, nylon, positively charged nylon
or filter paper, any of which may influence physicochemical
properties, including solubility properties, of the storage matrix,
as will be appreciated by those familiar with the art.
[0101] In certain of these and related embodiments, the first
solvent which is used to introduce the matrix material and/or the
biological sample to the biological sample storage device prior to
a drying step for dry sample storage may be the same as the second
solvent that is subsequently used to hydrate, rehydrate,
reconstitute or resuspend the dried sample/matrix combination, and
in other embodiments the second solvent may be different from the
first. Criteria for selection of a suitable solvent for dissolving
or dissociating the matrix material and/or the biological sample
will be known to those familiar with the relevant art based, for
example, on physicochemical properties of the particular matrix
material and sample being used, and on the structural or functional
properties (e.g., bioactivity) that are desirably retained during
dry storage and subsequent reconstitution, as well as on other
factors (e.g., compatibility with other storage device materials,
or liquid handling equipment, safety, etc.).
[0102] In certain preferred embodiments at least one solvent for
use in compositions and methods disclosed herein will be aqueous,
for example, a biocompatible solvent such as a biological fluid, a
physiological solution or an aqueous biological buffer solution
selected to support a biological structure and/or function of a
biomolecule by preserving for that biomolecule a favorable chemical
milieu that is conducive to the structure and/or function.
Non-limiting examples of such biocompatible solvents include
physiological saline (e.g., approximately 145 mM NaCl), Ringer's
solution, Hanks' balanced salt solution, Dulbecco's phosphate
buffered saline, Erle's balanced salt solution, and other buffers
and solutions and the like as will be known to those familiar with
the art, including those containing additives as may be desired for
particular biomolecules of interest.
[0103] According to other embodiments, however, the invention need
not be so limited and other solvents may be selected, for instance,
based on the solvent polarity/polarizability (SPP) scale value
using the system of Catalan et al. (e.g., 1995 Liebigs Ann. 241;
see also Catalan, 2001 In: Handbook of Solvents, Wypych (Ed.),
Andrew Publ., NY, and references cited therein), according to
which, for example, water has a SPP value of 0.962, toluene a SPP
value of 0.655, and 2-propanol a SPP value of 0.848. Methods for
determining the SPP value of a solvent based on ultraviolet
measurements of the
2-N,N-dimethyl-7-nitrofluorene/2-fluoro-7-nitrofluorene
probe/homomorph pair have been described (Catalan et al., 1995).
Solvents with desired SPP values (whether as pure single-component
solvents or as solvent mixtures of two, three, four or more
solvents; for solvent miscibility see, e.g., Godfrey 1972 Chem.
Technol. 2:359) based on the solubility properties of a particular
matrix material can be readily identified by those having
familiarity with the art in view of the instant disclosure.
Borate Compositions in the Dissolvable/Dissociatable Dry Storage
Matrix
[0104] According to non-limiting theory, the dissolvable or
dissociable matrix material may therefore comprise a borate
composition and at least one stabilizer as provided herein (e.g., a
compound of formula (I)-(VII) and/or an osmoprotectant compound,
which may include a compound in one of formulae (I)-(VII)) which,
by forming a matrix, creates a three dimensional space which allows
biological material of the biological sample to associate with the
matrix. The dissolvable or dissociable matrix material may be used
to introduce additional stabilizing agents such as salts and
buffers (e.g., pH buffers) under dehydrated (e.g., dried or
substantially solvent-free) conditions. By way of example but
without limitation, exemplary stabilizers include zwitterionic
compounds in certain preferred embodiments and may also include
non-zwitterionic compounds, and may in certain other preferred
embodiments be selected from hydroxyectoine, ectoine, homoectoine,
betaine, L-carnitine, sarcosine, N,N-Dimethylglycine,
triethylammonium acetate, glycerol phosphate, tricine, MOPSO,
pentaerythritol and
N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl sulfonate and
the like. The matrix also allows inclusion of components (e.g.,
buffers) for the adjustment of pH and other parameters for optimal
drying and storage conditions, and may optionally comprise one or a
plurality of detectable indicators as provided herein, such as
color-based pH indicators, and/or moisture indicators. According to
certain other embodiments, the dissolvable or dissociable matrix
material may be any suitable material having the compatible
characteristics for storing a particular type of biological sample
in a manner that satisfactorily preserves the desired structural
and/or functional properties, said characteristics including the
ability to dry in a manner that forms a matrix within the
interstices of which the biological molecules of interest are
deposited, and also including appropriate solvent (e.g., biological
buffer) compatibility further including an ability to be
redissolved or resuspended subsequent to dry storage in a manner
whereby the matrix molecules do not interfere with one or more
biological activities of interest in the sample.
[0105] Non-limiting examples of a borate compositions for use
according to certain presently contemplated embodiments include
sodium tetraborate (borax), boric acid, combinations of borate with
citrate, boric acid-glycerol, boric acid-1,3 propanediol and the
like, including dihydrogen borate, hydrogen borate, diborate,
triborate, tetraborate, metaborate, hydroxoborate (borax), borate
salt, boric acid-glycerol, boric anhydride (B.sub.2O.sub.3) and
boric-acid-1,3 propanediol. As used herein, boric acid salt and
borate salt are used interchangeably to refer to a salt such as
potassium borate, monoethanolamine borate, or another salt obtained
by, or that can be visualized as being obtained by, neutralization
of boric acid. The weight percent of a boric acid salt or borate
salt in a composition of these and related embodiments can be
expressed either as the weight percent of either the negatively
charged boron containing ion, e.g. the borate and/or boric acid
moieties, or as the weight percent of the entire boric acid salt,
e.g. both the negatively charged moiety and the positively charged
moiety. Preferably, the weight percent refers to the entire boric
acid salt. Weight percents of citric acid salts, or other acid
salts, can also be expressed in this manner, preferably with
reference to the entire acid salt. As used herein, the term "total
boron compound" refers to the sum of borate and boric acid
moieties. The borate salt may also include any of a variety of
salts of boric acid, for example, alkali metal salts or alkanol
amine salts. Examples of the use of borate as a stabilizing agent
for biological molecules include those found, for example, in US
Patent Publications 2006/0293212, 2006/0193968, 2007/0073039, and
2006/0177855, and in WO 2009/002568. Certain borate compositions as
disclosed herein may also possess functional attributes of pH
buffers and/or of biological or biochemical inhibitors as described
herein. For example, borate is known to inhibit nuclease digestion
reactions. (Maniatis, T. et al. 1982. Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor University Press, Cold Spring
Harbor, N.Y.)
[0106] Certain embodiments of the present invention are
contemplated that include the use of such combinations of a
dissolvable or dissociatable matrix material comprising a borate
composition and at least one such stabilizer of formula (I)_(VII)
(or an osmoprotectant compound as provided herein), along with a
second stabilizer that may or may not comprise a biological or
biochemical inhibitor. Certain other embodiments of the present
invention contemplate the use of such combinations of a dissolvable
or dissociatable matrix material comprising borate and at least one
stabilizer for substantially dry storage of biological samples such
as proteins, cells, viruses, bacteria, blood, tissues and
polynucleotides such as DNA, RNA, synthetic oligonucleotides,
genomic DNA, natural and recombinant nucleic acid plasmids and
constructs, and the like.
[0107] As described herein, according to certain embodiments, the
borate composition of the herein described dry storage matrix is
capable of non-covalent association with one or more stabilizers
that is a compound of formula (I)-(VII) or an osmoprotectant
compound, and that may be preferably hydroxyectoine, ectoine,
homoectoine, betaine, L-carnitine, sarcosine, N,N-dimethylglycine,
triethylammonium acetate, glycerol phosphate, tricine, MOPSO,
pentaerythritol and/or
N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl sulfonate, and
according to certain other non-limiting embodiments, the stabilizer
is capable of non-covalent association with one or more molecular
species present in the liquid-storable biological sample and having
origins in the subject or biological source (e.g., biomolecules
such as polypeptides, polynucleotides, naturally occurring
oligosaccharides, naturally occurring lipids, and the like).
[0108] Methodologies and instrumentation for the determination of
non-covalent associations between such components will be known to
those familiar with the art in view of the present disclosure, and
may include techniques such as electrospray ionization mass
spectrometry (Loo et al., 1989 Anal. Biochem. June; 179(2):404-412;
Di Tullio et al. 2005 J. Mass Spectrom. July; 40(7):845-865),
diffusion NMR spectroscopy (Cohen et al., 2005 Angew Chem Int Ed
Engl. January 14; 44(4):520-554), or other approaches by which
non-covalent associations between molecular species of interest can
be demonstrated readily and without undue experimentation (for
example, circular dichroism spectroscopy, scanning probe
microscopy, spectrophotometry and spectrofluorometry, and nuclear
magnetic resonance of biological macromolecules; see e.g., Schalley
C A et al. (Eds.) 2007 Analytical Methods in Supramolecular
Chemistry Wiley Publishers, Hoboken, N.J.; Sauvage and Hosseini
(Eds.). 1996. ComprehensivaFe Supramolecular Chemistry. Elsevier
Science, Inc. New York, London, Tokyo; Cragg, P J (Ed.). 2005 A
Practical Guide to Supramolecular Chemistry Wiley & Sons, Ltd.,
West Sussex, UK; James et al. (Eds.), 2001 and 2005 Nuclear
Magnetic Resonance of Macromolecules: Methods in Enzymology (vols.
338, 399 and 394) Academic Press, Ltd., London, UK).
Stabilizer
[0109] According to certain preferred embodiments described herein
there are provided compositions and methods that include a dry
storage matrix comprising a borate composition and at least one
stabilizer as provided herein. Exemplary and preferred stabilizers
may include a stabilizer compound according to any one or more of
structural formulae (I)-(VII), and/or an osmoprotectant compound
such as trimethylammonium acetate, glycerol phosphate, diglycerol
phosphate, N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(tricine), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, glyceric acid, malic acid, tartaric acid, lactic
acid, glycolic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate, and/or
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium. Certain
presently preferred embodiments contemplate inclusion in the matrix
of at least one stabilizer such as hydroxyectoine, ectoine,
homoectoine, betaine, L-carnitine, sarcosine, N,N-dimethylglycine,
triethylammonium acetate, glycerol phosphate,
N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine),
3-(N-Morpholino)-2-hydroxypropanesulfonic acid (MOPSO),
pentaerythritol, N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl
sulfonate, glycolic acid, lactic acid, malic acid, tartaric acid,
2-hydroxybutyric acid, 3-hydroxybutyric acid,
4-amino-3-hydroxybutyric acid, pyridine 2,5-dicarboxylic acid,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate,
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, and
4-[benzyl(2-hydroxyethyl)methylazaniumyl]butane-1-sulfonate, and/or
stabilizers such as those described in US/2008/0176209.
[0110] Stabilizers according to formulae (I)-(VII) include
compounds of the following formulae: [0111] (i) a compound of
formula I:
##STR00049##
[0111] wherein R.sub.1, R.sub.2, R.sub.3 are independently selected
from --H, --CH.sub.3 and --CH.sub.2--CH.sub.3, wherein when R.sub.1
and R.sub.2 are CH.sub.3 or CH.sub.2--CH.sub.3, R.sub.3 is either H
or absent, wherein X is selected from --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
##STR00050##
and wherein Y is selected from COO.sup.- and SO.sub.3.sup.-; [0112]
(ii) a compound of formula II:
##STR00051##
[0112] wherein R.sub.1 is selected from CH.sub.3 and
CH.sub.2CH.sub.3, and wherein when X is CH, Y is selected from H
and OH, and when X is CH.sub.2--CH, Y is H; [0113] (iii) a compound
of formula III:
##STR00052##
[0113] wherein R.sub.1 and R.sub.2 are independently selected from
--H, --CH.sub.3, and --CH.sub.2CH.sub.3, and wherein X is selected
from H, OH and SH; [0114] (iv) a compound of formula IV:
##STR00053##
[0114] wherein R.sub.1 is selected from aryl, arylaklyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, CH.sub.2CH.sub.2CH.sub.2CH.sub.2,
CH.sub.2CHOHCH.sub.2 and --CH.sub.2CHOHCHOHCH.sub.2--, wherein Y is
selected from COO.sup.- and SO.sub.3.sup.-, and wherein Z is
selected from --CH.sub.2--, --CHOH--, O and S; [0115] (v) a
compound of formula V:
##STR00054##
[0115] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylaklyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH;
and CH.sub.2CH.sub.2CH.sub.2OH, [0116] (vi) a compound of formula
VI:
##STR00055##
[0116] wherein R.sub.1 and R.sub.2 are each independently selected
from aryl, arylakyl, --H, --CH.sub.3--CH.sub.2--CH.sub.3,
--CH.sub.2CH.sub.2OH, CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH,
and CH.sub.2CH.sub.2CH.sub.2OH; [0117] (vii) a compound of formula
VII:
##STR00056##
[0117] wherein R.sub.1 is selected from aryl, arylalkyl, --H,
--CH.sub.3--CH.sub.2--CH.sub.3, --CH.sub.2CH.sub.2OH,
CH.sub.2CHOHCH.sub.3, CH.sub.2CHOHCH.sub.2OH, and
CH.sub.2CH.sub.2CH.sub.2OH, wherein X is selected from
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CHOH,
--CH.sub.2CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CHOHCH.sub.2--, and --CH.sub.2CHOHCHOHCH.sub.2--, wherein
Y is selected from CO.sub.2.sup.- and SO.sub.3.sup.-, and wherein Z
is selected from CH.sub.2, CHOH, O and S.
