U.S. patent application number 12/758708 was filed with the patent office on 2010-10-28 for compositions and method for storage of nucleic acid from bodily fluids.
This patent application is currently assigned to DNA GENOTEK INC.. Invention is credited to Chaim H. BIRNBOIM, Joanne Chartier, Rafal Iwasiow, Adele Jackson, Paul Lem.
Application Number | 20100273218 12/758708 |
Document ID | / |
Family ID | 36991239 |
Filed Date | 2010-10-28 |
United States Patent
Application |
20100273218 |
Kind Code |
A1 |
BIRNBOIM; Chaim H. ; et
al. |
October 28, 2010 |
COMPOSITIONS AND METHOD FOR STORAGE OF NUCLEIC ACID FROM BODILY
FLUIDS
Abstract
The present invention provides an aqueous composition comprising
SDS, Cyclohexanediamine tetraacetate, Tris-HCl and proteinase K for
the extraction of nucleic acid from a sample of bodily fluid, such
a saliva, wherein the extracted nucleic acid is stable for at least
fourteen days at room temperature The composition permits direct
use of the extracted and stored DNA in an amplification reaction
without further processing.
Inventors: |
BIRNBOIM; Chaim H.; (Ottawa,
CA) ; Jackson; Adele; (Stittsville, CA) ;
Iwasiow; Rafal; (Ottawa, CA) ; Chartier; Joanne;
(White Lake, CA) ; Lem; Paul; (Ottawa,
CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
DNA GENOTEK INC.
Ottawa
CA
|
Family ID: |
36991239 |
Appl. No.: |
12/758708 |
Filed: |
April 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11886360 |
Mar 28, 2008 |
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PCT/CA06/00380 |
Mar 14, 2006 |
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12758708 |
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60662510 |
Mar 16, 2005 |
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Current U.S.
Class: |
435/91.2 ;
435/219 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12N 15/1003 20130101 |
Class at
Publication: |
435/91.2 ;
435/219 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C12N 9/50 20060101 C12N009/50 |
Claims
1. An aqueous composition for extracting and preserving nucleic
acid from a sample of a bodily fluid such that said nucleic acid
within said sample remains stable for at least 14 days at room
temperature following mixture with said aqueous composition, said
aqueous composition comprising: (i) a denaturing agent (ii) a
chelator; (iii) a buffering agent at a pH of about pH 7 to about pH
11; and (iv) a protease; wherein said composition does not inhibit
nucleic acid amplification when said composition is added to an
amplification reaction mixture at an amount of at least 2%
(vol./vol.) of the total volume of said amplification reaction
mixture, and wherein the final concentration of said denaturing
agent in said amplification reaction mixture is less than about
0.01% (wt./vol.).
2. The aqueous composition of claim 1, wherein the bodily fluid is
saliva.
3. The aqueous composition of claim 1, wherein said denaturing
agent is SDS, said chelator is CDTA, said buffering agent is
TrisHCl, and said protease is proteinase K.
4. The aqueous composition of claim 3, which comprises 0.05% SDS,
20 mM CTDA, 200 mM TrisHCl pH 8.0, 400 mM NaOAc, and 10 .mu.g/ml
proteinase K.
5-9. (canceled)
10. The aqueous composition of claim 1, wherein said composition
does not inhibit nucleic acid amplification when added to said
amplification reaction mixture in an amount of between 2% (vol.)
and 10% (vol.) of the total volume of the amplification reaction
mixture.
11. The aqueous composition of claim 1, wherein said nucleic acid
remains stable at room temperature for at least three hundred and
sixty-five days.
12. A method of amplifying deoxyribonucleic acid (DNA) DNA directly
from a bodily fluid, comprising: (a) mixing a sample of the bodily
fluid with the aqueous composition of claim 1; and (b) diluting at
least 2% (vol./vol.) of the mixture formed in step (a) in an
amplification reaction mixture without prior extraction of nucleic
acid molecules present in said mixture and subjecting said
amplification reaction mixture to polymerase chain reaction (PCR)
amplification, wherein said PCR amplifies said nucleic acid
molecules.
13. The method of claim 12, wherein the bodily fluid is saliva.
14. The method of claim 12, wherein the mixture formed in step (a)
is stored at room temperature for at least fourteen days prior to
step (b).
15. The method of claim 12, wherein the mixture formed in step (a)
is stored at room temperature for at least three hundred and
sixty-five days prior to step (b).
