U.S. patent application number 12/338848 was filed with the patent office on 2009-06-25 for compositions and methods for obtaining nucleic acids from sputum.
Invention is credited to H. CHAIM BIRNBOIM.
Application Number | 20090162924 12/338848 |
Document ID | / |
Family ID | 29740816 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090162924 |
Kind Code |
A1 |
BIRNBOIM; H. CHAIM |
June 25, 2009 |
COMPOSITIONS AND METHODS FOR OBTAINING NUCLEIC ACIDS FROM
SPUTUM
Abstract
The present invention relates to compositions and methods for
preserving and extracting nucleic acids from saliva. The
compositions include a chelating agent, a denaturing agent, buffers
to maintain the pH of the composition within ranges desirable for
DNA and/or RNA. The compositions may also include a reducing agent
and/or antimicrobial agent. The invention extends to methods of
using the compositions of the invention to preserve and isolate
nucleic acids from saliva as well as to containers for the
compositions of the invention.
Inventors: |
BIRNBOIM; H. CHAIM; (Ottawa,
CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Family ID: |
29740816 |
Appl. No.: |
12/338848 |
Filed: |
December 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10455680 |
Jun 5, 2003 |
7482116 |
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12338848 |
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60386397 |
Jun 7, 2002 |
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60386398 |
Jun 7, 2002 |
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60386399 |
Jun 7, 2002 |
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Current U.S.
Class: |
435/270 ;
536/23.1 |
Current CPC
Class: |
B01L 2300/046 20130101;
B01L 2300/0832 20130101; C12Q 1/6806 20130101; B01L 2300/042
20130101; B01L 3/5082 20130101; C12N 15/1003 20130101; B01L
2300/0672 20130101; B01L 2300/047 20130101; B01L 3/502 20130101;
B01L 2400/0683 20130101; C12Q 1/6806 20130101; C12Q 2527/125
20130101; C12Q 2527/119 20130101 |
Class at
Publication: |
435/270 ;
536/23.1 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C12S 3/20 20060101 C12S003/20 |
Claims
1. A method for preserving nucleic acid contained in a biological
sample comprising the steps of: a) obtaining the biological sample
from a subject; and b) contacting said sample with a composition
comprising (i) at least one chelating agent selected from the group
consisting of chelating agents stronger than EDTA; and (ii) a
denaturing agent, wherein the pH of said composition is greater
than 5.0, to form a liquid mixture; and c) storing said mixture for
more than 1 day at room temperature, wherein said composition
stabilizes said nucleic acid for more than 1 day at room
temperature.
2. The method of claim 1, wherein the biological sample is tissue
or a bodily fluid.
3. The method of claim 2, wherein the bodily fluid is sputum.
4. The method of claim 3, wherein the sputum is saliva.
5. The method of claim 1, wherein said chelating agent is
cyclohexane diaminotetraacetate (CDTA), diethylenetriamine
pentaacetic acid (DTPA), tetraazacycylododecanetetraacetic acid
(DOTA), tetraazacyclotetradecanetetraacetic acid (TETA),
desferioximine or a chelator analog thereof.
6. The method of claim 1, wherein said denaturing agent is urea,
dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate,
perchlorate, an alcohol or a mixture thereof.
7. The method of claim 6, wherein the alcohol is ethanol.
8. The method of claim 1, wherein the chelating agent is CDTA and
the denaturing agent is SDS.
9. The method of claim 1, wherein the pH is between 7.0 and 10.0
inclusive.
10. The method of claim 1, wherein the biological sample is from a
mammal.
11. The method of claim 1, wherein the nucleic acid is from a
virus, a bacterium or from said subject.
12. The method of claim 1, further comprising the step of
recovering said nucleic acid by contacting said nucleic
acid-containing solution with a protease.
13. The method of claim 1, wherein the nucleic acid is DNA.
14. A method for recovering nucleic acid from a biological sample
comprising the steps of: a) obtaining the biological sample from a
subject; and b) contacting said sample with a composition
comprising (i) at least one chelating agent selected from the group
consisting of chelating agents stronger than EDTA; and (ii) a
denaturing agent, wherein the pH of said composition is greater
than 5.0, to form a liquid mixture; and c) storing said mixture for
more than 1 day at room temperature, wherein said composition
stabilizes said nucleic acid for more than 1 day at room
temperature; d) contacting said mixture with a protease; and e)
recovering said nucleic acid from said mixture.
15. The method of claim 14, wherein the biological sample is tissue
or a bodily fluid.
16. The method of claim 15, wherein the bodily fluid is sputum.
17. The method of claim 16, wherein the sputum is saliva.
18. The method of claim 14, wherein said chelating agent is
cyclohexane diaminotetraacetate (CDTA), diethylenetriamine
pentaacetic acid (DTPA), tetraazacycylododecanetetraacetic acid
(DOTA), tetraazacyclotetradecanetetraacetic acid (TETA),
desferioximine or a chelator analog thereof.
19. The method of claim 14, wherein said denaturing agent is urea,
dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate,
perchlorate, an alcohol or a mixture thereof.
20. The method of claim 19, wherein the alcohol is ethanol.
21. The method of claim 14, wherein the chelating agent is CDTA and
the denaturing agent is SDS.
22. The method of claim 14, wherein the pH is between 7.0 and 10.0
inclusive.
23. The method of claim 14, wherein the biological sample is from a
mammal.
24. The method of claim 14, wherein the nucleic acid is from a
virus, a bacterium or from said subject.
25. The method of claim 14, wherein the nucleic acid is DNA.
26. The method according to claim 14, wherein the protease is
proteinase K.
27. A method for preserving nucleic acid contained in a biological
sample comprising the steps of: a) obtaining the biological sample
from a subject; and b) contacting said sample with a composition
comprising (i) at least one chelating agent selected from the group
consisting of chelating agents stronger than EDTA; and (ii) a
denaturing agent, wherein the pH of said composition is greater
than 5.0, to form a liquid mixture; c) contacting said mixture with
a protease; d) storing said mixture for more than 1 day at room
temperature, wherein said composition stabilizes said nucleic acid
for more than 1 day at room temperature.
28. The method of claim 27, wherein the biological sample is tissue
or a bodily fluid.
29. The method of claim 28, wherein the bodily fluid is sputum.
30. The method of claim 29, wherein the sputum is saliva.
31. The method of claim 27, wherein said chelating agent is
cyclohexane diaminotetraacetate (CDTA), diethylenetriamine
pentaacetic acid (DTPA), tetraazacycylododecanetetraacetic acid
(DOTA), tetraazacyclotetradecanetetraacetic acid (TETA),
desferioximine or a chelator analog thereof.
32. The method of claim 27, wherein said denaturing agent is urea,
dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate,
perchlorate, an alcohol or a mixture thereof.
33. The method of claim 32, wherein the alcohol is ethanol.
34. The method of claim 27, wherein the chelating agent is CDTA and
the denaturing agent is SDS.
35. The method of claim 27, wherein the pH is between 7.0 and 10.0
inclusive.
36. The method of claim 27, wherein the biological sample is from a
mammal.
37. The method of claim 27, wherein the nucleic acid is from a
virus, a bacterium or from said subject.
38. The method of claim 27, wherein the nucleic acid is DNA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 10/455,680, entitled, "Compositions and
Methods for Obtaining Nucleic Acids from Sputum", filed Jun. 5,
2003, which claims the benefit of U.S. Application No. 60/386,397,
filed Jun. 7, 2002, U.S. Application No. 60/386,398, filed Jun. 75,
2002, and U.S. Application No. 60/386,399, filed Jun. 7, 2002, each
of which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to compositions and methods
for preserving nucleic acids at room temperature for extended
periods of time and for simplifying the isolation of nucleic
acids.