[0118] Syntheses of such stabilizers may be accomplished as
disclosed herein using reagents that are commercially available
(e.g., as described herein including in the Examples below, or
using other reagents from SigmaAldrich or Fluka, or Carbopol.RTM.
polymers from Noveon, Inc., Cleveland, Ohio, etc.) and/or according
to established procedures, such as those found in Fiesers' Reagents
for Organic Synthesis (T.-L. Ho (Ed.), Fieser, L. F. and Fieser,
M., 1999 John Wiley & Sons, NY). A number of stabilizers as
disclosed herein are commercially available, as will be appreciated
by those familiar with the relevant art.
[0119] Certain embodiments disclosed herein contemplate as a
stabilizer a zwitterionic compound that may be a betaine or betaine
analogue or other zwitterionic compound including carboxylates
and/or sulfonates, the preparation of which is described in
"Betaine Analogues and Related Compounds for Biomedical
Applications" (M. Vasudevamurthy, 2006 Doctoral Thesis, University
of Canterbury, Christchurch, New Zealand). Certain other
embodiments disclosed herein contemplate as a stabilizer a compound
that may be a quinuclidine derivative or a 3-quinuclidinol
derivative, for example,
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate and
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium, or other
quinuclidine or quinuclidinol derivatives such as those described
below, the preparation of which is described herein, and also
including, for example, such derivatives which may be obtained by
alkylation of a quinuclidine under reaction conditions such as
those described hereinbelow.
[0120] "Alkyl" as used herein means a straight chain or branched,
noncyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon
containing from 1 to 10 carbon atoms. Representative saturated
straight chain alkyls include methyl, ethyl, n-propyl, n-butyl,
n-pentyl, n-hexyl, and the like; while saturated branched alkyls
include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and
the like. Representative saturated cyclic alkyls include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like;
while unsaturated cyclic alkyls include cyclopentenyl and
cyclohexenyl, and the like. Cyclic alkyls are also referred to
herein as "homocycles" or "homocyclic rings." Unsaturated alkyls
contain at least one double or triple bond between adjacent carbon
atoms (referred to as an "alkenyl" or "alkynyl", respectively).
Representative straight chain and branched alkenyls include
ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,
1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,
2,3-dimethyl-2-butenyl, and the like; while representative straight
chain and branched alkynyls include acetylenyl, propynyl,
1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl,
and the like.
[0121] "Alkoxy" means an alkyl moiety attached through an oxygen
bridge (i.e., --O-alkyl) such as methoxy, ethoxy, and the like.
[0122] "Alkylthio" means an alkyl moiety attached through a sulfur
bridge (i.e., --S-alkyl) such as methylthio, ethylthio, and the
like.
[0123] "Alkylsulfonyl" means an alkyl moiety attached through a
sulfonyl bridge (i.e., --SO.sub.2-alkyl) such as methylsulfonyl,
ethylsulfonyl, and the like.
[0124] "Alkylamino" and "dialkylamino" mean one or two alkyl
moieties attached through a nitrogen bridge (i.e., --N-alkyl) such
as methylamino, ethylamino, dimethylamino, diethylamino, and the
like.
[0125] "Aryl" means an aromatic carbocyclic moiety such as phenyl
or naphthyl.
[0126] "Arylalkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with an aryl moiety, such as benzyl,
--(CH.sub.2).sub.2 phenyl, --(CH.sub.2).sub.3 phenyl,
--CH(phenyl).sub.2, and the like.
[0127] "Heteroaryl" means an aromatic heterocycle ring of 5- to 10
members and having at least one heteroatom selected from nitrogen,
oxygen and sulfur, and containing at least 1 carbon atom, including
both mono- and bicyclic ring systems. Representative heteroaryls
are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl,
indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl,
isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl,
imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, and quinazolinyl.
[0128] "Heteroarylalkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with a heteroaryl moeity, such as --CH.sub.2
pyridinyl, --CH.sub.2 pyrimidinyl, and the like.
[0129] "Halogen" means fluoro, chloro, bromo and iodo.
[0130] "Haloalkyl" means an alkyl having at least one hydrogen atom
replaced with halogen, such as trifluoromethyl and the like.
[0131] "Heterocycle" (also referred to as a "heterocyclic ring")
means a 4- to 7-membered monocyclic, or 7- to 10-membered bicyclic,
heterocyclic ring which is either saturated, unsaturated, or
aromatic, and which contains from 1 to 4 heteroatoms independently
selected from nitrogen, oxygen and sulfur, and wherein the nitrogen
and sulfur heteroatoms may be optionally oxidized, and the nitrogen
heteroatom may be optionally quaternized, including bicyclic rings
in which any of the above heterocycles are fused to a benzene ring.
The heterocycle may be attached via any heteroatom or carbon atom.
Heterocycles include heteroaryls as defined above. Thus, in
addition to the heteroaryls listed above, heterocycles also include
morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,
hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
[0132] "Heterocyclealkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with a heterocycle, such as --CH.sub.2
morpholinyl, and the like.
[0133] "Homocycle" (also referred to herein as "homocyclic ring")
means a saturated or unsaturated (but not aromatic) carbocyclic
ring containing from 3-7 carbon atoms, such as cyclopropane,
cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene,
and the like.
[0134] The term "substituted" as used herein means any of the above
groups (e.g., alkyl, alkenyl, alkynyl, homocycle) wherein at least
one hydrogen atom is replaced with a substituent. In the case of a
keto substituent ("--C(.dbd.O)--") two hydrogen atoms are replaced.
When substituted one or more of the above groups are substituted,
"substituents" within the context of this invention include
halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino,
alkyl, alkoxy, alkylthio, haloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, heterocycle and heterocyclealkyl, as well as
--NR.sub.aR.sub.b, --NR.sub.aC(.dbd.O)R.sub.b--,
NR.sub.aC(.dbd.O)NR.sub.aNR.sub.b,
--NR.sub.aC(.dbd.O)OR.sub.b--NR.sub.aSO.sub.2R.sub.b,
--C(.dbd.O)R.sub.a, --C(.dbd.O)OR.sub.a,
--C(.dbd.O)NR.sub.aR.sub.b, --OC(.dbd.O)NR.sub.aR.sub.b,
--OR.sub.a, --SR.sub.a, --SOR.sub.a, --S(.dbd.O).sub.2R.sub.a,
--OS(.dbd.O).sub.2R.sub.a and --S(.dbd.O).sub.2OR.sub.a. In
addition, the above substituents may be further substituted with
one or more of the above substituents, such that the substituent is
substituted alkyl, substituted aryl, substituted arylalkyl,
substituted heterocycle or substituted heterocyclealkyl. R.sub.a
and R.sub.b in this context may be the same or different and
independently hydrogen, alkyl, haloalkyl, substituted aryl, aryl,
substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,
substituted heterocycle, heterocyclealkyl or substituted
heterocyclealkyl.
[0135] In the formulae depicted herein, a bond to a substituent
and/or a bond that links a molecular fragment to the remainder of a
compound may be shown as intersecting one or more bonds in a ring
structure. This indicates that the bond may be attached to any one
of the atoms that constitutes the ring structure, so long as a
hydrogen atom could otherwise be present at that atom. Where no
particular substituent(s) is identified for a particular position
in a structure, then hydrogen(s) is present at that position. For
example, in the following structure:
##STR00057##
any carbon at positions 2, 3, 4, 5 and 6 of the piperidine ring may
be substituted with a --C(O)O.sup.- group, provided that only one
--C(O)O.sup.- substituent is on the piperidine ring. Ring atoms
that are not substituted with the --C(O)O.sup.- substituent are
understood to be substituted with hydrogen, unless indicated
otherwise. In those instances where the invention specifies that a
non-aromatic ring is substituted with one or more functional
groups, and those functional groups are shown connected to the
non-aromatic ring with bonds that bisect ring bonds, then the
functional groups may be present at different atoms of the ring, or
on the same atom of the ring, so long as that atom could otherwise
be substituted with a hydrogen atom.
[0136] According to certain embodiments as contemplated herein,
there is provided a matrix for substantially dry storage of a
biological sample which comprises a borate composition as provided
herein and at least one stabilizer that is a quinuclidine
derivative or a 3-quinuclidinol derivative that may be prepared
according to the methods described herein. Exemplary quinuclidine
or 3-quinuclidinol derivatives include:
4-(1-ammoniobicyclo[2.2.2]octan-1-yl)butanoate;
4-(1-ammoniobicyclo[2.2.2]octan-1-yl)butane-1-sulfonate;
3-(1-ammoniobicyclo[2.2.2]octan-1-yl)propane-1-sulfonate;
2-hydroxy-3-((1r,4r)-3-hydroxy-1-ammoniobicyclo[2.2.2]octan-1-yl)propane--
1-sulfonate; 3-(-1-ammoniobicyclo[2.2.2]octan-1-yl)acetate;
3-(1-ammoniobicyclo[2.2.2]octan-1-yl)propanoate;
2-((1r,4r)-3-hydroxy-1-ammoniobicyclo[2.2.2]octan-1-yl)acetate;
3-((1s,4s)-3-hydroxy-1-ammoniobicyclo[2.2.2]octan-1-yl)propanoate;
4-((1s,4r)-3-hydroxy-1-ammoniobicyclo[2.2.2]octan-1-yl)butanoate;
and
4-((1s,4r)-3-hydroxy-1-ammoniobicyclo[2.2.2]octan-1-yl)butane-1-sulfonate-
; such that the skilled person will recognize these and other
quinuclidine and 3-quinuclidinol derivatives may be employed as
stabilizers in the presently disclosed dry storage matrix
compositions and their related methods of use.
[0137] The dissolvable/dissociable matrix may also be prepared in
the sample storage device in a manner such that one or more wells
contain at least one stabilizer, and in certain embodiments at
least two stabilizers, which may include any agent that may
desirably be included to preserve, stabilize, maintain, protect or
otherwise contribute to the recovery from the biological sample
storage device of a biological sample that has substantially the
same biological activity as was present prior to the step of
contacting the sample with the sample storage device. The
stabilizer may in certain embodiments comprise a compound having a
structure according to one of formulae (I)-(VII) or an
osmoprotectant compound as described herein.
[0138] Stabilizers and osmoprotectant compounds may be used
interchangeably and may also include organic solutes accumulated by
cells/tissues in response to osmotic stress. In general,
osmoprotectants increase thermodynamic stability of folded proteins
and provide protection against denaturing stresses. Examples of
osmolytes that act as such osmoprotectants include, but are not
intended to be limited to, creatines, taurins, ectoins, their
derivatives and corresponding biologically compatible salts such as
hydroxyecotoine and homoectoine. Osmolytes are low molecular weight
organic compounds with no net charge. These include zwitterionic
compounds (compounds that contain charged species, but whose
overall charge is zero due to equal numbers of positive and
negative charges).
[0139] Additional examples of osmolytes contemplated for use in
presently contemplated dry storage matrices, including use as
stabilizers in combination with a borate composition as described
herein, include, but are not limited to, sugars (e.g., sucrose,
glucose, trehalose, fructose, xylose, mannitose, fucose), polyols
(e.g., glycerol, mannitol, sorbitol, glycol, inositol),
zwitterionic compounds (e.g., taurine), polyol sugars including
myo-inositol and trehalose, polyunsaturated fatty acids including 3
fatty acid docosahexaenoic acid and eicosapentaenoic acid, free
amino acids with no net charge (e.g., glycine, proline, valine,
leucine, alanine, glutamine), derivatives of amino acids (e.g.,
glycine betaine, alternatively referred to as betaine), and
trimethylamino N-oxide (TMAO). Betaine, betaine derivatives, and
TMAO are examples of zwitterionic tetra-substituted amine
derivatives, which are also contemplated as osmolytes for use in
certain of the presently disclosed formulations. Preferred
osmolytes include hydroxyectoine, ectoine, homoectoine, betaine,
L-carnitine, sarcosine, N,N-Dimethylglycine, triethylammonium
acetate, glycerol phosphate, tricine, MOPSO, pentaerythritol and
N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl sulfonate.