16. The method of claim 12, additionally comprising the step of
heating the mixture at a temperature of from about 45.degree. C. to
80.degree. C. for about 15 to 60 minutes prior to step (b).
17. A kit for amplifying deoxyribonucleic acid (DNA) from a bodily
fluid, comprising: (a) the aqueous composition of claim 1; and (b)
instructions for the use thereof.
18. The kit of claim 17, wherein said kit is capable of use with
saliva as the bodily fluid.
19. A method of amplifying deoxyribonucleic acid (DNA) directly
from a bodily fluid, comprising: (a) obtaining a sample comprising
a bodily fluid admixed with a composition comprising: (i) a
denaturing agent; (ii) a chelator; (iii) a buffering agent that
establishes a pH of said sample of about pH 7 to about pH 11; and
(iv) a protease, wherein nucleic acid molecules within said sample
remain stable for at least 14 days at room temperature; (b)
preparing an amplification reaction mixture comprising at least 2%
(vol./vol.) of the sample, wherein said amplification reaction
mixture is prepared without prior extraction of said nucleic acid
molecules from said sample and the final concentration of said
denaturing agent in said amplification reaction mixture is less
than about 0.01% (wt./vol.); and (c) subjecting said amplification
reaction mixture to polymerase chain reaction (PCR) amplification,
wherein said PCR amplifies said nucleic acid molecules.
20. The method of claim 19, wherein the bodily fluid is saliva.
21. The method of claim 19, wherein the sample is stored at room
temperature for at least fourteen days prior to step (b).
22. The method of claim 19, wherein the sample is stored at room
temperature for at least three hundred and sixty-five days prior to
step (b).
23. The method of claim 19, additionally comprising the step of
heating the sample at a temperature of from about 45.degree. C. to
80.degree. C. for about 15 to 60 minutes prior to step (b).
24. The aqueous composition of claim 1 further comprising said
bodily fluid.
25. The aqueous composition of claim 24, wherein said bodily fluid
is saliva.
26. The aqueous composition of claim 1, wherein said protease is
added with, or after addition of, said bodily fluid.
Description
FIELD OF THE INVENTION
[0001] The field of the invention generally relates to compositions
and methods for isolation and storage of nucleic acids from bodily
fluids, such as saliva, for detection of nucleotide sequences. More
specifically, the invention relates to compositions and methods
that do not require a separate step for extraction of nucleic acids
prior to use in nucleic acid amplification reactions.
BACKGROUND
[0002] Molecular-based techniques involving the amplification of
nucleic acids are increasingly being used in forensics, law
enforcement, the military, human medicine, veterinary medicine, and
research. 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. The use of
DNA-based tests is expected to increase in medicine, for example,
in 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. One technique
for the amplification of DNA is the polymerase chain reaction
(PCR).
[0003] PCR is a rapid, inexpensive and relatively simple means of
amplifying copies of DNA molecules from a variety of source
materials. However, a limitation of PCR is that DNA source
materials typically contain a variety of inhibitors, such as
pigments, proteins, saccharides and/or other impurities that
interfere with the amplification reaction. For example, a variety
of DNA polymerases, including Taq DNA polymerase (a typical
thermostable DNA polymerase derived from Thermus aquaticus) are
known to be inhibited by traces of body fluid-derived impurities in
a PCR mixture. To overcome the problem of inhibitors within the DNA
source material, there is typically a requirement for the
purification of the DNA from the source material prior to
amplification. However, purification procedures often involve
multiple steps that can be time-consuming and expensive.
[0004] DNA can be extracted from nearly every type of cell in the
human body and from a variety of cell-containing bodily fluids. The
term "bodily fluid", as used herein, can refers to a naturally
occurring fluid from an animal, such as saliva, sputum, serum,
plasma, blood, urine, mucus, gastric juices, pancreatic juices,
semen, products of lactation or menstruation, tears, or lymph. A
typical source of bodily fluid for extraction of DNA is white blood
cells in venous blood. However, the use of blood as a source of DNA
has many disadvantages. Collection of blood is not a trivial
procedure. Taking of venous blood requires trained personnel.
Furthermore, it is an invasive procedure, which frequently causes a
degree of distress and pain to the donor. Precautions are needed to
minimize exposure of blood collecting personnel to blood-borne
pathogens. Once collected, the blood sample must be either frozen
or quickly transported to a laboratory for extraction 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 haeme derived from haemoglobin in red blood
cells can inhibit some types of DNA analysis.