[0003] DNA can be extracted from virtually every type of cell in
the human body, with the exception of red blood cells. The usual
source of bodily samples for extraction of DNA is venous blood,
since the number of nucleated white blood cells (principally
neutrophils and lymphocytes) is relatively high and quite
consistent: the normal range is about 5 to 10 million white blood
cells per milliliter of blood. The DNA content of human cells is
about 6 micrograms per million cells, so 1 milliliter can
theoretically yield from 30 to 60 micrograms of DNA. However, there
are about 5 billion red blood cells per milliliter of blood, which,
since they contain no DNA, must be removed to obtain pure DNA.
Furthermore, the use of blood as a source of DNA has many other
disadvantages. Collection of blood is not a trivial procedure.
Taking of venous blood requires trained personnel. It is an
invasive procedure, which frequently causes some distress and pain
to the donor. Precautions are needed to minimize exposure of
personnel to blood-borne pathogens. Once collected, the blood
sample must be either frozen or quickly transported to a laboratory
for extraction of DNA. For these reasons, venous blood is not the
ideal source of DNA. A simpler procedure for obtaining blood is to
collect a few drops after a finger prick and blotting it onto a
piece of filter paper. Less training of personnel is required. Once
dried, the DNA is quite stable. The amount of DNA recovered is
small but sufficient for many forensic purposes. However, a finger
prick is still an invasive procedure and heme derived from
hemoglobin in blood can inhibit some types of DNA analysis.
[0004] Swabbing the inside of the cheek with a brush (a buccal
swab) is another source of cells that contain DNA. It is much less
invasive than taking of blood and can be collected by individuals
with less training than is required in the collection of blood.
Once collected, the time that useable DNA can be recovered can be
extended by either drying the swab or wiping onto filter paper and
drying it. However, as the inside of the mouth is not a sterile
source (as compared to blood) and microbes can degrade the quality
of the DNA after a period of time. The number of cells recovered by
this procedure is not large and typically less than 1-2 micrograms
of DNA can be expected in the entire sample.
[0005] Saliva is a fairly clear, colorless fluid secreted
principally by the major salivary glands (parotid, submandibular,
and sublingual). Its function is to lubricate and cleanse the oral
cavity, as well as to initiate the process of digestion. The
parotid gland primarily secretes serous (watery) saliva, while the
other glands secrete a mixture of serous and mucinous (sticky)
saliva. Components of saliva include albumin, globulin, mucins, and
digestive enzymes. It has long been known that cellular DNA is
present in saliva and that this DNA is suitable for forensic
purposes. Forensic use is typically limited to victim or suspect
identification, using the tiny amounts of DNA from saliva that may
recovered at a crime scene or from the back of a postage stamp. The
notion that saliva may be a reliable source of genomic DNA and a
rival to venous blood samples for this purpose has been
investigated more recently in a scientific publication (van Schie,
et al., J. Immunol. Methods 208:91-101, 1997). The authors used
freshly collected or frozen saliva samples and purified the DNA by
a fairly complex extraction procedure. Estimates of the quantity of
DNA recovered were based upon light absorption at 260 nm, a
procedure known to be an unreliable method since other common
biological macromolecules, such as RNA, have essentially the same
ultraviolet light absorption spectrum. Nevertheless, these authors
showed that quality genomic DNA was indeed present by gel
electrophoretic analysis and polymerase chain reaction analysis for
certain allelic polymorphisms. Another communication (Terasaki, et
al., Hum. Immunol. 59:597-598, 1998) reported similar results about
the suitability of saliva as a source of DNA for HLA typing by
polymerase chain reaction analysis. Although the amount of DNA
recovered was reported, the method used to measure DNA was not.
These authors provided 3 examples where saliva dried on filter
paper yielded DNA suitable for analysis.
[0006] With the increasing use of DNA-based analysis in forensics,
law enforcement, military, human medicine, veterinary medicine, and
research, there is a need for a product that would allow saliva to
become a standard reliable source of DNA from an individual (to
replace blood, the current standard). In forensic, military and
mass disaster situations, for example, DNA samples are now
routinely taken from living persons thought to be relatives of
unidentified victims of accident or foul play, to aid in
identification of the dead. Military personnel or other individuals
who expect to encounter hazardous situations where their lives may
be at risk may wish to store DNA samples prior to exposing
themselves to these hazards. In the law enforcement area, convicted
felons in both Canada and the United States are now required to
provide DNA samples. DNA-based tests are expected to increase in
medicine, such as testing for cystic fibrosis, cytochrome P450
isotypes, polymorphisms affecting susceptibility to infectious and
autoimmune diseases, HLA typing, paternity issues, to name but a
few. In clinical studies, an example would be to screen populations
for colon cancer-predisposing genes or family members of a breast
cancer victim for breast cancer predisposing genes. In all of these
cases, there are significant advantages to providing a saliva
sample rather than providing a blood sample as a source of DNA. All
donors would prefer donating saliva rather than blood because of
the discomfort, pain, or apprehension associated with phlebotomy or
pin-pricks. Saliva has a further advantage of not requiring
specialized personnel thereby reducing cost where mass sample
collection is being carried out. The risk of blood-borne infection
is likewise decreased.
[0007] In addition to the problem of developing a standard
collection and preservation method for DNA in saliva, there remains
an ongoing need to improve methods of overcoming problems specific
to the recovery of nucleic acids from saliva. The problem of
extraction of high molecular weight DNA and RNA from mammalian
cells has been partially addressed by Birnboim in Methods of
Enzymology 216:154-160, 1993, but this work was not extended to the
recovery of nucleic acids from mucin-containing bodily fluids.
[0008] Multimeric proteins called mucins are high molecular weight
glycosylated proteins that form a major part of a protective
biofilm on the surface of epithelial cells, where they can provide
a barrier to particulate matter and bind microorganisms. These
glycoproteins contribute greatly to the viscoelastic nature of
saliva. The major high-molecular-weight mucin in salivary
secretions is MUC5B, one of four gel-forming mucins that exist as
multimeric proteins with molecular weights greater than 20-40
million daltons. MUC5B is a large oligomeric mucin composed of
disulphide-linked subunits.
[0009] It is known that reagents that reduce disulfides also reduce
the viscosity of mucin, such as that found in sputum or saliva.
Reducing agents, in particular sulfur-containing chemicals such as
.beta.-mercaptoethanol and dithiothreitol, are widely used in
biochemistry. However, many biochemically relevant reducing agents
are capable of reacting in solution with dissolved oxygen. This is
known are autooxidation (also called autoxidation or
auto-oxidation), where 1-electron reduction intermediates of oxygen
are formed, viz., superoxide (O.sub.2.sup.-.), hydrogen peroxide
(H.sub.2O.sub.2) and hydroxyl radical (OH.). In addition,
transitional metal cations function as catalysts and O.sub.2.sup.-.
has been demonstrated to be an intermediate. Unfortunately,
reducing agents and reducing compositions of the prior art have a
relatively short shelf life, especially in basic solutions, and
stock solutions that contain reducing agents cannot be prepared and
stored under ambient conditions for an extended period time,
usually not more than a day or two.
[0010] Therefore, in addition to a need for a means to collect
sputum or saliva, and subsequently preserving the nucleic acids
contained therein by contacting them with a stabilizing
composition, there is a need for the inclusion of a stable reducing
agent into the composition, such that nucleic acids can be
conveniently recovered from it, especially after extended periods
of time in the presence of oxygen at neutral or mildly alkaline
pH.
SUMMARY OF THE INVENTION
[0011] The present inventor has developed a composition, which,
when mixed with a mucin-containing bodily fluid, preserves the
nucleic acids at room temperature under ambient conditions for
extended periods of time. There is no requirement for freezing of
the samples before nucleic acid recovery and purification. The
properties of this composition are that it (a) chemically
stabilizes nucleic acids, (b) inhibits nucleases that may be
present in the saliva, and (e) is compatible with proteolytic
enzymes and other reagents used to purify/amplify oligo- or
polynucleotides. A fourth and novel property of this composition is
that it contains an agent that rapidly reduces the viscous
properties of mucin, greatly facilitating the extraction of nucleic
acids contained within.