[0140] The addition of osmolytes often results in an observed
increase in stability of the native structure for some proteins.
The stabilization effect is observed with various osmolytes and
small electrolytes such as sucrose, glycerol, trimethylamine
N-oxide (TMAO), potassium glutamate, arginine and betaine (Wang et
al., (1997) Biochemistry 36, 9101-9108; De-Sanctis et al. (1996) J.
Protein. Chem. 15, 599-606; Chen et al., (1996) J. Pharm. Sci. 85,
419-426; Zhi et al., (1992) Protein Science 1, 552-529, the
disclosures of which are incorporated herein by reference). This
effect is based on the exclusion of osmolytes from hydration shells
and crevices on protein surface (Timasheff (1992) Biochemistry 31,
9857-9864, the disclosure of which is incorporated herein by
reference) or decreased solvation (Parsegian et al., (1995).
Methods. Enzymol. 259, 43-94, the disclosure of which is
incorporated herein by reference). In a series of quantitative
studies, Wang and Bolen have shown that the osmolyte-induced
increase in protein stability is due to a preferential burial of
the polypeptide backbone rather than the amino acid side chains
(Wang et al. (1997) Biochemistry 36, 9101-9108). Because native
protein conformations are stabilized, proper folding reactions are
also enhanced in the presence of osmolytes (Frye, K. J. and Royer,
C. A. (1997) Protein. Sci. 6: 789-793; Kumar et al., (1998)
Biochem. Mol. Biol. Int. 46, 509-517; Baskakov, I. and Bolen, D. W.
(1998) J. Biol. Chem. 273: 4831-4834, the disclosures of which are
incorporated herein by reference). Osmolytes usually affect protein
stability and folding at physiological concentration range of 1-4 M
(Yancey et al., (1982) Science 217, 1214-1222, the disclosure of
which is incorporated herein by reference). However, it is apparent
that the degree of stabilization depends on both the nature of the
osmolyte and the protein substrate (Sola-Penna et al., (1997) Eur.
J. Biochem. 248, 24-29, the disclosure of which is incorporated
herein by reference) and, in some instances, the initial
aggregation reaction can actually accelerate in the presence of
osmolytes (Voziyan, P. A. and Fisher M. T. (2000) Protein Science,
Volume 9, 2405-2412).
[0141] Osmolytes such as ectoine, glutamine, hydroxyectoine and
betaine have been used to stabilize a variety of samples of
biological origin for various uses. For example, DE-A-198 34 816
relates to the use of ectoine or ectoine derivatives in cosmetic
formulations. It is disclosed that the mentioned compounds protect
and stabilize nucleic acids of the human skin cells from physical,
chemical and biological influences, such as radiation, especially
ultraviolet radiation, denaturing substances, enzymes, especially
endonucleases and restriction enzymes, and viruses, especially
herpes viruses. U.S. Pat. No. 5,039,704 discloses a method of
treating a catabolic dysfunction in an animal, wherein a
therapeutically effective amount of glutamine or an analogue of
glutamine is administered. U.S. Pat. No. 5,684,045 relates to the
treatment of a catabolic gut-associated pathological process,
especially intestinal mucosal and pancreatic atrophy, enhanced gut
permeability and other diseases. These diseases are treated with a
therapeutically effective amount of glutamine or an analogue
thereof. U.S. Pat. No. 5,428,063 relates to a pharmaceutical
composition in food supplements for the treatment or prevention of
liver diseases. This involves the administration of high doses of
betaine. U.S. Pat. No. 5,827,874 relates to the use of proline for
the treatment of inflammations and pain, especially for the
treatment of inflammatory conditions, rheumatic and non-rheumatic
pain, and for post-surgical and post-traumatic pain. Knapp et al.
in Extremophiles (1999), 3(3), 191-8, describe a
temperature-stabilizing effect of compatible solutes. Ectoine,
hydroxyectoine and betaine are mentioned. Sauer et al.,
Biotechnology and Bioengineering (1998), 57 (3), 306-13, disclose a
temperature-stabilizing effect of the compatible solutes ectoine,
hydroxyectoine and betaine. EP-A-0 915 167 relates to a method for
the in-vivo recovery of components from cells by alternating
conditions to which the cells are subjected. Ectoine and
hydroxyectoine are described as an effective additive for the
cryoprotection of biologically active substances. US 20050196824
relates to chaperonin and osmolyte protein folding and related
screening methods.
[0142] Additional examples of stabilizing effects of hydroxyectoine
include US 2003/0175232, US 2003/0157088 for use of hydroxyectoine
in stabilizing enzymes in cosmetic products; US 2003/0199446 that
discloses use of ectoin or ectoin derivatives for stabilizing p53
genes and gene products; WO/2004/112476 for use in reagents and
methods for biomaterial preservation. Examples of the stabilizing
effects of ectoine include U.S. Pat. No. 7,150,980 and US
2003/0138805 that discloses use of ectoine for use in methods for
DNA amplification and sequencing by increasing the thermostability
of DNA polymerases at elevated temperatures; U.S. Pat. No.
5,789,414 wherein ectoine is used to protect against freezing,
drying and heating of pharmaceutical compositions; US 2003/0022148
and U.S. Pat. No. 6,475,716 that discloses ectoine as a
biostabilizing substance for use in methods and reagents for organ
preservation; and US 20080187924 describing methods for treatment
of a sample containing biomolecules; and US 20020081565 that
discloses use of ectoine as a stabilizer in the process of
producing freeze dried competent cells and uses thereof. None of
the disclosures cited above teaches or suggests the presently
disclosed combinations of a borate composition and a stabilizer as
described herein.
[0143] Other stabilizers contemplated for use according to certain
embodiments disclosed herein may comprise an agent that is a
biological inhibitor or a biochemical inhibitor, as provided
herein. Accordingly, in certain embodiments the biological sample
dry-storage matrix comprises at least one stabilizer that is such
an inhibitor, for example, an anti-microbial agent such as (but not
limited to) an anti-fungal and/or antibacterial agent capable of
inhibiting or suppressing bacterial or fungal growth, viability
and/or colonization, to inhibit microbial contamination of the
wells and the stored sample during long-term storage. Stabilizers
which may also be useful in the methods of this invention include
polycations (see for example Slita et al., J Biotechnol. 2007 Jan.
20; 127(4):679-93. Epub 2006 July 27), reducing agents (for
example, dithiothreitol, 2-mercaptoethanol, dithioerythritol or
other known thiol-active reducing agents, or the like); Scopes, R.
K. 1994 Protein Purification: Principals and Practices. Third
edition, Springer, Inc., New York), steric stabilizers (such as
alkyl groups, PEG chains, polysaccharides, alkyl amines; U.S. Pat.
No. 7,098,033), amino acids and polyamino acids (see for example
U.S. Pat. Nos. 7,011,825 and 6,143,817) including their derivatives
(see for example U.S. Pat. No. 4,127,502), and buffers (Scopes, R.
K. 1994 Protein Purification: Principals and Practices. Third
edition, Springer, Inc., New York; Current Protocols, Protein
Sciences, Cell Biology, Wiley and Sons, 2003). Non-limiting
examples of amino acid stabilizers include serine, threonine,
glycine, proline, carnitine, betaine and the like (see for example
U.S. Pat. Nos. 7,258,873, 6,689,353 and 5,078,997). The stabilizer
may in certain embodiments comprise a, a detergent, a polyol, an
osmolyte, an organic solvent, an electrostatic reagent, a metal
ion, a ligand, an inhibitor, a cofactor or substrate, a chaperonin,
a redox buffer, disulfide isomerase or a protease inhibitor, which
may facilitate dissolution of certain biological samples, such as
proteins (see for example U.S. Pat. No. 6,057,159; Scopes, R. K.
1994 Protein Purification: Principals and Practices. Third edition,
Springer, Inc., New York; Current Protocols, Protein Sciences, Cell
Biology, Wiley and Sons, 2003).
[0144] Certain embodiments of the present invention are
contemplated that expressly exclude particular dissolvable or
dissociatable matrix materials such as soluble cationic polymers
(e.g., DEAE-dextran) or anionic polymers (e.g., dextran sulphate)
or agarose when used, absent other components of the herein
described embodiments, with a di- or trisaccharide stabilizer
(e.g., trehalose, lactitol, lactose, maltose, maltitol, sucrose,
sorbitol, cellobiose, inositol, or chitosan) as disclosed for dry
protein storage, for example, in one or more of U.S. Pat. No.
5,240,843, U.S. Pat. No. 5,834,254, U.S. Pat. No. 5,556,771, U.S.
Pat. No. 4,891,319, U.S. Pat. No. 5,876,992, WO 90/05182, and WO
91/14773; and certain of the present embodiments may also expressly
exclude the use of certain stabilizers that are disclosed in
references cited above; but certain other embodiments of the
present invention contemplate the use of such combinations of a
dissolvable or dissociatable matrix comprising a borate composition
and at least one stabilizer, including such first di- or
trisaccharide stabilizer, along optionally with a second stabilizer
that comprises a biological or biochemical inhibitor which may be a
.beta.-galactosidase inhibitor selected from the group consisting
of D-galactono-1,4-lactone, L-arabinose, L-fucose, fructose,
sucrose, D-galactose, dextrose, maltose, raffinose, xylose,
melibiose, D-arabinose, cellobiose, D-glucose, and galactose.
Certain other embodiments of the present invention contemplate the
use of such combinations of a dissolvable or dissociatable matrix
comprising a borate composition and at least one stabilizer (e.g.,
a compound of formula (I)-(VII) or an osmoprotectant compound) for
substantially dry storage of biological samples other than
proteins, for example, polynucleotides such as DNA, RNA, synthetic
oligonucleotides, genomic DNA, natural and recombinant nucleic acid
plasmids and constructs, and the like. Certain other embodiments of
the present invention contemplate the use, for substantially dry
storage of a biological sample as provided herein without
refrigeration, of a matrix comprising a borate composition and at
least one stabilizer that dissolves in a biocompatible solvent and
which comprises a matrix material that dissolves in a biocompatible
solvent and at least one stabilizer that dissolves in a
biocompatible solvent.
[0145] As described herein, an added advantage of the herein
described dissolvable matrix is that the storage container can be
directly used as a reaction chamber after dissolving the matrix and
rehydration of the material. The stability and activity of proteins
in liquid form may be dependent on activity requirements such as
pH, salt concentration, and cofactors. The stability of many
proteins may in some cases be extremely labile at higher
temperatures and the drying of proteins at ambient (e.g., room)
temperature may therefore provide a stabilizing environment.
Typically, in certain embodiments that relate to a dry-storable
cell sample, the intact cell or virus may be present in an aqueous
liquid that comprises a first solvent, for example as a cell or
particle suspension or slurry that can be contacted with the matrix
for substantially dry storage through the use of liquid handling
instruments as appropriate for the type and quantity of cells or
viruses to be stored.
[0146] Water comprises an exemplary first solvent and any of a
number of aqueous liquids may be suitable aqueous liquids, such as
well known buffered salt solutions, osmolar solutions or cell
growth media including microbiological growth media (e.g., normal
saline or physiological saline, phosphate-buffered saline, Tris,
HEPES, carbonate, glycine or other buffered media, Hanks balanced
salt solution, Ringer's solution, Luria broth, etc.), whereby
following the step of contacting the sample with the matrix a step
of drying is performed during which some or all of the solvent is
removed. Preferably, the cell or virus is stored dry at room
temperature for a period of time long enough to ensure subsequent
recovery and isolation of nucleic acid when the step of
redissolving or resuspending is performed, as opposed to recovery
of viable cells or infectious viral particles (see, e.g., U.S.
application Ser. No. 11/291,267, which typically will involve dry
storage periods of shorter duration), which as noted herein may
vary as a function of the particular cell or virus type being
stored and which in any event can be determined as described herein
routinely through pilot studies in which various storage periods
are employed and the recovered material is subsequently tested for
nucleic acid recovery and/or residual cell viability.
[0147] The nucleic acid from the cell or virus is isolated
following resuspending or redissolving of the dried sample. The
solvent used for resuspending or redissolving the dried sample may
be the same or different from the first solvent used to contact the
sample with the storage matrix. Preferably, the solvent used to
resuspend or redissolve the sample comprises an aqueous solvent,
and more preferably the solvent used in the step of resuspending or
redissolving to isolate nucleic acid is water. The isolated nucleic
acid is in certain preferred embodiments DNA, and may be genomic
DNA or plasmid DNA, depending on the source from which it is
extracted (e.g. bacteria, virus, yeast, eukaryotic cell, etc.). As
disclosed herein, following dry storage, and subsequent to
resuspending or redissolving the composition that comprises the
matrix material and the cell(s) or virus(es), thereby to isolate
nucleic acid, the isolated nucleic acid is then ready for use,
without the need for further purification, in downstream
applications that may include, but need not be limited to, PCR
amplification, cellular transformation, polynucleotide sequencing,
rolling circle amplification, site-directed mutagenesis, T7
transcript generation, restriction enzyme analysis and other
applications practiced by those skilled in the art (see for
example, Maniatis, T. et al. 1982. Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor University Press, Cold Spring Harbor,
N.Y.; Ausubel et al., 1993 Current Protocols in Molecular Biology,
Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston,
Mass.).