[0005] Swabbing the inside of the cheek with a brush (a buccal
swab) is another method of obtaining cells that contain DNA. This
procedure is much less invasive than taking blood and permits
collection by individuals with less training than is required in
the collection of blood. Once collected, the time that useable DNA
can be recovered is relatively short. Microbes in the mouth can
degrade the DNA. However, this time can be extended by either
drying the swab or wiping it onto filter paper and drying it.
[0006] 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 mucins, and digestive
enzymes.
[0007] 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 viscous nature of saliva.
[0008] 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 small 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 by van Schie, et al. (van Schie et al., (1997) J.
Immunol. Methods. 208: 91-101). van Schie et al. 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. Terasaki et al. (Terasaki et al. (1998) Hum Immunol.
59: 597-598) 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.
[0009] There are significant advantages to providing a saliva
sample rather than a blood sample as a source of DNA. Donors
generally 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. However, it will be clear to the
skilled worker that while saliva is a preferred source of DNA,
other bodily fluids, including blood, can be used.
[0010] More recently, it has been discovered that saliva can be
used directly for real-time PCR without any DNA purification
procedure. French et al. (French et al. (2002) Molecular And
Cellular Probes. 16: 319-326) diluted fresh whole saliva 1:1 with
water and used this mixture immediately, or following storage at
4.degree. C. (2-3 days) or -20.degree. C., for real-time PCR with a
LightCycler instrument (Roche Diagnostics). PCR reaction volumes
were typically 20 .mu.l, containing 2 .mu.l of saliva (diluted to
50% in water). The calculated concentration of DNA available for
PCR was found to be between 0.1 ng/.mu.l and 3.5 ng/.mu.l, varying
between samples obtained from different individuals and on
different days. The authors commented that the amount of DNA
available for amplification in crude saliva may not account for all
of the DNA present in saliva samples, where a quantity of the total
DNA may still reside within buccal cells or be too degraded to
permit target amplification. A significant reduction in assay
efficiency was not observed with saliva samples stored at 4.degree.
C. (2-3 days) or -20.degree. C.
[0011] With the increasing use of DNA-based analysis in forensics,
law enforcement, military, human medicine, veterinary medicine, and
research, there is a need for compositions and methods that allow
bodily fluids such as saliva to become a standard reliable source
of DNA from an individual (to replace blood, the current standard).
Desirably, it would be possible to use such compositions and
methods for detecting DNA without requiring a separate step for
extraction and purification of DNA from the saliva. Furthermore, it
would be desirable to be able to store the bodily fluid at ambient
temperature for several days. This would be especially advantageous
when shipping of the saliva sample is required and/or a source of
refrigeration is not available. In addition, it would be desirable
if the concentration of genomic DNA in the saliva sample was high
enough for both traditional PCR and real-time PCR without requiring
additional steps. Traditional PCR usually requires DNA template in
amounts>10 ng. According to the Roche Molecular Biochemicals PCR
Application Manual (2.sup.nd edition, 1999, Roche Diagnostics),
traditional PCR with low amounts of template (<10 ng human
genomic DNA) requires special reaction modifications, such as
changes in cycle number, redesign of primers, use of "Hot Start",
etc. In contrast, real-time PCR is much more sensitive than
traditional PCR. For example, the LightCycler real-time PCR
instrument (Roche Diagnostics) has 100% sensitivity for detecting
30 pg of control human genomic DNA (LightCycler Control Kit DNA
manual, version 3, 2003).
[0012] This background information is provided for the purpose of
making known information believed by the applicant to be of
possible relevance to the present invention. No admission is
necessarily intended, nor should be construed, that any of the
preceding information constitutes prior art against the present
invention.
SUMMARY OF THE INVENTION
[0013] An object of the present invention is to provide a
composition and method for storage of DNA from a bodily fluid such
as saliva, that permits direct use of the DNA sample in a nucleic
acid amplification reaction.
[0014] In accordance with one aspect of the invention, there is
provided an aqueous composition for extracting nucleic acid from a
sample of a bodily fluid such that said nucleic within said sample
remains stable for at least 14 days at room temperature, wherein
said composition does not inhibit nucleic acid amplification when
said composition is added to an amplification reaction mixture at
an amount of at least 2% of the total volume of said amplification
reaction mixture.
[0015] In accordance with another aspect of the invention, there is
provided a method of amplifying DNA directly from a bodily fluid,
comprising: (a) mixing a sample of the bodily fluid with an equal
volume of an aqueous composition according to the present
invention; (b) subjecting a portion of the mixture formed in step
(a) to PCR amplification.