[0012] Accordingly, a first aspect of the invention features a
composition for preserving nucleic acids that includes a chelating
agent, and a denaturing agent, where the pH of the composition is
greater than 5.0. In one embodiment, the composition is an aqueous
solution.
[0013] In another embodiment, the composition also includes a
reducing agent. For example, it can include one or more of the
following: ascorbic acid, dithionite, erythiorbate,
N-acetylcysteine, cysteine, glutathione, dithiothreitol,
2-mercaptoethanol, dierythritol, a resin-supported thiol, a
resin-supported phosphine, vitamin E, and trolox, or salts thereof.
Desirably, the reducing agent is ascorbic acid, erythiorbate,
N-acetylcysteine, dithiothreitol, or 2-mercaptoethanol, and most
desirably, the reducing agent is ascorbic acid. In another
embodiment, the composition does not contain ascorbic acid. In yet
another embodiment, the concentration of the reducing agent in the
composition is greater than or equal to 50 millimolar.
[0014] Antioxidant free-radical scavengers are also desirable
reducing agents for the composition of the present invention.
Examples include antioxidant vitamins, antioxidant hormones,
antioxidant enzymes, thiols, and phenols.
[0015] Desirably, the reducing agent retains reducing activity for
at least 46 days in the presence of one or more of the following:
oxygen, ambient air, ambient light, and alkaline pH.
[0016] The chelating agent of the composition can be selected from
the group consisting of: ethylenediamine tetraacetic acid (EDTA),
cyclohexane diaminetetraacetate (CDTA), diethylenetriamine
pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid
(DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), and
desferrioximine, or chelator analogs thereof. Desirably, the
chelating agent is cyclohexane diaminetetraacetate (CDTA),
diethylenetriamine pentaacetic acid (DTPA),
tetraazacyclododecanetetraacetic acid (DOTA), or desferrioximine,
and most desirably, the chelating agent is cyclohexane
diaminetetraacetate (CDTA).
[0017] In another embodiment, the chelating agent of the
composition inhibits metal redox cycling. By "inhibits metal redox
cycling" is meant the inhibition of metal-based oxidation/reduction
cycles that produce reactive oxygen free-radical species. Examples
of redox ion pairs involved in such cycles include
Fe.sup.2+/Fe.sup.3+, Cu.sup.1+/Cu.sup.2+, and various oxidation
states of molybdenum, vanadium, nickel, and cobalt. Chelators that
bind one or both ions of a redox ion pair can inhibit the
production of reactive oxygen species such as, for example,
hydroxyl radical (HO.), hydroperoxyl radical (HOO.), superoxide
radical (O.sub.2.sup.-.), nitric oxide radical (NO.), or
peroxynitrite radical (ONO.sub.2.sup.-.).
[0018] The nucleic acid to be preserved by the composition can be
DNA or RNA, including mRNA or viral RNA.
[0019] The pH of the composition can between from about 5-0 and
about 11.0, desirably from about 6.5 to about 7.5, and most
desirably, about 7.0. For the preservation of DNA, a pH from about
7.0 to about 10.0 can be used. Depending on other components of the
compositions, desirable pHs are about 7.5, about 8.0, or a pH range
from about 8.0 to about 9.0. A buffer, such as HEPES, TRIS, or
carbonate buffer can be added to the composition to maintain the pH
in a constant range. For the preservation of RNA, a pH from about
5.0 to about 7.0, desirably from about 6.5 to about 6.8 can be
used. Again, a buffer, such as BES, can be used to maintain the pH
in a constant range.
[0020] The denaturing agent of the composition can be selected from
the group consisting of: urea, dodecyl sulfate, guanidinium
chloride, guanidinium thiocyanate, perchlorate, and an alcohol.
Desirably, the denaturing agent is urea, dodecyl sulfate, or an
alcohol, wherein the alcohol is 10%-60% of the total composition
volume. The alcohols can be methanol, ethanol, n-propanol,
isopropanol, n-butanol, trifluoroethanol, phenol, or
2,6-di-tert-butyl-4-methylphenol.
[0021] In another embodiment, the composition includes an
antimicrobial agent. By "antimicrobial agent" is meant a substance
or group of substances which reduces the rate of growth of an
organism compared to the rate of growth of the organism in their
absence. A reduction in the rate of growth of an organism may be by
at least 5%, more desirably, by at least 10%, even more desirably,
by at least 20%, 50%, or 75%, and most desirably, by 90% or more.
The definition also extends to substances which affect the
viability, virulence, or pathogenicity of an organism. An
antimicrobial agent can be natural (e.g., derived from bacteria),
synthetic, or recombinant. An antimicrobial agent can be
bacteriostatic, bactericidal or both. An antimicrobial agent is
bacteriostatic if it inhibits cell division without affecting the
viability of the inhibited cell. An antimicrobial agent is
bactericidal if it causes cell death. Cell death is commonly
detected by the absence of cell growth in liquid growth medium
(e.g., absence of turbidity) or on a solid surface (e.g., absence
of colony formation on agar). Those of skill in the art know that a
substance or group of substances which is bacteriostatic at a given
concentration may be bactericidal at a higher concentration.
Certain bacteriostatic substances are not bactericidal at any
concentration. Desirably, the composition of the invention includes
an alcohol as an antimicrobial agent, and most desirably the
composition includes ethanol.
[0022] In another embodiment, the composition also includes an
inhibitor of ribonuclease. Desirable inhibitors are selected from
the group consisting of: heparin, heparan sulfate,
oligo(vinylsulfonic acid), poly(vinylsulfonic acid),
oligo(vinylphosphonic acid), and poly(vinylsulfuric acid), or salts
thereof. The inclusion of an inhibitor of ribonuclease in the
composition of the invention is particularly desirable when the
nucleic acid to be preserved is RNA, desirably mRNA, or when the
nucleic acid to be preserved is from a virus or a bacterium.
[0023] A second aspect of the invention features a method of
reducing the viscosity of a mucin-containing bodily fluid or tissue
by reducing disulfide bonds inherent to mucin, wherein the bodily
fluid or tissue is mixed with a composition of the invention that
includes a reducing agent. In one embodiment, the bodily fluid is
sputum, desirably saliva. By "sputum" is meant that mucoid matter
contained in or discharged from the nasal or buccal cavity of an
animal, including saliva and discharges from the respiratory
passages, including the lungs. In another embodiment, the method
includes the recovery of a nucleic acid.
[0024] A third aspect of the invention features a method of
preserving a nucleic acid contained in sputum that includes the
steps of obtaining sputum from a subject, and contacting the sputum
with a composition of the invention, thus preserving the nucleic
acid.
[0025] In one embodiment, when the nucleic acid is DNA, the DNA is
stable for more than 14 days, desirably more than 30 days, and more
desirably more than 60 days. In another embodiment, when the
nucleic acid is DNA and the composition does not contain ascorbic
acid, the DNA is stable for more than 60 days, and desirably more
than 360 days.
[0026] A fourth aspect of the invention features a method of
recovering a nucleic acid from sputum that includes the steps of:
i) obtaining sputum from a subject, ii) contacting the sputum with
a composition of the invention to form a mixture, iii) contacting
the mixture with a protease, and iv) recovering the nucleic acid
from the mixture. Desirably, the protease is proteinase K or
pronase.
[0027] In one embodiment of any of the second, third, or fourth
aspects, the sputum is saliva. In another embodiment, the sputum is
from a mammal, desirably a human. In yet another embodiment, the
nucleic acid is DNA or RNA. If the nucleic acid is RNA, desirably
it is mRNA or viral RNA. The nucleic acid can be from a source
foreign to the subject from which the sputum sample is taken. For
example, the nucleic acid can be from a bacterium or a virus that
is residing in the buccal, nasal, or respiratory passages of the
subject.