[0148] As also described herein (including in the Examples) and in
U.S. Application No. 20060099567, the presence of the disaccharide
trehalose, believed to contribute to the stabilization of
biological samples (e.g., Garcia de Castro et al., 2000 Appl.
Environ. Microbiol. 66:4142; Manzanera et al., 2002 Appl. Environ.
Microbiol. 68:4328), was not sufficient under certain conditions to
support recovery of enzymatic activity in a protein following dry
storage. As a brief background, trehalose is the natural substrate
of trehalase, an enzyme that cleaves disaccharides. Trehalose is
known to stabilize organic material such as proteins (e.g.,
PCT/GB86/00396), but when present under suboptimal conditions may
be disadvantageous for long term storage of proteins at ambient
temperatures, since it is a natural energy source for fungi and
bacteria.
[0149] Additional stabilizers contemplated for use according to
certain other embodiments of the present invention may be present
in a dry storage matrix but are not covalently linked to the
polymeric matrix material as disclosed herein, and may include
small molecules that comprise structures (i)-(xv), including
several known amino acid side chains and mono-, di- and
polysaccharides such as:
##STR00058## ##STR00059##
wherein R is selected from --H, --OH, --CH.sub.2OH, --NHAc and
--OAc. Such compositions are known in the art and are readily
available from commercial suppliers.
[0150] In certain further embodiments at least one stabilizer may
be selected from trehalose, lactitol, lactose, maltose, melezitose,
maltitol, mannitol, gentibiose, raffinose, sucrose, sorbitol,
cellobiose, melibiose, inositol, chitosan, hydroxyectoine, ectoine,
homoectoine and/or, where, as also noted above, according to
certain of such embodiments a chelator as described herein is also
present to protect against metal ion contamination from the applied
biological sample and additionally or alternatively according to
certain other of such embodiments a herein disclosed matrix
material is also present. The presently disclosed embodiments
include several that contemplate the use, as modified according to
the descriptions herein, of certain dry storage compositions of
U.S. Pat. No. 5,240,843, U.S. Pat. No. 5,834,254, U.S. Pat. No.
5,556,771, U.S. Pat. No. 4,891,319, WO 87/00196, WO 89/00012, WO
89/06542, U.S. Pat. No. 5,876,992, U.S. Pat. No. 4,451,569, EP
0448146A1, WO 90/05182, and WO 91/14773, while certain other
presently disclosed embodiments are contemplated that expressly
exclude one or more components of the dry storage compositions of
these publications.
[0151] Other exemplary stabilizers are commercially available and
have structures that are well known, and include the following:
##STR00060## ##STR00061## ##STR00062##
[0152] Screening assays for identifying stabilizers are also
provided by the present disclosure. More specifically, according to
certain related embodiments, it is contemplated that the unexpected
discovery disclosed herein, that biological activity of an isolated
nucleic acid sample can be recovered following unrefrigerated
substantially dry storage of the nucleic acid sample in a matrix
that comprises a matrix material and a stabilizer, may be exploited
to provide a method of identifying, from amongst one or a
pluralithy of candidate agents, a stabilizer for stabilizing a
substantially dry-storable nucleic acid sample as provided herein.
Similarly, it is also contemplated that the surprising discovery as
disclosed herein, that cellular nucleic acid can be readily
recovered following unrefrigerated substantially dry storage of a
cell sample prepared by drying a dry-storage matrix after
contacting it with one or a plurality of isolated intact cells that
contain nucleic acid (e.g., cellular nucleic acid), may be
exploited to provide a method of identifying, from amongst one or a
pluralithy of candidate agents, a stabilizer for stabilizing
cellular nucleic acid in a substantially dry-storable cell sample
as provided herein.
[0153] According to these and related embodiments, the dry-storage
matrix may be prepared (i) with a known stabilizer as provided
herein (e.g., as a positive control), or (ii) with one or more
candidate stabilizers to prepare dry storage matrices to be tested
for effectiveness of the candidate stabilizer(s) at contributing to
the ability of biological activity of an isolated nucleic acid
sample to be recovered from a resuspended or redissolved sample
following unrefrigerated substantially dry storage of the sample,
or (iii) with no stabilizer (e.g., as a negative control lacking
any protective contribution from a stabilizer to retention of
biological activity).
[0154] Following the steps of contacting the sample with each such
matrix either in the presence or absence of a candidate agent
(e.g., fluidly contacting an isolated nucleic acid with a matrix
material that is dissolved or dissociated in a first biocompatible
solvent; or contacting one or a plurality of isolated intact cells
that contain nucleic acid with a matrix material that is dissolved
or dissociated in a first biocompatible solvent), substantially
drying the matrix, and maintaining the substantially dried matrix
without refrigeration for at least one day, isolated nucleic acid
may be recovered from each such sample as described herein, and the
biological activity recovered from each dry-stored sample can be
determined. Biological activity of the recovered nucleic acid from
a sample that has been dried in the presence of a candidate
stabilizer can be compared to that of a sample that has been dried
in the absence of the stabilizer, such that as provided herein
retention of substantially all activity by the sample dried with
stabilizer present and substantial loss of activity by the sample
dried in the absence of stabilizer, indicates the candidate agent
acts as a stabilizer and has therefore been identified as such by
the present method.
Detectable Indicator
[0155] Detectable indicators include compositions that permit
detection (e.g., with statistical significance relative to an
appropriate control, as will be know to the skilled artisan) or
similar determination of any detectable parameter that directly
relates to a condition, process, pathway, induction, activation,
inhibition, regulation, dynamic structure, state, contamination,
degradation or other activity or functional or structural change in
a biological sample, including but not limited to altered enzymatic
(including proteolytic and/or nucleolytic), respiratory, metabolic,
catabolic, binding, catalytic, allosteric, conformational, or other
biochemical or biophysical activity in the biological sample, and
also including interactions between intermediates that may be
formed as the result of such activities, including metabolites,
catabolites, substrates, precursors, cofactors and the like.
[0156] A wide variety of detectable indicators are known to the art
and can be selected for inclusion in the presently disclosed
compositions and methods depending on the particular parameter or
parameters that may be of interest for particular biological
samples in particular sample storage applications. Non-limiting
examples of parameters that may be detected by such detectable
indicators include detection of the presence of one or more of an
amine, an alcohol, an aldehyde, water, a thiol, a sulfide, a
nitrite, avidin, biotin, an immunoglobulin, an oligosaccharide, a
nucleic acid, a polypeptide, an enzyme, a cytoskeletal protein, a
reactive oxygen species, a metal ion, pH, Na.sup.+, K.sup.+,
Cl.sup.-, a cyanide, a phosphate, selenium, a protease, a nuclease,
a kinase, a phosphatase, a glycosidase, and a microbial
contaminant, and others.
[0157] Examples of a broad range of detectable indicators
(including colorimetric indicators) that may be selected for
specific purposes are described in Haugland, 2002 Handbook of
Fluorescent Probes and Research Products--Ninth Ed., Molecular
Probes, Eugene, Oreg.; in Mohr, 1999 J. Mater. Chem., 9: 2259-2264;
in Suslick et al., 2004 Tetrahedron 60:11133-11138; and in U.S.
Pat. No. 6,323,039. (See also, e.g., Fluka Laboratory Products
Catalog, 2001 Fluka, Milwaukee, Wis.; and Sigma Life Sciences
Research Catalog, 2000, Sigma, St. Louis, Mo.) A detectable
indicator may be a fluorescent indicator, a luminescent indicator,
a phosphorescent indicator, a radiometric indicator, a dye, an
enzyme, a substrate of an enzyme, an energy transfer molecule, or
an affinity label. In certain preferred embodiments the detectable
indicator may be one or more of phenol red, food dyes, ethidium
bromide, dyes which do not appreciably interfere in quantitative
PCR reactions, a DNA polymerase, an RNase inhibitor, a restriction
endonuclease (e.g., a restriction enzyme used as a restriction
nuclease such as a site- or sequence-specific restriction
endonuclease), cobalt chloride (a moisture indicator that changes
from blue color when water is present to pink when dry),
Reichardt's dye (Aldrich Chemical) and a fluorogenic protease
substrate.
[0158] According to certain embodiments herein described, drying
the cells or viruses after the step of contacting with the
dry-storage matrix can be performed at ambient temperatures on the
lab bench, in a laminar flow hood, dessicating chamber, or under
reduced atmospheric pressure including under vacuum (e.g. with
vacuum pump such as a SpeedVac.RTM.). Other methods of drying are
also contemplated and include for example without limitation,
radiant heat drying, drying under a light source, dessicating,
drying under nitrogen or other gas (e.g., preferably under a stream
of a flowing inert gas), use of drying solvents or other chemicals,
for example volatile organic solvents such as lower alcohols, lower
alkanes and haloalkanes (e.g., pentanes, hexanes, methylene
chloride, chloroform, carbon tetrachloride), ethers (e.g.,
tetrahydrofuran), ethyl acetate, acetonitrile, trifluoroacetic
acid, pyridine, acetone or other solvents (where such solvents may
in certain other embodiments comprise a second solvent in which a
biological sample may be resuspended or redissolved), preferably in
anhydrous form, air pressure, freeze-drying and other methods to
facilitate and accelerate evaporation.
[0159] Drying of the sample can be determined by simple visual
inspection or touch (i.e. tapping with a pipette tip) to ensure all
moisture has been evaporated or removed; samples should not look or
feel tacky from residual moisture). In some embodiments, a moisture
indicator may be preferably included to ascertain a degree of
drying has been achieved at which rehydration will effect nucleic
acid isolation. For example, cobalt chloride may optionally be
included as a detectable (by visible color-change or colorimetry)
indicator of moisture content in a sample. A moisture indicator
such as an electronic device that measures the dielectric content
of material to determine moisture content (e.g. Aqua-Spear.TM.,
Mastrad Limited, Douglas, UK) is also contemplated for use in
certain of these and related embodiments. A drying agent such as
calcium sulfate (i.e. Drierite.RTM., W.A. Hammond Drierite Co.,
Xenia, Ohio) or phosphorus pentoxide with a moisture indicator is
also contemplated for use in certain embodiments of the present
disclosure.
[0160] A detectable indicator in certain embodiments may comprise a
polynucleotide polymerase and/or a suitable oligonucleotide, either
or both of which may be employed as an indicator or, in certain
other embodiments, as components of other nucleic acids-based
applications of the compositions and methods described herein.
Polymerases (including DNA polymerases and RNA polymerases) useful
in accordance with certain embodiments of the present invention
include, but are not limited to, Thermus thermophilus (Tth) DNA
polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermologa
neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA
polymerase, Thermococcus litoralis (Tli or VENT.TM.) DNA
polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT.TM.
DNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillus
sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca)
DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase,
Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus
(Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase,
Thermus brockianus (DYNAZYME.TM.) DNA polymerase, Methanobacterium
thermoautotrophicum (Mth) DNA polymerase, mycobacterium DNA
polymerase (Mtb, Mlep), and mutants, and variants and derivatives
thereof. RNA polymerases such as T3, T5 and SP6 and mutants,
variants and derivatives thereof may also be used in accordance
with the invention.
[0161] Polymerases used in accordance with the invention may be any
enzyme that can synthesize a nucleic acid molecule from a nucleic
acid template, typically in the 5' to 3' direction. The nucleic
acid polymerases used in the present invention may be mesophilic or
thermophilic, and are preferably thermophilic. Preferred mesophilic
DNA polymerases include T7 DNA polymerase, T5 DNA polymerase,
Klenow fragment DNA polymerase, DNA polymerase III and the like.
Preferred thermostable DNA polymerases that may be used in the
methods of the invention include Taq, Tne, Tma, Pfu, Tfl, Tth,
Stoffel fragment, VENT.TM. and DEEPVENT.TM. DNA polymerases, and
mutants, variants and derivatives thereof (U.S. Pat. No. 5,436,149;
U.S. Pat. No. 4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No.
5,079,352; U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S.
Pat. No. 5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No.
5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; Barnes, W. M.,
Gene 112:29-35 (1992); Lawyer et al., PCR Meth. Appl. 2:275-287
(1993); Flaman et al., Nucl. Acids Res. 22(15):3259-3260
(1994)).
[0162] Other detectable indicators for use in certain embodiments
contemplated herein include affinity reagents such as antibodies,
lectins, immunoglobulin Fc receptor proteins (e.g., Staphylococcus
aureus protein A, protein G or other Fc receptors), avidin, biotin,
other ligands, receptors or counterreceptors or their analogues or
mimetics, and the like. For such affinity methodologies, reagents
for immunometric measurements, such as suitably labeled antibodies
or lectins, may be prepared including, for example, those labeled
with radionuclides, with fluorophores, with affinity tags, with
biotin or biotin mimetic sequences or those prepared as
antibody-enzyme conjugates (see, e.g., Weir, D. M., Handbook of
Experimental Immunology, 1986, Blackwell Scientific, Boston;
Scouten, W. H., Methods in Enzymology 135:30-65, 1987; Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988; Haugland, 2002 Handbook of Fluorescent Probes and
Research Products--Ninth Ed., Molecular Probes, Eugene, Oreg.;
Scopes, R. K., Protein Purification: Principles and Practice, 1987,
Springer-Verlag, NY; Hermanson, G. T. et al., Immobilized Affinity
Ligand Techniques, 1992, Academic Press, Inc., NY; Luo et al., 1998
J. Biotechnol. 65:225 and references cited therein).
[0163] The dissolvable (or dissociable) matrix may be applied to
storage containers, storage vessels or the like for biological
samples, for example, by contacting or administering a matrix
material that dissolves or dissociates in a solvent to one or a
plurality of sample wells or vessels or the like of a storage
device as described herein. For instance, the dissolvable matrix
material may readily adhere to tubes and plates made of glass or
plastic such as polypropylene, polystyrene or other materials. The
dissolvable material is dried, which may by way of non-limiting
illustration be accomplished by air drying at ambient temperature
(typically within the range 20.degree. C.-30.degree. C. such as at
22.degree. C., 23.degree. C., 24.degree. C., 25.degree. C.) and/or
at an appropriately elevated temperature, and/or under reduced
atmospheric pressure (e.g., partial or full vacuum) and/or under a
suitable gas stream such as a stream of filtered air, CO.sub.2 or
an inert gas such as nitrogen or other suitable drying gas, or by
other drying means including lyophilization (i.e., freeze-drying
under reduced pressure whereby frozen solvent sublimation to the
gas phase transpires).
[0164] After the step of drying to achieve a matrix that is
substantially dry, which may be complete drying (e.g., with
statistical significance, all or substantially all detectable
solvent has been removed) or, if desired, to achieve only partial
drying, the dissolvable/dissociable matrix material is ready to
accept the biological sample to be stored. In certain preferred
embodiments a matrix that is substantially dry is provided for
substantially dry storage of a biological sample, which includes
storage of a matrix that has been combined with a sample and from
which, with statistical significance, all or substantially all
detectable solvent has been removed. Preferably and in certain
embodiments which may vary according to the nature of the sample to
be stored and its intended uses, greater than 75%, 80%, 82%, 84%,
86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of
detectable solvent has been removed for purposes of substantially
dry storage.
[0165] Biological material provided in or derived from a biological
sample may also be added to the wells, tubes, vessels or the like
in combination with the storage matrix in liquid form (e.g., by
simultaneously contacting the sample well with the sample and the
matrix dissolved or dissociated in a solvent), allowing the drying
of the biological material and the matrix material to proceed at
the same time, for example, to arrive at a matrix for substantially
dry storage as provide herein. The dissolvable matrix does not, in
preferred embodiments, interfere with biochemical reactions such
that purification steps may not be required to separate the matrix
from the biological sample prior to further processing of the
sample, for instance, prior to performance of biochemical
reactions, such as assays or the like, in the wells of the sample
storage device.
[0166] For example, certain preferred embodiments as disclosed
herein relate to a method for isolating a nucleic acid from a cell,
wherein the cellular nucleic acid is DNA or RNA that is naturally
occurring or the result of genetic engineering, the method
comprising contacting a biological sample that comprises a cell
with a dry-storage matrix in a container such as a sample well or
vessel to obtain a composition comprising the matrix material and
the cell; drying the container; maintaining the dried container
(for instance, a dried sample well as part of a biological sample
storage device that is maintained without refrigeration); and
resuspending or redissolving the matrix material and the cell in a
solvent, thereby isolating the nucleic acid. As described herein,
these and related embodiments provide a surprisingly simple and
fast method to isolate and recover from cells, with minimal
manipulation, genomic (e.g., chromosomal) and epigenomic (e.g.,
plasmid) nucleic acid molecules, following unrefrigerated dry
storage under conditions in which the nucleic acid molecules are
unusually stable to temperature, ultraviolet radiation, and other
potential environmental insults.
[0167] The buffer conditions in the dissolvable matrix may be
adjusted such that greater than at least 70-75%, 75-80%, 80-85%,
85-90%, at least 90 percent, preferably greater than 95 percent,
more preferably greater than 96, 97, 98 or 99 percent of the
biological activity (e.g., enzymatic or affinity activity, or
structural integrity or other biological activity as described
herein and known to the art) of the biological sample is maintained
upon solvent reconstitution (e.g., rehydration with water),
eliminating the need to laboriously remove the sample from the
storage container and transfer it to a reaction buffer in a
separate container. Certain such invention embodiments
correspondingly provide the unexpected advantage of eliminating the
need to separately aliquot and/or calibrate certain biological
reagents each time a stored sample is to be assayed.
[0168] Other non-limiting examples of matrix materials comprising a
borate composition and at least one stabilizer as provided herein,
that may be used in conjunction with dry storage matrix materials,
including additional materials that comprise one or more of
polycarbonate, cellulose (e.g., cellulose papers such as FTA.TM.
paper, Whatman Corp., Florham Park, N.J.), cellulose acetate,
cellulose nitrate, nitrocellulose, agarose, crosslinked agarose
such as 2,3-dibromopropanol-crosslinked agarose,
3,6-anhydro-L-galactose, dextrans and other polysaccharides
including chemically crosslinked polysaccharides such as
epichlorohydrin-crosslinked dextran or N,N'-methylene
bisacrylamide-crosslinked dextran, borosilicate microfiber glass,
fiberglass, asbestos, polymers and plastics such as polypropylene,
polystyrene, polyvinylidene fluoride (PVDF), nylon, polysulfone,
polyethersulfone, polytetrafluoroethylene, and derivatives of these
materials (e.g., U.S. Pat. No. 5,496,562) as well as other similar
materials as are known in the art, or as can readily be determined
to be suitable for use in the devices and methods described herein
based on the present disclosure. See also, for example, U.S. Pat.
No. 5,089,407, U.S. Pat. No. 4,891,319, U.S. Pat. No. 4,806,343,
and U.S. Pat. No. 6,610,531.
[0169] The matrix material may be treated for the storage and
preservation of biological materials. It is well documented that
the adjustment of buffer conditions and the addition of chemicals
and enzymes and other reagents can stabilize DNA and RNA (for
example, Sambrook et al., 1989; Current Protocols, Nucleic Acid
Chemistry, Molecular Biology, Wiley and Sons, 2003) and/or
proteins, enzymes and/or other biological materials (for example,
blood, tissue, bodily fluids) against degradation from enzymes,
proteases and environmental factors (for example, Current
Protocols, Protein Sciences, Cell Biology, Wiley and Sons, 2003).
Matrix compositions for dry storage and methods for their use that
combine certain chemical components to provide beneficial effects
on the biological sample are also contemplated and may vary
according to particular samples and uses thereof.
[0170] Various such chemical components and compounds may include
but are not limited to a buffer capable of maintaining a desired pH
level as may be selected by those familiar with the art, for
example, buffers comprising Tris, Bis-Tris
(Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol or
2,2-Bis(hydroxymethyl)-2,2',2''-nitrilotriethanol), citrate,
acetate, phosphate, borate, HEPES, MES, MOPS, PIPES, carbonate
and/or bicarbonate or other buffers (see, e.g., Calbiochem.RTM.
Biochemicals & Immunochemicals Catalog 2004/2005, pp. 68-69 and
pages cited therein, EMD Biosciences, La Jolla, Calif.) and
suitable solutes such as salts (e.g., KCl, NaCl, CaCl.sub.2,
MgCl.sub.2, etc.) for maintaining, preserving, enhancing,
protecting or otherwise promoting one or more biological sample
components (e.g., biomolecules), or activity buffers that may be
selected and optimized for particular activities of specific
biomolecules such as nucleic acid hybridization or activities of
enzymes, antibodies or other proteins, or other buffers, for
instance, Tris buffer (THAM, Trometanol,
2-amino-2-(hydroxymethyl)-1,3-propane diol), Tris-EDTA buffer (TE),
sodium chloride/sodium citrate buffer (SSC), MOPS/sodium
acetate/EDTA buffer (MOPS), ethylenediamine tetraacetic acid
(EDTA), sodium acetate buffer at physiological pH, and the
like.
[0171] Additional and/or exemplary pH buffers include 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), and Tricine
(N-[Tris(hydroxymethyl)methyl]glycine).
[0172] Chelators may also be optionally included in dry storage
matrices, for instance, ethylenediaminetetraacetic acid (EDTA),
ethylene glycol tetraacetic acid (EGTA),
diethylenetriaminepentaacetic acid (DTPA),
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). and other known chelators familiar to
those skilled in the art, including salts thereof. For example,
certain of the presently disclosed embodiments contemplate
substantially drying a borate composition/stabilizer solution that
includes a chelator, to obtain a matrix material for substantially
dry storage of a biological sample, such as a solution that
comprises about 1-50 mM sodium borate, about 1-50 mM tetraborate,
about 10-100 mM hydroxyectoine, and about 0.05-0.5 mM EDTA, DTPA,
EGTA or NTA.
[0173] Other chemical components that may be included in dry
storage matrices include human placental ribonuclease inhibitor,
bovine ribonuclease inhibitor, porcine ribonuclease inhibitor,
diethyl pyrocarbonate, ethanol, formamide, guanidinium thiocyanate,
vanadyl-ribonucleoside complexes, macaloid, proteinase K, heparin,
hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate,
dithiothreitol (DTT), beta-mercaptoethanol or specific inhibiting
antibodies.
[0174] Accordingly, certain invention embodiments contemplate a
matrix for substantially dry storage of a biological sample,
comprising a matrix material that dissolves or dissociates in a
solvent, at least one stabilizer, and a sample treatment
composition. The sample treatment composition may comprise an
activity buffer as described below, and/or the sample treatment
composition may comprise one or more of a cell lysis buffer, a free
radical trapping agent, a sample denaturant, a solubilization
agent, surfactant, and a pathogen-neutralizing agent. As provided
by these embodiments, the dry storage matrix may thus comprise a
set of components prepared to effect a desired treatment on a
biological sample when the sample is introduced to the matrix, for
example, in embodiments wherein the step of contacting the sample
with the matrix occurs simultaneously with, or immediately prior
to, rehydration or solvent reconstitution of the dried matrix.
Moreover, in certain contemplated embodiments any buffer (including
an activity buffer, a cell lysis buffer, etc.), additives, sample
treatment composition or dry storage matrix described herein may be
designed and/or configured such that after drying the storage
matrix, only water may be added to obtain a functional,
reconstituted biocompatible solvent from which to recover the
biological sample.
[0175] An activity buffer may comprise a solvent or solution in
liquid form, including a concentrate, or one or more dry
ingredients which, when reconstituted with, dissolved in and/or
diluted with one or more appropriate solvents (e.g., water
typically, or additionally or alternatively, an alcohol such as
methanol, ethanol, n-propanol, isopropanol, butanol, etc., an
organic solvent such as dimethylsulfoxide, acetonitrile, phenol,
chloroform, etc. or other solvent) as appropriate for the intended
use, results in a liquid that is suitable for a desired use of the
biological sample, such as a functional or structural
characterization of one or more components of the sample.
[0176] Non-limiting examples of such uses may include determining
one or more enzyme activities, determining intermolecular binding
interactions, detecting the presence of a specific polynucleotide
or amino acid sequence or of an immunologically defined epitope or
of a defined oligosaccharide structure, detection of particular
viruses or of microbial cells or of animal cells (including human),
determining particular metabolites or catabolites, etc., all of
which can be accomplished using methodologies and conditions that
are defined and known to those skilled in the relevant art,
including suitable conditions that can be provided through
contacting the sample with an appropriate activity buffer.