[0028] In a fifth aspect, the invention features a method of
preserving and/or recovering a nucleic acid from a bodily fluid
that includes, placing the bodily fluid into a first region of a
container, placing a composition of the invention into a second
region of the container, which is separated from the first region
by a barrier, closing the container, and disturbing the integrity
of the barrier such that the composition and the bodily fluid are
brought into contact.
[0029] In one embodiment, the disestablishment of the barrier is
coupled to the closing of the container when a lid is placed on it.
In one example, the barrier is punctured. In a desirable example,
the barrier is in the form of a pivoting sealing disc. In this
example, attachment of the lid to the container forces the disc to
pivot from its original position of spanning the space between the
first region and the second region to a position in which both
regions are exposed to each other, thereby forming a mixture
between a composition of the invention and the bodily fluid is
allowed. Desirably, the bodily fluid is sputum, and most desirably,
saliva.
[0030] In a sixth aspect, the invention features a device for
preserving and/or isolating a nucleic acid obtained from a
biological sample. The device includes: a container that has a
first region for collecting a biological sample and a second region
containing a composition for preserving a nucleic acid, a barrier
between the first region the second region that keeps the
biological sample and the composition separate, a means for closing
the container, and a means for disturbing the integrity of the
barrier such that the composition is capable of contacting the
biological sample. The first region can have an opening of from 2.0
to 7.0 cm, desirably from 2.5 to 3.5 cm, and most desirably 3.0 cm.
Desirably, the biological sample is sputum, and most desirably,
saliva.
[0031] In one embodiment of the sixth aspect, the nucleic
acid-preserving composition is a composition of the present
invention. In another embodiment, the means for closing the
container is coupled to the means for disturbing the integrity of
the barrier. In yet another embodiment, the means for closing the
container is an airtight lid.
[0032] In a seventh aspect, the invention features a method of
manufacturing a device for preserving a nucleic acid in a
biological sample that includes: providing a container that has a
first region and a second region, with the first region suitable
for containing a composition of the invention and the second region
having an opening suitable for the application of a biological
sample; placing the composition into the first region; and applying
a barrier to the container between the first region and the second
region, with the barrier being impermeable to the composition and
capable of being disestablished.
[0033] In an embodiment of either the sixth or seventh aspect, the
barrier can be a pivoting disc, where in a first position, the disc
spans the compartment and separates the first and second areas.
Pivoting the disc to a second position (e.g., by connecting a
screw-on lid to a plunger mechanism which contacts the disc,
causing it to pivot when the lid is screwed on) disestablishes the
barrier and allows the biological sample contained in the first
region to contact the composition that is contained in the second
region.
[0034] By "about" is meant +/-10% of the stated value or a chemical
or obvious equivalent thereof.
[0035] By "alcohol" is meant a water-miscible organic compound
containing a hydroxyl group, including water-miscible mixtures of
hydroxyl-containing organic compounds.
[0036] By "antioxidant free-radical scavenger" is meant a substance
that reduces a reactive oxygen free radical species. Such free
radicals include, for example, hydroxyl radical (HO.), hydroperoxyl
radical (HOO.), superoxide radical (O.sub.2.sup.-.), nitric oxide
radical (NO.), or peroxynitrite radical (ONO.sub.2.sup.-.).
[0037] By "aqueous solution" is meant a solution or suspension that
contains 30% or more water by volume.
[0038] By "bodily fluid" is meant a naturally occurring fluid from
an animal, such as saliva, serum, plasma, blood, urine, mucus,
gastric juices, pancreatic juices, semen, products of lactation or
menstration, tears, or lymph.
[0039] By "biological sample" is meant any sample containing
nucleic acids that has been obtained from or deposited by an
animal. Non-limiting examples include skin, hair, bodily fluids,
fecal matter, and tissue.
[0040] By "chelator analog" is meant a derivative chelator compound
with the same backbone structure and having the same general
properties as the parent chelator compound.
[0041] By "denaturing agent" is meant a substance that alters the
natural state of that to which it is added.
[0042] By "mucin" is meant any mucoprotein that raises the
viscosity of the medium surrounding the cells that secrete it.
[0043] By "mucoid" is meant any bodily fluid containing mucin
[0044] By "nucleic acid" is meant a chain of the nucleotides,
including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),
typically found in chromosomes, mitochodria, ribosomes, bacteria,
or viruses.
[0045] By "nucleic acid-preserving composition" is meant any
composition of the present invention, unless otherwise
specified.
[0046] When referring to a nucleic acid, by "stable" is meant that
at least about 50% of the initial amount of high molecular weight
nucleic acid (DNA, RNA, mRNA, or viral RNA) contained in a sample
is still present after storing the sample at ambient temperature
(i.e., 20.degree. C. to 25.degree. C.) for the specified time
period. The amount of high molecular weight DNA in a sample can
quantified by densitometry analysis of the high molecular weight
DNA band from an agarose gel (see FIG. 1 and Example 4).
[0047] By "resin-supported phosphine" is meant a polymer that
contains a multiplicity of covalently-bound phosphine groups.
[0048] By "resin-supported thiol" is meant is meant a polymer that
contains a multiplicity of covalently-bound sulfhydryl groups.
[0049] By "saliva" is meant the secretion, or combination of
secretions, from any of the salivary glands, including the parotid,
submaxillary, and sublingual glands, optionally mixed with the
secretion from the buccal glands.
[0050] By "sputum" is meant that mucoid matter contained in or
discharged from the nasal or buccal cavity of a mammal, including
saliva and discharges from the respiratory passages, including the
lungs.
[0051] By "subject" is meant any animal. Desirably, the subject is
a mammal that can produce saliva for the purposes of nucleic acid
extraction. Most desirably, the subject is a human.
[0052] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples, while indicating preferred embodiments of the
invention are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 is an electrophoresis agarose analysis of DNA
isolated from saliva using the capacity of methods of one
embodiment of the invention.
[0054] FIG. 2 is a graph illustrating real time PCR of stimulated
saliva DNA of Example 5.
[0055] FIG. 3 is a graph illustrating real time PCR of unstimulated
saliva DNA of Example 6.
[0056] FIG. 4 is an electrophoresis agarose analysis of the DNA in
saliva samples mixed with compositions of the invention, the
mixtures having been incubated for various times at various
temperatures.
[0057] FIG. 5 shows structures of (oxidized) ascorbate anion,
(reduced) dehydroascorbic acid, and a free radical intermediate
[0058] FIG. 6 is a compilation of two spectrophotometric scans of
sodium ascorbate (100 .mu.M) in CB (1 mM CDTA, 10 mM BES, pH 7.4),
prepared under aerobic conditions over 30 minutes at room
temperature (scan 1) and 3 minutes after addition of a few crystals
of MnCl.sub.2.(scan 2), as per Example 8.
[0059] FIG. 7 is a compilation of spectrophotometric scans, at the
indicated times, of the 100 .mu.M sodium ascorbate prepared in CB
of Example 8. The solution was exposed to ambient atmosphere and
temperature between scans but was not contacted with MnCl.sub.2
(see Example 9).
[0060] FIG. 8 is a graph of absorbances at 265 mm, obtained at the
indicated times, of a solution of sodium ascorbate (250 mM)
containing 30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM CDTA, mixed
with 50 mL of CB, as per Example 10. The stock solution was
maintained at room temperature and no precaution was taken to
exclude ambient atmosphere or ambient light.
[0061] FIG. 9 is a compilation of spectrophotometric scans of the
46 day-old solution prepared in Example 10. Scan 1 (t=46 days) was
taken before the addition of MnCl.sub.2. Scan 2 was taken 2 minutes
after the addition MnCl.sub.2. Scan 3 was taken 8 minutes after the
addition MnCl.sub.2. Scan 4 was taken 27 minutes after the addition
MnCl.sub.2.