[0177] A cell lysis buffer may be any composition that is selected
to lyse (i.e., disrupt a boundary membrane of) a cell or organelle,
and many such formulations are known to the art, based on
principles of osmotic shock (e.g., hypotonic shock) and/or
disruption of a cell membrane such as a plasma membrane through the
use of a surfactant such as a detergent (e.g., Triton.RTM. X-100,
Nonidet.RTM. P-40, any of the Tween.RTM. family of surfactants,
sodium dodecyl sulfate, sodium lauryl sulfate, deoxycholate,
octyl-glucopyranoside, betaines, or the like) and/or solute (e.g.,
urea, guanidine hydrochloride, guanidinium isothiocyanate, high
salt concentration) system. Numerous cell lysis buffers are known
and can be appropriately selected as a function of the nature of
the biological sample and of the biomolecule(s), biological
activities or biological structures that are desirably recovered,
which may also in some embodiments include the selection of
appropriate pH buffers, biological or biochemical inhibitors and
detectable indicators.
[0178] Sample denaturants similarly may vary as a function of the
biological sample and the dry storage matrix, but may include an
agent that non-covalently alters (e.g., with statistical
significance relative to an appropriate control such as an
untreated sample) at least one of the three-dimensional
conformation, quarternary, tertiary and/or secondary structure,
degree of solvation, surface charge profile, surface hydrophobicity
profile, or hydrogen bond-forming capability of a biomolecule of
interest in the sample. Examples of sample denaturants include
chaotropes (e.g., urea, guanidine, thiocyanate salts), detergents
(e.g., sodium dodecyl sulfate), high-salt conditions or other
agents or combinations of agents that promote denaturing
conditions.
[0179] Free radical trapping agents for use in certain embodiments
may include any agent that is capable of stably absorbing an
unpaired free radical electron from a reactive compound, such as
reactive oxygen species (ROS), for example, superoxide,
peroxynitrite or hydroxyl radicals, and potentially other reactive
species, and antioxidants represent exemplary free radical trapping
agents. Accordingly a wide variety of known free radical trapping
agents are commercially available and may be selected for inclusion
in certain embodiments of the presently disclosed compositions and
methods. Examples include ascorbate, beta-carotene, vitamin E,
lycopene, tert-nitrosobutane, alpha-phenyl-tert-butylnitrone,
5,5-dimethylpyrroline-N-oxide, and others, as described in, e.g.,
Halliwell and Gutteridge (Free Radicals in Biology and Medicine,
1989 Clarendon Press, Oxford, UK, Chapters 5 and 6); Vanin (1999
Meth. Enzymol. 301:269); Marshall (2001 Stroke 32:190); Yang et al.
(2000 Exp. Neurol. 163:39); Zhao et al. (2001 Brain Res. 909:46);
and elsewhere.
[0180] As noted above, certain embodiments contemplate inclusion of
a pathogen-neutralizing agent in the presently disclosed
compositions and methods, which includes any agent that is capable
of completely or partially, but in any event in a manner having
statistical significance relative to an appropriate control,
neutralizing, impairing, impeding, inhibiting, blocking,
preventing, counteracting, reducing, decreasing or otherwise
blocking any pathogenic effect of a pathogen such as a bacterium,
virus, fungus, parasite, prion, yeast, protozoan, infectious agent
or any other microbiological agent that causes a disease or
disorder in humans or vertebrate animals. Persons familiar with the
relevant art will recognize suitable pathogen-neutralizing agents
for use according to the present disclosure. Exemplary agents
include sodium azide, borate, sodium hypochlorite, hydrogen
peroxide or other oxidizing agents, sodium dichloroisocyanurate,
ethanol, isopropanol, antibiotics, fungicides, nucleoside
analogues, antiviral compounds, and other microbicides; these or
others may be selected according to the properties of the
particular biological sample of interest.
[0181] As elaborated upon below, each well of a typical biological
sample storage device in which the presently described dry storage
matrix may be used holds about 5 .mu.l to about 100, 200 or 300
.mu.l of liquid sample material, preferably about 10 .mu.l to about
30 .mu.l of liquid sample material. Sample amounts can vary from
about 0.01 .mu.g to about 1000 .mu.g of DNA, RNA, protein, blood,
urine, feces, virus, bacteria, cells, tissue, cell extract, tissue
extract, metabolites, chemicals, or other materials. Sample
application is through direct application and can be automated. The
applied wells may be provided with a detectable indicator such as a
color indicator that changes color indicating an occupied well.
Color change may be achieved by adding a color agent. For example,
Ponceau red dye, Nitrazine yellow, Bromthymol Blue, Bromophenyl
blue, Bromocresol Green, Methyl Orange, Congo red,
Bromochlorophenol can be deposited with or prior to subsequent to
the sample material, or by treating the matrix material before or
after deposition of sample material into the well. A pH-dependent
color reagent can be applied that changes color after deposition of
a sample with a biological pH of 6.5 to 8.5 onto the matrix within
the well. Applied wells dry within about 1 to about 20 minutes at
ambient temperature or within about 0.1 to about 10 minutes at
elevated temperature. DNA can be retrieved through re-hydration of
the well for up to about 50 to about 80 times. The re-hydration
reagent may be a solution or sample buffer, for example, one having
a biological pH of 6.5-8.5, such as Tris buffer, Tris-EDTA buffer
(TE), sodium chloride/sodium citrate buffer (SSC), MOPS/sodium
acetate/EDTA buffer (MOPS), sodium acetate buffer, or another
buffer as described herein and known in the art. The dry storage
device design is applicable without further modifications for the
storage of biological samples, including, for example, purified
genomic DNA from bacterial, yeast, human, animals, plants and other
sources. With additional modification, such as but not limited to
coating the filters with denaturing agents for proteases, the dry
storage device can be also used for bacteria, buccal swabs or
samples, biopsy tissue, semen, urine, feces, blood, proteins and
other samples.
[0182] Related embodiments are directed to kits that comprise the
biological sample storage device as described herein, along with
one or more ancillary reagents that may be selected for desired
uses. Optionally the kit may also include a box, case, jar, drum,
drawer, cabinet, carton, carrier, handle, rack, tray, pan, tank,
bag, envelope, sleeve, housing or the like, such as any other
suitable container. Ancillary reagents may include one or more
solvents or buffers as described herein and known to the art, and
may in certain embodiments include an activity buffer.
[0183] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
[0184] As used herein, in particular embodiments, the terms "about"
or "approximately" when preceding a numerical value indicates the
value plus or minus a range of 5%. In other embodiments, the terms
"about" or "approximately" when preceding a numerical value
indicates the value plus or minus a range of 10%. In yet other
embodiments, the terms "about" or "approximately" when preceding a
numerical value indicates the value plus or minus a range of
15%.
[0185] For example, in one embodiment, an oligonucleotide of
approximately 20 nucleotides in length is equivalent to
oligonucleotides that range from 19 to 21 nucleotides in length. In
another embodiment, an oligonucleotide of approximately 20
nucleotides in length is equivalent to oligonucleotides that range
from 18 to 22 nucleotides in length. In yet another embodiment, an
oligonucleotide of approximately 20 nucleotides in length is
equivalent to oligonucleotides that range from 17 to 23 nucleotides
in length.
[0186] Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements. By "consisting of" is
meant including, and limited to, whatever follows the phrase
"consisting of." Thus, the phrase "consisting of" indicates that
the listed elements are required or mandatory, and that no other
elements may be present. By "consisting essentially of" is meant
including any elements listed after the phrase, and limited to
other elements that do not interfere with or contribute to the
activity or action specified in the disclosure for the listed
elements. Thus, the phrase "consisting essentially of" indicates
that the listed elements are required or mandatory, but that no
other elements are required and may or may not be present depending
upon whether or not they affect the activity or action of the
listed elements. As used herein, the term "each" when used in
reference to a collection of items is intended to identify one or
more individual items in the collection but does not necessarily
refer to every item in the collection unless the content clearly
dictates otherwise.
[0187] The practice of the present invention will employ, unless
indicated specifically to the contrary, conventional methods of
chemistry, biochemistry, organic chemistry, molecular biology,
microbiology, recombinant DNA techniques, genetics, immunology,
cell biology, stem cell protocols, cell culture and transgenic
biology that are within the skill of the art, many of which are
described below for the purpose of illustration. Such techniques
are explained fully in the literature. See, e.g., Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (3.sup.rd Edition, 2001);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2.sup.nd
Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory
Manual (1982); Ausubel et al., Current Protocols in Molecular
Biology (John Wiley and Sons, updated July 2008); Short Protocols
in Molecular Biology: A Compendium of Methods from Current
Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-interscience; Glover, DNA Cloning: A Practical Approach, vol.
I & II (IRL Press, Oxford, 1985); Anand, Techniques for the
Analysis of Complex Genomes, (Academic Press, New York, 1992);
Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology
(Academic Press, New York, 1991); Oligonucleotide Synthesis (N.
Gait, Ed., 1984); Nucleic Acid Hybridization (B. Hames & S.
Higgins, Eds., 1985); Transcription and Translation (B. Hames &
S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed.,
1986); Perbal, A Practical Guide to Molecular Cloning (1984); Fire
et al., RNA Interference Technology: From Basic Science to Drug
Development (Cambridge University Press, Cambridge, 2005);
Schepers, RNA Interference in Practice (Wiley-VCH, 2005); Engelke,
RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology
(DNA Press, 2003); Gott, RNA Interference, Editing, and
Modification: Methods and Protocols (Methods in Molecular Biology;
Human Press, Totowa, N.J., 2004); Sohail, Gene Silencing by RNA
Interference: Technology and Application (CRC, 2004); Clarke and
Sanseau, microRNA: Biology, Function & Expression (Nuts &
Bolts series; DNA Press, 2006); Immobilized Cells And Enzymes (IRL
Press, 1986); the treatise, Methods In Enzymology (Academic Press,
Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H.
Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory);
Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and CC Blackwell, eds., 1986); Riott, Essential Immunology,
6th Edition, (Blackwell Scientific Publications, Oxford, 1988);
Embryonic Stem Cells: Methods and Protocols (Methods in Molecular
Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell
Protocols: Volume I: Isolation and Characterization (Methods in
Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem
Cell Protocols Volume II: Differentiation Models (Methods in
Molecular Biology) (Kurstad Turksen, Ed., 2006); Human Embryonic
Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen
Ed., 2006); Mesenchymal Stem Cells: Methods and Protocols (Methods
in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and
Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols
(Methods in Molecular Medicine) (Christopher A. Klug, and Craig T.
Jordan Eds., 2001); Hematopoietic Stem Cell Protocols (Methods in
Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells:
Methods and Protocols (Methods in Molecular Biology) (Leslie P.
Weiner Ed., 2008); Hogan et al., Methods of Manipulating the Mouse
Embryo (2.sup.nd Edition, 1994); Nagy et al., Methods of
Manipulating the Mouse Embryo (3.sup.rd Edition, 2002), and The
zebrafish book. A guide for the laboratory use of zebrafish (Danio
rerio), 4th Ed., (Univ. of Oregon Press, Eugene, Oreg., 2000).
[0188] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
EXAMPLES
Example 1
Preparation of Matrix for Biological Sample Storage
[0189] This example describes preparation of a dissolvable matrix
comprising a borate composition and a stabilizer as provided
herein, for storage of biological sample material, including
unrefrigerated substantially dry storage. Unless otherwise noted,
all reagents to which reference is made in these Examples were from
Sigma-Aldrich (St. Louis, Mo.). 191 mg of sodium tetraborate
decahydrate was placed in a 50 mL conical tube and then 158 mg of
hydroxyectoine was added. RNase- and DNase-free 18.2 megaOhm water
was added to bring the total volume to 50 mL. The mixture was
stirred until the solids were completely dissolved.
[0190] The matrix in liquid form was applied to sample wells of a
96-well plate and dried completely at room temperature either under
standard pressure or under vacuum in a vacuum chamber. The drying
time for a 20-50 .mu.l volume of the borate-stabilizer matrix was
overnight, and under vacuum a shorter drying time was required. The
plates were then ready for the storage of biological material.
[0191] Additional storage additives such as one or more of EDTA or
other chelators known in the art and described herein (e.g.,
ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic
acid (EGTA), diethylenetriaminepentaacetic acid (DTPA),
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), and salts thereof), Na-Acetate,
cysteine, dithiothreitol (DTT, Cleland's reagent), potassium
acetate, K.sub.2HPO.sub.4, glycerol, Triton X-100.RTM., sodium
dodecyl sulfate (SDS), sodium azide, protease inhibitors (e.g.,
PMSF, aminoethylbenzenesulfonyl fluoride, pepstatin, E64, bestatin,
leupeptin, aprotinin), 2-mercaptoethanol, polyethylene glycol
(PEG), bovine serum albumin (BSA), nicotinic adenine dinucleotide
(NAD), ATP may also be added directly into the storage matrix for
stabilization and activation after rehydration, depending on the
biological sample to be stored and the bioactivity to be recovered
and/or tested. For biological material associated with biological
activity such as enzymes, the reaction conditions may be adjusted
directly in the storage matrix. In some cases the only substance to
be added for rehydration prior to an activity reaction is water.
The matrix can also include one or more inhibitors such as
antibacterial and/or antifungal agents. The borate-stabilizer dry
storage matrix can be sterilized through sterile filtration or
autoclaving prior to aliquoting the matrix into the individual
storage wells. The autoclaved matrix is applied in aliquots to the
storage wells either in single tubes or in multiwell plates at a
liquid volume of 10 to 100 .mu.l per well in the case of a 96-well
plate.
Example 2
Dry Storage of Nucleic Acids
[0192] Biological sample storage devices were prepared with dried
borate/hydroxyectoine matrices as described in Example 1. General
molecular biology materials and methods were used, as described.
(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 2001; Ausubel
et al., 1993 Current Protocols in Molecular Biology, Greene Publ.
Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.).
Stability tests were performed for plasmids, oligonucleotides, DNA
fragments in the form of a 1 kB ladder, PCR products, genomic DNA
(feline and human) and RNA. Recovery and stability tests were
performed using electrophoretic gel-based, polymerase chain
reaction (PCR), and transformation rate analyses.
A. Genomic Human DNA
[0193] A total of 100 ng of human genomic DNA (Novagen/EMD4
Biosciences, San Diego, Calif.) in Tris-EDTA (TE) buffer-pH 8 was
applied directly into wells of a 96-well plate that either
contained the borate/stabilizer matrix material or lacked any
borate-stabilizer matrix (non-protected control sample). The borate
composition matrix materials in different wells contained various
amounts of different stabilizers as provided herein, including
hydroxyectoine, ectoine, homoectoine, betaine, L-carnitine,
sarcosine, N,N-Dimethylglycine, triethylammonium acetate, glycerol
phosphate, tricine, MOPSO, pentaerythritol and
N-ethyl-N,N-bis-(2-hydroxyethyl)ammonium-N-4-butyl sulfonate,
prepared in 10 mM sodium tetraborate decahydrate or 10 mM sodium
borate (prepared from boric acid). Identical aliquots were also
distributed into empty wells of a second 96-well plate for storage
at -20.degree. C., for use as reference control samples. The
genomic DNA was dried overnight and stored at room temperature.
Control DNA samples were stored frozen at -20.degree. C. The dried
samples were then stored for 18 days at 85.degree. C.
[0194] Following dry storage, wells containing the genomic human
DNA were rehydrated with 10 .mu.l water and then 5 .mu.l was mixed
with DNA electrophoretic gel loading buffer and applied to wells of
an 0.8% agarose gel cast with ethidium bromide to assess the
integrity of the recovered DNA following storage at elevated
temperatures. PCR reactions contained 1.times.PCR buffer, 2 human
.beta.-actin specific primers at a concentration of 10 .mu.M and
dNTPs, and were allowed to proceed for 15 minutes under standard
conditions. The rehydrated reaction mixture was transferred into
PCR tubes and Taq polymerase was added. The reaction was cycled for
35 cycles and analyzed on a 1% agarose gel. The fragment of the
human .beta.-actin gene of expected size (1.2 kb) was amplified
without a decrease in amplification compared to frozen stored
genomic DNA.
Example 3
Storage of Human Genomic DNA for Three Months at 85.degree. C.
[0195] Human genomic DNA samples prepared as described in Example 2
were analyzed after 90 days of dry storage at 85.degree. C. Samples
were rehydrated with sample loading buffer and applied to an 0.8%
agarose gel and electrophoresed at a constant 150 V for 40 min. Gel
images were obtained using a KODAK-100 gel imager (Eastman Kodak,
Rochester, N.Y.) using an ethidium bromide filter and excitation of
the DNA bands with 302 nm UV light with exposure set at 0.15
seconds. Highly intact DNA was recovered from borate/stabilizer
matrix-containing wells and could be observed migrating in gel
electrophoresis with an apparent molecular mass greater than the 23
kb fragment of the reference standard DNA ladder for the samples
with which the borate/stabilizer matrix was used to protect the
DNA. Unprotected DNA (i.e., DNA that had been dry-stored in wells
in the absence of any matrix) was degraded to the point where it
could not be visualized by this method. Borate/stabilizer matrices
protected DNA from degradation even after significantly longer
(e.g., one-year) dry storage periods at 85.degree. C. (Example 4,
FIG. 2).
Example 4
Preparation and Use of Matrix for Unrefrigerated Dry Biological
Sample Storage
[0196] This example describes preparation of the dissolvable matrix
for storage of material. 191 mg of sodium tetraborate decahydrate
was placed in a 50 mL conical tube and then 395 mg of
hydroxyectoine was added. RNase- and DNase-free 18.2 MS.OMEGA.
water was added to bring the total volume to 50 mL for a final
composition of 50 mM hydroxyectoine/10 mM sodium tetraborate
decahydrate. The mixture was stirred until the solids were
completely dissolved. Additional formulations were prepared as
described above, containing 100, 25 or 10 mM hydroxyectoine in 10
mM sodium tetraborate decahydrate. All reagents were from
Sigma-Aldrich (St. Louis, Mo.) unless otherwise noted.
[0197] The borate/hydroxyectoine compositions in liquid form were
applied to sample wells of a 96 well SampleGard.TM. plate
(Biomatrica Inc., San Diego, Calif.)), 20 .mu.L/well, and dried
completely at room temperature overnight in a biological safety
cabinet. Human genomic DNA isolated from 293T cells was diluted in
NEB (New England Biolabs, Beverly, Mass.) DNase- and RNase-free
water to give a final concentration of 5 .mu.g/.mu.L. 20 .mu.L of
the genomic DNA solution was applied to each matrix-containing well
and also to several control wells that did not receive any of the
borate/stabilizer matrix preservative composition. The solution in
each well was mixed to ensure complete dissolution of the matrix
(where present) and then the plate was allowed to dry overnight in
a biological safety cabinet. Additionally, several 20 .mu.L
aliquots of DNA were placed into Axygen (Axygen Scientific, Inc.,
Union City, Calif.) DNAse-free sterile 1.7 mL microfuge tubes,
closed and stored at -20.degree. C. After drying the plates were
transferred to a Thermo Scientific (ThermoFischer Scientific,
Waltham, Mass.) oven set at 85.degree. C. and stored there until
removed for analysis by agarose gel electrophoresis. FIG. 2 shows
the electrophoretic profile of DNA that was recovered after one
year of dry storage at 85.degree. C. on borate/stabilizer matrix
material and analyzed after redissolving the contents of individual
wells in 10 .mu.L of gel loading buffer and applying the
redissolved material to individual wells of a 0.8% agarose gel
containing ethidium bromide. The gel was electrophoresed at a
constant 150V for 45 min and imaged using UV transillumination at
302 nm on a KODAK 100 gel imager equipped with an ethidium bromide
filter.
Example 5
Preparation of
N-Ethyl-N,N-Bis(2-Hydroxyethyl)Ammonium-4-Butylsulfonate, an
Exemplary Stabilizer
[0198] This example describes synthesis of
N-ethyl-N,N-bis(2-hydroxyethyl)ammonium-4-butylsulfonate, an
exemplary stabilizer, according to the methodologies of M.
Vasudevamurthy ("Betaine Analogues and Related Compounds for
Biomedical Applications", Doctoral thesis, Univ. of Canterbury,
Christchurch, New Zealand, 2006). 2.66 grams of
N-ethyldiethanolamine (Sigma-Aldrich, cat#112062) was added to a 15
mL ace glass pressure tube that had been equipped with a stir bar.
2.72 g of 1,4-butane sultone (Sigma-Aldrich, cat# B85501) was added
and the Teflon tube plug with o-ring was attached and tightened.
The tube was mounted upright in a silicone oil bath and heated to
90.degree. C. and held at 90.degree. C. for 16 hours. The mixture
was stirred until it became a solid mass. After 16 hours the tube
was cooled and 10 mL of ethyl acetate was added. The solid mass was
broken up using a stir bar and then filtered using a 30 mL medium
porosity fitted glass funnel. The solid was washed 3.times. with 20
mL of ethylacetate and dried at 70.degree. C. in a drying oven to
give 4.864 g of
N-ethyl-N,N-bis(2-hydroxyethyl)ammonium-4-butylsulfonate. The
structure was confirmed by positive mode electrospray mass
spectroscopy ESI/MS=290.1 m/z (M+Na), confirmed by .sup.1H NMR.
##STR00063##
Example 6
Preparation of Stabilizers
[0199] This example describes the synthesis and characterization of
several exemplary stabilizers as provided herein.
Compound VIII. Synthesis of
1-ethyl-1-(4-sulfonatobutyl)piperidin-1-ium
##STR00064##
[0201] 1.13 grams of N-ethylpiperidine was weighed into a 15 mL Ace
Glass pressure tube containing a stir bar. 1.36 grams of was added
dropwise to the pressure tube and the tube sealed with a teflon
plug and o-ring. The tube was placed in a silicone oil bath and the
temperature raised to 90.degree. C. using a VWR heating stir plate
(VWR Scientific, West Chester, Pa.) with temperature control and
maintained at 90.degree. C. After 16 hours the tube was allowed to
cool to room temperature and 10 mL of ethyl acetate was added to
the tube and the solid broken up using a spatula. The solid was
collected by vacuum filtration using a medium porosity glass fitted
funnel and washed with 30 mL of ethyl acetate. The solid was dried
in a drying chamber containing Drierite.RTM. to give 1.59 g of a
white solid. The structure was confirmed by positive mode
electrospray mass spectroscopy (M+H)=250.2 and (M+Na) 272.4 m/z and
.sup.1H NMR.
Compound IX. Synthesis of
4-ethyl-4-(4-sulfonatobutyl)morpholin-4-ium
##STR00065##
[0203] 1.15 grams of N-ethylmorpholine was alkylated with
1,4-butanesultone in similar fashion to the previous example to
give 1.09 grams of a white solid. The structure was confirmed by
positive mode electrospray mass spectroscopy (M+H)=252.6 and (M+Na)
274.2 m/z and .sup.1H NMR.
Compound X. Synthesis of
3-(1-azoniabicyclo[2.2.2]oct-1-yl)propane-1-sulfonate
##STR00066##
[0205] 1.91 grams of 3-quinuclidinol was added to a 100 mL round
bottom flask containing a stir bar. 50 mL of chloroform was added
to the flask and mixture stirred to dissolve the quinuclidinol. The
solution was cooled to near 0.degree. C. in an ice bath. 1.83 grams
of propane sultone was dissolved in 8 mL of chloroform in a test
tube and then added dropwise to the solution of 3-quinuclidinol
with stirring. During the addition of the propane sultone a white
solid began to precipitate from solution. Following the addition
the ice bath was removed and the solution allowed to warm to room
temperature. The mixture was allowed to stir overnight then diluted
with 50 ml of acetone and the solid collected on a fine porosity
fritted glass funnel. The white precipitate was washed with 30 mL
of acetone and the solid dried in a drying chamber containing
Drierite.RTM. to obtain 3.42 g of a white solid. The structure was
confirmed by positive mode electrospray mass spectrometry
(M+Na)=286.2 and .sup.1H NMR.
Compound XI. Synthesis of
1-(2-carboxylatoethyl)-1-azabicyclo[2.2.2]octan-1-ium
##STR00067##
[0207] 1.91 grams of 3-quinuclidinol was added to a 100 mL round
bottom flask containing a stir bar. 50 mL of dichloromethane was
added to the flask and the mixture stirred to dissolve the
quinuclidinol. 1.08 grams of acrylic acid was dissolved in 8 mL of
dichloromethane in a test tube and then added dropwise to the
solution of 3-quinuclidinol with stirring. The mixture was allowed
to stir for 48 hours at room temperature during which time about
half of the dichloromethane evaporated leaving a white slurry which
was diluted with ethyl acetate and the solid collected in a medium
porosity glass fitted funnel. The solid was washed with 30 mL of
ethyl acetate and the resulting solid dried in a 85.degree. C.
drying oven to give 2.37 grams of a white solid. The structure was
confirmed by positive mode electrospray mass spectrometry
(M+H)=199.3 m/z and .sup.1H NMR.
Compound XII. Synthesis of
4-[tris(2-hydroxyethyl)azaniumyl]butane-1-sulfonate
##STR00068##
[0209] 1.49 grams of triethanolamine was added to a 100 mL round
bottom flask equipped with a stir bar. 40 mL of dichloromethane was
added to the flask and the mixture stirred thoroughly. The solution
was cooled to near 0.degree. C. using an ice water bath. 1.36 grams
of 1,4-butanesultone was dissolved in 8 mL of dichloromethane and
the butanesultone solution added dropwise to the stirred
triethanolamine solution. Following complete addition the ice water
bath was removed and the solution allowed to warm to room
temperature. The mixture was stirred for 16 hours during which a
flocculent precipitate formed. The solid was filtered using a
medium porosity glass fritted funnel. The white solid was washed
using 30 mL of ethyl ether and the solid dried in a dessicator over
Drierite.RTM. to give 2.52 grams of a white solid. The structure
was confirmed by positive mode electrospray mass spectrometry
(M+Na)=308.3 m/z and .sup.1H NMR.