[0062] FIG. 10 is an exploded view of a sample container of the
invention. Included in the figure is a cross-sectional top view
taken at line 1-1 of container 3 showing plunger 4 and plunger
channel 5. Also shown is a cross-sectional top view taken at line
2-2 of container 3, showing supports 6 for sealing disc 7 (not
shown in this figure but shown in FIG. 11).
[0063] FIG. 11 is a side view of the sample container of FIG. 10,
now showing sealing disc 7.
DETAILED DESCRIPTION
[0064] The following standard abbreviations are used herein: DNA,
deoxyribonucleic acid; RNA, ribonucleic acid; mRNA, messenger RNA;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BES,
N,N-bis[2-hydroxyethyl]-2-aminomethane-sulfonic acid; TRIS,
tris(hydroxymethyl)aminomethane, CDTA, cyclohexane
diaminetetraacetate; DTPA,
N,N-bis(2-(bis(carboxymethyl)amino)ethyl)glycine; DOTA,
1,4,7,10-tetrazacyclododecanetetraacetic acid; and TETA,
1,4,8,11-tetraazacyclotetradecanetetraacetic acid.
Compositions of the Invention
[0065] The present inventors have developed compositions that
render sputum as a viable option to the use of blood as a source of
nucleic acids. The compositions provide the advantageous properties
of chemical stabilization of nucleic acids and the inhibition of
nucleases, including deoxyribonucleases, and microbial growth.
Chemical stabilization of the nucleic acids in a saliva sample is
achieved through the use of buffers to maintain an appropriate pH,
as well as the use of chelating agents to prevent the phenomenon of
metal redox cycling or the binding of metal ions to the phosphate
backbone of nucleic acids. The chelating agents of the invention
also participate in the inhibition of deoxyribonucleases and
microbial growth, which can be additionally inhibited by the
inclusion of denaturing agents and/or other suitable antimicrobial
substances, such as ethanol, into the compositions of the
invention. The compositions of the invention can also include one
or more reducing agents, which can reduce sample viscosity, thereby
making nucleic acid recovery an easier process.
[0066] Accordingly, the present invention features a composition
for preserving and/or recovering nucleic acids from sputum,
desirably saliva, that includes one or more chelators and one or
more denaturing agents, wherein the pH of the composition is
greater than 5, desirably within a pH range of about 6 to about 11,
more desirably within a pH range of about 7.5 to about 10.0, and
most desirably, within a pH of about 7.0.
[0067] The chemical backbone and the purine bases of DNA are most
stable at slightly alkaline pH, with an optimal stability generally
recognized as being within a pH range of about 7-11, and desirably
a pH of about 8. Below a pH of about 6, depurination (i.e.,
spontaneous loss of purine bases from the deoxyribose-phosphate
backbone) can occur. Above a pH of about 10, spontaneous loss of
amino groups from cytosine nucleotides may occur, thereby
converting cytosine to uracil. Above a pH of about 12, DNA is
denatured, converting it from the double-strand form to the
single-strand form. In contrast, RNA is most stable in the pH range
of 5.0 to 7.0, desirably a pH of from 6.5 to 6.8. Accordingly, the
pH of the composition may be adjusted using pH buffers, desirably
those that best control the pH within the range of about 5 to about
11. Examples of pH buffers with desirable properties include, but
not limited to, TRIS hydrochloride, HEPES and BES.
[0068] DNA has a strong affinity for metal ions, some of which,
such as the common transition metals iron or copper, can catalyze
the formation of reactive oxygen species. Therefore, a composition
of the invention includes one or more chelators that can form
complexes with metal ions to prevent them from binding to DNA,
remove metal ions that that have already bound to DNA, or bind to
metal ions (e.g., Fe(II)/Fe(III) or Cu(I)/Cu(II)) strongly enough
to inhibit their redox cycling, and hence, the formation of
reactive oxygen species. EDTA, a commonly used chelator in
biological reagents, can be of some use for either of these
purposes. More desirable are stronger chelators (i.e., chelators
with a higher dissociation constant than EDTA when bound to a
metal), used alone or in combination, that include, but are not
limited to, CDTA, DTPA, DOTA, TETA, and desferioximine, or chelator
analogs thereof. The amount or concentration of chelator will
depend upon the strength of the chelator, which would need to be
determined empirically. For CDTA, concentrations in the 1-20 mM
range are sufficient, however other concentrations would work, and
the compositions of the invention are not intending to be limited
to this range.
[0069] Deoxyribonucleases and ribonucleases are enzymes that
breakdown DNA or RNA, respectively. Their main source in the
digestive tract is secretions of the pancreas, although lower
levels may be present in cells of the salivary gland and buccal
mucosa. In addition, microorganisms resident in the mouth or from
recently ingested foods may contain deoxyribonucleases or
ribonucleases. Pancreatic deoxyribonuclease is known to require
divalent metal ions such as Mg(IT), Mn(II) and/or Ca(II) for
enzymatic activity. The strong chelators described above, in
addition to providing chemical stability to the nucleic acids, will
inhibit this class of metal ion-requiring deoxyribonucleases. The
action of deoxyribonucleases and ribonucleases can also be
inhibited by denaturing agents that will destroy the complex
structures of these enzymes (proteins). Hence, denaturing agents
are included in the nucleic acid preserving composition of the
invention. Examples of denaturing agents that may be used (alone or
in combination) include, but not limited to, urea, soluble salts of
dodecyl sulfate and other strong detergents, guanidinium chloride,
guanidinium thiocyanate, soluble salts of perchlorate, alcohols,
such as ethanol, above 10%. Other reagents, such as heparin,
heparan sulfate, or oligo(vinylsulfonic acid) (Smith, et al., J.
Biol. Chem. Mar. 20 2003; [epub ahead of print]) are known to
inhibit the action of deoxynucleases and/or ribonucleases.
[0070] Low specificity proteases such as proteinase K are
frequently used in the purification of nucleic acids. Since
proteases are themselves proteins, their action can be inhibited by
denaturing agents. Thus, a balance must be struck between the
concentration of denaturing agents that will, on the one hand,
inhibit deoxyribonucleases or ribonucleases and denature other
proteins in saliva and, on the other hand, not significantly
inhibit the proteolytic enzymes. At later stages in DNA
purification, the DNA is often concentrated by precipitation with
alcohol. Thus, salts, buffers, chelators and other components of
the nucleic acid preserving/recovery solution must be chosen so as
not to precipitate when concentrations of alcohol over 50% are
added to precipitate the DNA.
[0071] The viscosity of sputum and saliva depends upon the presence
of very high molecular weight glycoproteins complexes called
mucins, particular the gel-forming mucins (Offner, et al., Adv.
Dent. Res. 14:69-75, 2000; Seregni, et al., Tumori 83:625-632,
1997). It has been found that the inclusion of a reducing agent
into a composition of the invention has the effect of markedly
reducing the viscosity of saliva, especially "unstimulated" saliva,
thereby facilitating the recovery of nucleic acids. Accordingly, in
one embodiment, a composition of the invention further includes one
or more reducing agents. The reducing agents are desirably at high
concentration (greater than 0.05 M). While not wishing to be
limited by theory, it is presumed that the reducing agent reduces
the viscosity of the saliva by breaking disulfide bonds that hold
together chains of mucin, and that any reducing agent that has the
appropriate redox potential to reduce disulfide bonds in proteins
would be suitable. Desirably, the reducing agent is selected from
the group consisting of: ascorbic acid, dithionite, erythiorbate,
N-acetylcysteine, cysteine, glutathione, dithiothreitol,
2-mercaptoethanol, dierythritol, a resin-supported thiol, a
resin-supported phosphine, vitamin E, and trolox, or salts
thereof.