Compound XIII. Synthesis of
4-[benzyl(2-hydroxyethyl)methylazaniumyl]butane-1-sulfonate
##STR00069##
[0211] Compound XIII was synthesized in like manner to the example
of Compound XI. 1.65 grams of N-benzyl-N-methylethanolamine was
alkylated with 1.36 grams of 1,4-butanesultone to give 1.26 g of a
white solid. The structure was confirmed by positive mode
electrospray mass spectrometry (M+H)=302.4 m/z, (M+Na)=324.3 and
.sup.1H NMR.
Example 7
Dry Storage of Nucleic Acids on Matrix Comprising Borate
Composition and Zwitterionic Stabilizer
[0212] This example shows characterization by quantitative
polymerase chain reaction (qPCR) of DNA following dry storage in a
borate-stabilizer matrix as disclosed herein, including a
comparison of sample recovery following dry storage at 85.degree.
C. to sample recovery following dry storage at ambient (room)
temperature.
[0213] The following compositions were prepared and spotted into a
96 well SampleGard.TM. (Biomatrica Inc., San Diego, Calif.) plate
at 20 uL/well:
[0214] #1=50 mM hydroxyectoine, 10 mM boric acid, 0.4 mM DTPA, pH
8.3.
[0215] #2=50 mM hydroxyectoine, 10 mM boric acid, 1 mM sodium
tetraborate, 0.4 mM DTPA, pH 8.3.
[0216] #3=25 mM hydroxyectoine, 10 mM boric acid, 0.4 mM DTPA, pH
8.3.
[0217] #4=25 mM hydroxyectoine, 10 mM boric acid, 1 mM sodium
tetraborate, 0.4 mM DTPA, pH 8.3.
[0218] The plates were kept in the laminar flow hood and the
solutions allowed to evaporate overnight. Genomic DNA isolated from
human 293T cells was diluted to 1 ng/ul and then 10 uL applied to
each well. The plates were mixed on a plate mixer for 10 min at
1000 rpm and then placed into a laminar flow hood and allowed to
dry overnight. The next day one plate was placed in an 85.degree.
C. oven and the other kept at room temperature. The -20.degree. C.
controls were prepared by placing 10 uL of the 1 ng/uL stock of
293T DNA into DNAse-free microfuge tubes. The tubes were stored at
-20.degree. C., thawed and used at the respective time points. qPCR
analysis was performed on an ABI 7300 instrument using ABI's
TaqMan.TM. Universal PCR MasterMix.TM. according to the
manufacturer's recommendations (ABI/Life Technologies, Carlsbad,
Calif.). The default program was employed using the following
primer and TaqMan.TM. probe:
TABLE-US-00001 beta actin Forward primer: [SEQ ID NO: 1] 5'TCA CCC
ACA CTG TGC CCA TCT ACG A3' beta actin Reverse primer: [SEQ ID NO:
2] 5'CAG CGG AAC CGC TCA TTG CCA ATG G3' beta actin probe: [SEQ ID
NO: 3] 5'(6-FAM)ATG CCT CCC CCA TGC CAT CCT GCG T (BHQ1a-Q)3'
[0219] Quantitative sample recoveries are depicted in FIGS. 3 and
4.
Example 8
Stabilization of RNA Extracted from 293T Cells
[0220] Various formulations of borate composition
(inhibitor)/stabilizer (osmoprotectant) matrix materials were
prepared according to Tables 1 and 2; where indicated the pH was
adjusted with 1 N NaOH or 1 N HCl to achieve the final pH as shown
below.
TABLE-US-00002 TABLE 1 Borate Composition/Stabilizer Combinations
and Concentrations Stabilizer/ Sol'n Osmoprotectant Inhibitor 1
Inhibitor 2 Final pH 1 25 mM 25 mM Boric None Hydroxyectoine acid 2
50 mM 25 mM Boric None Hydroxyectoine acid 3 25 mM 25 mM Boric 5 mM
Sodium Hydroxyectoine acid tetraborate 4 50 mM 25 mM Boric 5 mM
Sodium Hydroxyectoine acid tetraborate 5 20 mM None 10 mM Sodium
5.5 Malic acid tetraborate 6 20 mM None 10 mM Sodium 6.6
Dipicolinic acid tetraborate
[0221] 20 .mu.A of each of the formulations shown in Table 1 were
applied to each of 24 wells in SampleGard.TM. plates (Biomatrica
Inc., San Diego, Calif.). The plates were kept in a laminar flow
hood overnight to allow the matrix to dry. The next day the
concentration of purified RNA was adjusted with RNase free water to
give 27.5 ng of RNA/.mu.L base of the absorbance at 260 nm. 20
.mu.L of diluted RNA was spotted into each and mixed with the
matrix for a total of 550 ng of RNA per well. The plates were kept
in the laminar flow hood and allowed to dry overnight. The plates
were transferred to a 60.degree. C. Bender oven and maintained at
60.degree. C. for three days prior to analysis. After 72 hours the
samples were removed from the oven and rehydrated in 18 .mu.l of
gel loading buffer and applied to a 1.2% agarose gel prepared with
1.times.TAE buffer and containing 20 .mu.g of ethidium bromide/100
mL of gel. The gel was electrophoresed for 40 min at a constant
150V and imaged on a UV light box equipped with a Kodak Gel-100
imager (FIG. 5).
TABLE-US-00003 TABLE 2 Additional Borate Composition/Stabilizer
(Osmoprotectant) Combinations and Concentrations Stablilizer/
Stabilizer/ Final Sol'n Osmoprotectant 1 Inhibitor 1 Osmoprotectant
2 pH 7 50 mM 10 mM Sodium 20 mM Hydroxyectoine tetraborate
Dipicolinic acid 8 50 mM 10 mM Sodium 20 mM Malic Hydroxyectoine
tetraborate acid 9 25 mM L-Carnitine 25 mM Boric acid 5.8 10 25 mM
Threonine 25 mM Boric acid 5.6 11 40 mM Threonine 10 mM Sodium 6.6
tetraborate
[0222] 20 .mu.L of each of the formulations shown in Table 2 were
applied to each of 24 wells in SampleGard.TM. plates (Biomatrica
Inc., San Diego, Calif.). The plates were kept in a laminar flow
hood overnight to allow the matrix to dry. The next day the
concentration of purified RNA was adjusted with RNase-free water to
give 27.5 ng of RNA/.mu.L base of the absorbance at 260 nm. 20
.mu.L of diluted RNA was spotted into each and mixed with the
matrix for a total of 550 ng of RNA per well. The plates were kept
in the laminar flow hood and allowed to dry overnight. The plates
were transferred to a 60.degree. C. Bender oven and maintained at
60.degree. C. for three days prior to analysis. After 72 hours the
samples were removed from the oven and rehydrated in 18 .mu.L of
gel loading buffer and applied to a 1.2% agarose gel prepared with
1.times.TAE buffer and containing 20 .mu.g of ethidium bromide/100
mL of gel. The gel was electrophoresed for 40 min at a constant
150V and imaged on a UV light box equipped with a Kodak Gel-100
imager (FIG. 6).
Example 9
Syntheses of Stabilizer Compounds
Synthesis of 4-(2-Ethoxy-2-oxoethyl)-4-methylmorpholin-4-ium
bromide
[0223] 3.0 g (29.6 mmol) of 4-methylmorpholine was dissolved in 50
mL dichloromethane and cooled to 0.degree. C. in an ice bath. To
this solution 5.0 g of ethyl bromoacetate (29.6 mmol) was added
dropwise with continuous stirring and the mixture was then allowed
to warm to room temperature. The clear solution started to form
white precipitate after 5 minutes of stirring at room temperature.
The resulting mixture was stirred for another 3 h. The precipitate
was isolated by vacuum filtration using a 30 mL glass fritted
funnel and washed 3 times with 30 mL of acetone to give 8.0 g of
the product as a white solid. Positive mode ESI/MS: m/z=188 (M+,
minus Br); confirmed by .sup.1H NMR.
Synthesis of 2-(4-methylmorpholino-4-ium)acetate
[0224] To a solution of
4-(2-ethoxy-2-oxoethyl)-4-methylmorpholin-4-ium bromide, 1 (3.0 g,
11.2 mmol) in water (45 mL) was added conc. H.sub.2SO.sub.4 (1.0
mL). Dowex.RTM. (1.times.8, 200-400 mesh `Cl`) (3.0 g) was added to
this solution. The resulting mixture was heated to reflux for 18 h.
The resin was filtered off and the aqueous solution was
concentrated. The resultant light yellow oil was triturated with
isopropanol to afford 1.7 g, 95% yield of the desired product as
white solid. Positive mode ESI/MS: m/z=160.1 (M+H); confirmed by
.sup.1H NMR.
Synthesis of 1-(2-Ethoxy-2-oxoethyl)-1-ethylpiperidinium
bromide
[0225] To a solution of 1-ethylpiperidine (2.5 g, 22.1 mmol) in
dichloromethane (4 mL) was added ethylbromoacetate (3.7 g, 22.1
mmol) via a syringe. The mixture was stirred at room temperature
overnight. The resulting white precipitate was washed with hexane
and ethylacetate to give 6.1 g, 98.6% yield of the product as a
white solid. Positive mode ESI/MS: m/z=200.4 (M+, minus Br);
confirmed by .sup.1H NMR.
Synthesis of 2-(1-ethylpiperidinium-1-yl)acetate
[0226] To a solution of 1-(2-ethoxy-2-oxoethyl)-1-ethylpiperidinium
bromide (3) (3.0 g, 10.7 mmol) in water (45 mL) was added conc.
H.sub.2SO.sub.4 (1.0 mL). Dowex.RTM. (1.times.8, 200-400 mesh `Cl`)
(3.0 g) was added to this solution. The resulting mixture was
heated to reflux for 18 h. The resin was filtered off and the
aqueous solution was concentrated. The resultant light yellow oil
was triturated with isopropanol and did not form a precipitate. The
yellow oil was dried under reduced pressure to give 1.6 g, 93%
yield of the desired product as yellow oil. Positive mode ESI/MS:
m/z=172.1 (M+H); confirmed by .sup.1H NMR.
Synthesis of 3-(Ethoxycarbonyl)-1,1-dimethylpiperidinium iodide
(11)
[0227] Ethylnipecotate (4.0 mL, 25.6 mmol) was added dropwise to a
round bottom flask containing methyliodide (6.4 mL, 102.4 mmol) and
25 mL in an ice bath. The reaction mixture was stirred at room
temperature overnight. The resulting precipitate was isolated by
filtration. The precipitate was washed with dichloromethane several
times to give 2.7 g, 34% yield of the desired product as a white
solid. Positive mode ESI/MS: m/z=186.2 (M+, minus iodide);
confirmed by .sup.1H NMR
Synthesis of 1,1-Dimethylpiperidinium-3-carboxylate (12)
[0228] To a solution of 3-(ethoxycarbonyl)-1,1-dimethylpiperidinium
iodide (5) (2.2 g, 6.9 mmol) in water (45 mL) was added conc.
H.sub.2SO.sub.4 (1.0 mL). Dowex.RTM. (1.times.8, 200-400 mesh `Cl`)
(2.0 g) was added to this solution. The resulting mixture was
heated to reflux for 18 h. The resin was filtered off and the
aqueous solution was concentrated. The resultant brown oil was
triturated with isopropanol to form an off-white precipitate. The
precipitate was washed with isopropanol until the color became
white. The resulting white precipitate was dried under reduced
pressure to give 1.0 g, 93% yield of the desired product. Positive
mode ESI/MS: m/z=158 (M+H); confirmed by .sup.1H NMR.
[0229] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent
application publications, U.S. patent applications, foreign
patents, foreign patent applications and non-patent publications
referred to in this specification and/or listed in the Application
Data Sheet are incorporated herein by reference, in their entirety.
Aspects of the embodiments can be modified, if necessary to employ
concepts of the various patents, applications and publications to
provide yet further embodiments.
[0230] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
[0231] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
3125DNAArtificial SequencePrimer Sequence 1tcacccacac tgtgcccatc
tacga 25225DNAArtificial SequencePrimer Sequence 2cagcggaacc
gctcattgcc aatgg 25325DNAArtificial SequenceProbe Sequence
3atgcctcccc catgccatcc tgcgt 25
* * * * *