[0072] In another embodiment, a composition of the invention that
includes a reducing agent maintains reducing capacity at room
temperature in a sealed container in the presence of ambient
oxygen, and/or in the presence of ambient light for more than a
week, desirably for up to about 46 days, and most desirably for at
least 46 days. This embodiment combines the nucleic acid
stabilization provided by a strong chelator a denaturing agent, and
a reducing agent in a composition with a pH within the range of
about 6 to about 11, and desirably a pH of about 8.0.
[0073] A particularly desirable reducing agent is sodium ascorbate.
As well as an important dietary antioxidant micronutrient, ascorbic
acid (vitamin C) is a non-thiol reducing agent and is inexpensive,
non-toxic, and stable in the presence of the chelators and
denaturing agents that are included in the compositions of the
invention. The structures of (oxidized) ascorbate anion, (reduced)
dehydroascorbic acid, and a free radical intermediate are shown in
FIG. 5. The most thoroughly studied oxidation reaction of ascorbate
is its oxidation by oxygen. As with many other reducing agents,
trace amounts of transitional metals such as iron or copper can
promote autooxidation (Buettner, Free Radic. Res. Commun. 1:349-53,
1986; Buettner and Jurkiewicz Radiat. Res. 145:532-41, 1996;
Miller, et al., Free Radic. BioL Med. 8:95-108, 1990). Metal
cation-catalyzed oxidation of ascorbate can be conveniently
monitored as a decrease in absorbance at 265 nm (Buettner Free
Radic. Res. Commun. 10:5-9, 1990), as described in Example 8 and
shown in FIGS. 5, 6, and 8. Certain chelating agents can
appreciably slow down autooxidation of ascorbate at pH 7.0 or lower
(Buettner J. Biochem. Biophys. Methods 16:27-40, 1988), as
described in Example 10 and shown in FIG. 8.
[0074] In another embodiment, a composition of the present
invention includes one or more chelators, one or more denaturing
agents, and one or more antimicrobial agents, wherein the pH of the
composition is within a pH range of about 6.0 to about 11.0,
desirably at a pH of about 8.0. Microbial growth may also be
inhibited by the strong chelators and denaturing agents, for
example, ethanol, described above. Therefore, in a further
embodiment of the present invention, a composition for preserving
and/or recovering DNA from sputum includes one or more chelators
and one or more denaturing agents, wherein at least one or more of
the denaturing agents and/or chelating agents is present in amounts
to act as an antimicrobial agent.
[0075] Reagents that indicate when a biological sample has been
contacted with a composition of the invention can also be included
as part of the composition. Desirable are those reagents that
result in a visual color change of the composition solution upon
mixing with the added sample. These reagents can function by
reacting with any number of functional groups that are contained in
biological samples, including, for example, amines, thiols, or
glycosyl groups. Such colorimetric reagents are known to those
skilled in the art and are chosen in such a manner that other
components of the composition do not interfere with their effective
usage.
METHODS OF THE INVENTION
[0076] The present invention features methods of collecting,
preserving, and recovering nucleic acids from sputum using a
composition of the invention. The methods of the invention involve
contacting a sputum sample from a subject with a composition of the
invention and optionally mixing the resulting solution with a
protease, such as pronase or proteinase K. Furthermore, some
compositions of the invention feature a reducing agent that can
facilitate the recovery of nucleic acids from composition/sample
mixtures by decreasing the viscosity of these mixtures.
[0077] Accordingly, one aspect of the invention features a method
of preserving a nucleic acid contained in sputum that includes the
steps of obtaining sputum from a subject, and contacting the sputum
with a composition of the invention, thus preserving the nucleic
acid. Examples 1 and 2 describe the collection of saliva, both from
subjects that can follow instructions and from those that can
not.
[0078] The sputum is typically contacted with a composition of the
invention upon collection or immediately after it is collected, and
preferably not much later than about 1 hour after collection. This
time can vary depending on storage conditions of the sputum after
collection. For example, it could be indefinite if stored frozen or
perhaps 1-2 days if stored at 4.degree. C. A reducing agent can be
in the preserving composition used, or added at a later time prior
to nucleic acid isolation. Desirable reducing agent-containing
compositions are those that are stable and retain a reducing
capacity for more than a week, desirably for up to about 46 days,
and most desirably for at least 46 days.
[0079] In an example (see Example 5), the results of which are
presented in Table 1, saliva was collected and mixed with
approximately an equal volume of a composition of the invention
(see Example 3 for preparation), and analyzed for DNA content by
PCR analysis at later timepoints.
TABLE-US-00001 TABLE 1 Estimated amounts of DNA in saliva samples*
Donor # 1 2 3 4 5 6 7 8 9 10 11 Stim. saliva collected on 02 Feb
26, analyzed 64 days by the DNase method 21.2 21.4 16.6 16.0 28.8
44.8 22.2 16.6 Unstim. saliva collected on 02 Mar 25, analyzed 15
days later by DNase method 64.2 80.6 24.4 27.2 69.0 *DNA content in
nanograms per microliter
[0080] To collect the sputum from the subject it is preferred that
the mouth be rinsed before sampling. Food particles can introduce
foreign DNA and saliva transferred by kissing can be a source of
foreign human DNA. The mouth can be rinsed with about 50 mL of
tepid water by vigorous swishing or by brushing with a tooth brush
without tooth paste. Unstimulated saliva is usually of the mucinous
type and is secreted at a slow rate. Stimulated saliva
(anticipation of tasty food, sweet or sour candy) is of the serous
(watery) type and secreted at a faster rate. It has been found (see
Table 2) that there is more DNA in 2 mL of unstimulated saliva than
2 mL of stimulated saliva. After rinsing of the mouth and waiting
about two or three minutes, the donor may spit a volume (for
example, about 2 mL) of "unstimulated" saliva into the receiving
tube. If this proves to be difficult, saliva flow can conveniently
be stimulated with a cube of table sugar, or any other such
saliva-stimulatory substance that does not interfere with DNA
recovery or purification.
TABLE-US-00002 TABLE 2 Comparison of DNA content of unstimulated
and stimulated saliva Donor #7 unstimulated stimulated Collected on
2002 Apr. 6, analyzed 36.2* 21.8* 2 days later by the DNase method
*Estimated amount of DNA in ng per .mu.L of original undiluted
saliva sample
[0081] Another aspect of the invention features a method of
reducing the viscosity of a mucin-containing bodily fluid or tissue
by reducing disulfide bonds inherent to mucin, wherein the bodily
fluid or tissue is mixed with a composition of the invention that
includes a reducing agent. In one embodiment, the bodily fluid is
sputum, desirably saliva.
[0082] Yet another aspect of the invention features a method of
recovering a nucleic acid from sputum that includes the steps of:
i) obtaining sputum from a subject, ii) contacting the sputum with
a composition of the invention to form a mixture, iii) contacting
the mixture with a protease, and iv) recovering the nucleic acid
from the mixture.
[0083] Suitable proteases include, for example, proteinase K or
pronase. The protease may suitably be in a dry form that would
become activated once mixed with sputum and a composition of the
invention. In one embodiment, the protease is deposited onto an
interior surface of the collection device. This can be accomplished
by dissolving the protease in a solution made up of equal volumes
of 5% sucrose in water and 5% glycerol in ethanol and then, after
placing the solution on the surface, removing the volatiles under a
controlled vacuum to leave the protease bound to the surface as a
sticky residue. If the composition does not contain a reducing
agent (or even if it does), a reducing agent can be added at any
time prior to isolation of the nucleic from the sample, desirably
prior to or concurrently with contacting the sample with a suitable
protease.
[0084] When sputum is mixed with a composition of the present
invention, cells are disrupted, nucleic acids are liberated from
the cells, membranous material is solubilized, proteins are
stripped from the nucleic acids, and protein digestion begins. If
present, a reducing agent in the composition reduces the viscosity
of the gel-forming mucin. Incubation can be at room temperature
over a relatively long period of time (days or weeks) while samples
are being shipped to a laboratory for analysis. If transferred to a
laboratory soon after collection, incubation at 55.degree. C. for 4
to 16 hours is sufficient to allow the activated protease to digest
the majority of protein to small peptides or amino acids. Under
such conditions, nucleic acids and polysaccharides remain
relatively intact.
[0085] Once digestion is complete, nucleic acid isolation can be
performed using any technique known in the art (Short Protocols in
Molecular Biology, 5th Edition Frederick M. Ausubel, Roger Brent,
Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith
(Editor), Kevin Struhl (Editors). ISBN: 0-471-25092-9. 2002. John
Wiley and Sons). In one example, in which SDS is used as a
denaturant component of the composition, a "precipitation solution"
consisting of, for example, potassium chloride may be added to a
portion of the sputum-composition mixture resulting in the
precipitation of potassium dodecyl sulfate, after standing on ice
to cool the solution. Following a short period of centrifugation to
remove the precipitate and any residual insoluble material, the
supernatant is collected. At this stage, the supernatant is
expected to contain as much as 10-30 nanograms per microliter of
DNA. For analyses where as little as 1 nanogram of DNA is
sufficient, the sample can be diluted.
[0086] When larger amounts of DNA are required, the DNA in the
supernatant can be precipitated by the addition of alcohol and
redissolved in any suitable buffer. This step has the effect of
removing inhibitory components of the composition, which are
present to preserve the nucleic acids during transport to the
laboratory.
[0087] If more highly purified DNA is required, then other known
purification steps can be used (Short Protocols in Molecular
Biology, 5th Edition Frederick M. Ausubel, Roger Brent, Robert E.
Kingston, David D. Moore, J. G. Seidman, John A. Smith (Editor),
Kevin Struhl (Editors). ISBN: 0-471-25092-9. 2002. John Wiley and
Sons), such as extraction with phenol or solid-phase extraction. It
should be noted that, because the DNA is in a relatively pure state
using the procedures described above, any additional purification
steps are made easier when compared to analogous purifications of
DNA originating from a blood sample.
[0088] The methods of the present invention can be used to isolate
nucleic acids from sputum for any application requiring a nucleic
acid sample. For example, some specific applications of the methods
of the present invention include, but are not limited to, forensic
applications, medical applications (including genetic screening and
disease typing), and paternity testing.
[0089] Another aspect of the invention features a method of
preserving and/or recovering a nucleic acid from a bodily fluid
that includes, placing the bodily fluid into a first region of a
container, placing a composition of the invention into a second
region of the container, which is separated from the first region
by a barrier, closing the container, and disturbing the integrity
of the barrier such that the composition and the bodily fluid are
brought into contact. Collection devices of the invention, which
also can serve as containers for bring the compositions and nucleic
acid-containing bodily fluids together are described below.
Collection Devices
[0090] The invention also provides a novel collection device useful
for collecting a biological sample from a subject, and subsequently
mixing the collected sample with a composition intended to
stabilize, preserve, or facilitate the recovery of components of
the sample. Such components may include, without limiting the
invention, nucleic acids, proteins, peptides, toxins, chitins,
fatty acids, and glycogens. Non-limiting examples of biological
samples are skin, hair, fecal matter, bodily fluids, and
tissue.
[0091] Desirably, the invention features a device for preserving
and/or recovering a nucleic acid obtained from a biological sample.
The device includes: a container that has a first region for
collecting a biological sample and a second region containing a
composition for preserving a nucleic acid, a barrier between a
first region and a second region that keeps the sample and
composition separate, a means for closing the container, and a
means for disturbing the integrity of the barrier, such that the
composition is capable of contacting the bodily sample. In one
embodiment, the composition is a composition of the present
invention. In another embodiment, the sample is a biological
fluid.
[0092] The collection device of the invention simultaneously serves
several functions. Some of the desirable features of this
collection vessel include one or more of the following:
[0093] a) it may be constructed of a sturdy breakage-resistant
plastic, desirably a biocompatible plastic. Desirably, the
container would be constructed from a material that would not leach
chemicals into the container's contents;
[0094] b) it would have a broad mouth that would make it relatively
simple for a subject to place the required volume of fluid sample,
desirably expectorated sputum, and most desirably expectorated
saliva, into the device's container;
[0095] c) the bottom part of the container would be narrow to
reduce the overall volume of the container to make it easier to
collect the small volume (1-2 milliliters) of fluid that would be
expected from a routine sampling, in particular, when the sample is
an expectorate. Optionally, the device would contain markings to
allow for an estimate of the sample volume collected;
[0096] d) the means for closing the container may be a cap that is
designed to lock once tightened to become tamper-resistant;
[0097] e) the means for closing the container may be a cap that is
designed to provide a liquid-tight and/or airtight seal for the
container once the cap is fixed into place;
[0098] f) the barrier may be a septum or plastic bag compartment
that would separate the composition from the fluid until the septum
or bag compartment is pierced or the contents otherwise
released;
[0099] g) the barrier may be in the form of a pivoting partition.
In this embodiment, attachment of the lid to the container forces
the partition to pivot from its original position of spanning the
space between the first region and the second region to a position
in which both regions are exposed to each other and contact between
the composition contained in one space and the bodily fluid
contained in the other space is allowed;
[0100] h) the barrier can be press fit, glued, or heat fit into
place;
[0101] i) the means for closing the container may be coupled to the
disestablishment of the barrier; and
[0102] j) an antimicrobial agent that coats the outside of the
device.
[0103] A device of the invention is shown in FIGS. 10 and 11. With
cap 1 not attached to the device, a biological sample (not shown)
is applied to a first region 8 of container 3, which is separated
from a second region 9 by sealing disc 7. After sample application,
cap 1 is placed onto the device and secured via a screw thread
mechanism to a tight fit, thereby sealing container 3. As the cap
is twisted on (shown by dotted line and arrow 10, ram 2, which is
attached to cap 1, moves downward as shown by dotted line arrow 11.
This downward movement forces plunger 4, which is contained in
plunger barrel 5, downward as indicated by dotted line and arrow
12. The downward movement of plunger 4 forces sealing disc 7 to
pivot, as shown by dotted line and arrow 13. Pivoting of disc 7
disestablishes the barrier between regions 8 and 9, thereby
permitting contact between the sample and a composition of the
invention, shown as a dotted solution contained in region 9.
Kits
[0104] The present invention also features kits for performing the
methods of the invention that include a device of the invention
containing a composition of the invention, with instructions for
stabilizing, preserving, or facilitating the recovery of nucleic
acids from a biological sample by using the device to bring a
biological sample into contact with the composition.
EXAMPLES
Example 1
Protocol for Obtaining Saliva Samples from Subjects Capable of
Following Instructions
[0105] The subject is instructed to wait for a period of 20-30
minutes before last eating. The subject will brush his teeth
without using toothpaste, if possible. The subject will rinse his
mouth vigorously with 50 mL of cool or tepid water. The subject
will then spit saliva into the special collection tube until the
level of saliva reaches the 2 mL mark. This may take several
minutes. If the subject finds that he is unable to deliver
sufficient saliva, he will be given a cube of table sugar to chew,
and told not to be concerned if some of the sugar is spit into the
tube.
[0106] When the required amount of saliva is collected, it is mixed
with 2 mL of a nucleic acid-preserving composition. The precise way
this will be introduced will depend upon the container design.
[0107] Once the composition is introduced, the cap is attached to
the container and tightened to seal it securely. The container is
then vigorously shaken and the process is complete. The DNA is now
in an intermediate preserved state. It can be maintained in a
frozen state or at any temperature up to about 60.degree. C.
[0108] The container can be mailed back to the testing lab at room
temperature.
Example 2
Protocol for Obtaining Saliva Samples from Babies, Very Young
Children and Infirm Adults Incapable of Following Instructions
[0109] A rubber or plastic tube or nipple will be introduced into
the mouth, attached to a sponge, suction bulb or small syringe, and
kept in the mouth for several minutes until visible drooling
occurs. A bit of sugar cube will be placed in the mouth to
stimulate saliva if necessary. The responsible adult will wear
disposable gloves provided for the purpose to avoid contamination
with his/her DNA. The responsible adult will draw saliva into the
bulb or syringe and transfer it into the collection container. The
DNA preserving/extraction composition is introduced and the
container is capped and sealed. The tube is vigorously shaken for 1
minute.
Example 3
Preparation of a Nucleic Acid-Preserving Composition
[0110] The composition of the nucleic acid-preserving solution used
in Examples 4-6 is 33 mM TRIS-HCl, 0.67 M urea, 0.67 M LiCl, 0.6%
sodium dodecyl sulfate, 3.3 mM CDTA, 30% ethanol, and 0.25 M sodium
ascorbate, all adjusted to a final pH of 8.0. In the examples, the
composition is mixed with an equal volume of saliva. Subsequent to
these experiments, it has been found that a composition which is
0.3 M TRIS-HCl, 0.67 M urea, 0.67 M NaOAc, 0.6% sodium dodecyl
sulfate, 3.3 mM CDTA, 30% ethanol, and 0.1 M sodium ascorbate, all
adjusted to a final pH of 8.0, stabilizes DNA for longer periods of
time.
Example 4
Extration of Minimally Purified Chromosomal DNA from the Stimulated
Saliva of 8 Different Donors
[0111] After collection of saliva in an equal volume of the
composition as noted in Example 3, followed by 14 days storage at
room temperature, a 0.25 mL portion of each donor's sample was
treated with proteinase K, centrifuged briefly to remove insoluble
material and the DNA therein was precipitated with 2 volumes of
ethanol. The precipitate was dissolved in 0.05 mL of water, and an
8 .mu.L aliquot (equivalent to about 20 .mu.L of undiluted saliva)
was analyzed by electrophoresis on a 0.8% agarose gel, stained with
ethidium bromide to visualize the DNA (see FIG. 1). Of note is the
characteristic band of chromosomal DNA present in all samples at
the position of the arrow, that corresponds to the position of
chromosomal DNA extracted from white blood cells (data not
shown).
Example 5
"Real Time" Polymerase Chain Reaction Using DNA from Stimulated
Saliva
[0112] Stimulated saliva samples collected on 26 Feb. 2002 (see
Table 1) and stored at room temperature were analyzed 62 days
later. Minimally purified DNA was prepared as follows: an aliquot
was centrifuged to remove insoluble material; to the clarified
supernatant was added 2 volumes of ethanol; the precipitate
containing DNA was collected by centrifugation and redissolved in
water. A volume of the redissolved DNA equivalent to 0.05
microliters of each of the original saliva samples was used for
analysis. Real time PCR was carried out using a Roche Light Cycler
instrument, where the fluorescent dye SYBR green I was added to
follow the reaction (see results of FIG. 2). The primers were
designed to detect the human Clotting Factor IX gene (Grant, et
al., J. Immunol Methods 225:61-6, 1999). C=control, highly purifed
white blood cell DNA. Each curve represents results using saliva
DNA from different donors, represented by a number. These results
using real time PCR demonstrate the suitability of minimally
purified saliva DNA from different donors for PCR analysis.
Example 6
"Real Time" Polymerase Chain Reaction Using DNA from Unstimulated
Saliva
[0113] FIG. 3 is a graph showing saliva DNA samples collected on
2002 Mar. 25 (see Table 1) and analyzed on 30 days later in
accordance with FIG. 1. Minimally purified DNA was used Polymerase
chain reaction and other conditions as described in Examples 4 and
5 except saliva collection was done under unstimulated conditions.
Numbers refer to individual donors. C is control DNA, a highly
purified sample of DNA purified from blood.
[0114] Tables 1 and 2 show estimates of DNA recovered from saliva
samples. In all cases, the individual donor has been identified by
a unique number. These data show that the amount of DNA that can be
recovered from this group of donors ranges from 16 micrograms per
milliliter of saliva and higher. Estimation of the amount of DNA by
chemical methods such as DABA presents some problems and the DNase
method provides most reliable results.
Example 7
Stability Studies on DNA from Saliva
[0115] Saliva was mixed with an equal volume of the indicated
composition and the mixture was incubated for the indicated time
period at the indicated temperature (see Table 3). After
incubation, approximately 40 .mu.L of mixture was digested briefly
with ribonuclease to remove the majority of the RNA present in the
sample, then applied to the indicated lane of a 0.8% agarose gel.
Following electrophoresis, the gel was stained with ethidium
bromide as in Example 4.
TABLE-US-00003 TABLE 3 Lane Incubation No. Composition Conditions 1
0.5M NaOAc, 0.2M TRIS-HCl, 0.15M 70.degree. C. for 3 days, then Na
ascorbate, 10 mM CDTA, 1% SDS, 50.degree. C. for 16 days 30% (v/v)
ethanol, pH = 9.5 2 0.5M NaOAc, 0.2M TRIS-HCl, 10 mM 50.degree. C.
for 21 days CDTA, 1% SDS, 30% (v/v) ethanol, pH = 9.5 3 0.5M NaOAc,
0.2M TRIS-HCl, 10 mM 70.degree. C. for 3 days, then CDTA, 1% SDS,
30% (v/v) ethanol, 50.degree. C. for 31 days pH = 9.5 4 0.67M LiCl,
33 mM TRIS-HCl, 0.67 M 20.degree. C.-25.degree. C. for urea, 0.6%
SDS, 3.3 mM CDTA, 30% 15 months (v/v) ethanol, pH = 8.0 5 0.67M
LiCl, 33 mM TRIS-HCl, 0.67 M 20.degree. C.-25.degree. C. for urea,
0.6% SDS, 3.3 mM CDTA, 30% 15 months (v/v) ethanol, pH = 8.0 6
Control chromosomal DNA prepared from white blood cells
Example 8
Rapid Autooxidation of Ascorbate in the Presence of a Transition
Metal Ion
[0116] A solution of sodium ascorbate (100 .mu.M) in CB (10 mM BES,
pH 7.4, containing 1 mM CDTA) was freshly prepared under aerobic
(equilibrated with ambient air) conditions. Several
spectrophotometric scans over 30 minutes at room temperature showed
no change in the absorbance profile (all similar to scan (1)). Scan
(2) was taken 3 minutes after addition of a few crystals of
MnCl.sub.2. The results can be seen in FIG. 6. As shown, 100 .mu.M
ascorbate at neutral pH has an absorbance (.lamda.max=265 nm) of
about 1.25 (corresponding to the expected molar extinction
coefficient (A.sub.M) of about 12,500. Upon addition, the
transition metal, manganous chloride, catalyzed the autooxidation
of ascorbate, which can conveniently be monitored by a decrease in
absorbance at .lamda.=265 nm (Buettner, Free Radic. Res. Commun.
10:5-9, 1990).
Example 9
Spontaneous Autooxidation of Ascorbate
[0117] Repeated scans at the indicated time points were taken of an
aliquot of the 100 .mu.M sodium ascorbate solution prepared in
Example 8, before the addition of MnCl.sub.2. The sample was
exposed to air and maintained at room temperature between scans.
The results are illustrated in FIG. 7, and indicate that
autooxidation of ascorbate occurs at pH 7.4 can occur over an
extended period of time in the presence of low concentrations (1
mM) of CDTA, a "strong" chelator.
Example 10
Stability of Sodium Ascorbate in a Nucleic Acid-Preserving
Composition
[0118] A stock solution of sodium ascorbate (250 mM) was prepared
in a solution containing 30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM
CDTA. 20 .mu.L was removed at the indicated times, mixed with 50 mL
of CB (see Example 8) and the absorbance at 265 nm was read
immediately. The stock solution was maintained at room temperature.
The results are shown in FIG. 8.
[0119] While the present invention has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the invention is not limited
to the disclosed examples. To the contrary, the invention is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims.
[0120] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
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