[0016] In accordance with another aspect of the invention, there is
provided a kit for amplifying DNA from a bodily fluid, comprising:
(a) an aqueous composition according to the present invention; and
(b) instructions for the use thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows an agarose gel stained with ethidium bromide
following electrophoresis of PCR products and depicts direct
amplification of DNA from saliva mixed with an aqueous composition
and stored at room temperature for one day, fourteen days, or 365
days. Prior to PCR, the samples were incubated at 60.degree. C. for
1 hour. The contents of the lanes in the agarose gel are as
follows:
TABLE-US-00001 Lane # Description 1 100-bp marker 2 Day 1 3 Day 14
4 Day 365 5 100-bp marker
[0018] FIG. 2 shows an agarose gel stained with ethidium bromide
following electrophoresis of PCR products and depicts direct
amplification of DNA from saliva mixed with an equal volume of the
aqueous composition shown in the following table following storage
at room temperature for 1 day, 7 days, 14 days, 21 days, or 365
days. The contents of the lanes in the agarose gel are as
follows:
TABLE-US-00002 Lane # Description 1 100-bp marker 2 Water 3 100 mM
sodium hydroxide 4 100 mM potassium hydroxide 5 200 mM sodium
carbonate 6 100-bp marker
[0019] FIG. 3 shows an agarose gel stained with ethidium bromide
following electrophoresis of PCR products and depicts the effect of
graded concentrations of NaOH on a PCR amplification reaction
containing purified human DNA. The contents of the lanes in the
agarose gel are as follows:
TABLE-US-00003 Final concentration of Lane # NaOH (mM) 1 100-bp
marker 2 0 3 1 4 2 5 4 6 6 7 8 8 10 9 12 10 14 11 100-bp marker
[0020] FIG. 4 shows an agarose gel stained with ethidium bromide
following electrophoresis of PCR products and depicts the effect of
increasing volumes of a NaOH/saliva mixture on a 50 .mu.L PCR
amplification reaction. The contents of the lanes in the agarose
gel are as follows:
TABLE-US-00004 Volume of NaOH plus Lane # saliva (.mu.L) 1 100-bp
marker 2 0 3 1 4 2 5 3 6 4 7 5 8 6 9 7 10 8 11 9 12 10 13 12 14 14
15 16 16 18 17 20 18 100-bp marker
[0021] FIG. 5 shows an agarose gel stained with ethidium bromide
following electrophoresis of PCR products and depicts the
inhibitory effect of Oragene.TM. compared to other compositions on
a PCR amplification reaction. The contents of the lanes in the
agarose gel are as follows:
TABLE-US-00005 Lane # Description 1 100-bp marker 2 Water 3 100 mM
sodium hydroxide 4 100 mM potassium hydroxide 5 200 mM sodium
carbonate 6 Oragene solution 7 100-bp marker
DETAILED DESCRIPTION OF THE INVENTION
[0022] As will be described in more detail below, the present
invention relates to aqueous compositions and methods for
extraction and storage of DNA from bodily fluids such as saliva,
wherein the DNA in the resulting composition remains stable for at
least fourteen days at room temperature. The composition of the
present invention permits DNA released from saliva, or other bodily
fluid, to be used directly in nucleic acid amplification reactions
without any additional processing steps.
[0023] The term "about", as used herein, refers to +/-10% of the
stated value of a chemical or obvious equivalent thereof.
[0024] The term "bodily fluid", as used herein, refers to a
naturally occurring fluid from an animal, such as saliva, sputum,
serum, plasma, blood, urine, mucus, gastric juices, pancreatic
juices, semen, products of lactation or menstruation, tears, or
lymph.
[0025] The term "mucin", as used herein, refers to any mucoprotein
that raises the viscosity of the medium surrounding the cells that
secrete it.
[0026] The term "mucoid", as used herein, refers to any bodily
fluid containing mucin.
[0027] The term "nucleic acid", as used herein, refers to a chain
of nucleotides, including deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), typically found in nature in chromosomes,
chromatin, mitochondria, cytoplasm, ribosomes, bacteria, fungi
and/or viruses.
[0028] The term "DNA polymerase", as used herein, refers to an
enzyme that catalyzes a deoxyribonucleic acid synthesis via a
primer binding and subsequent incorporation of nucleotides.
Suitable polymerases include, but are not limited to, DNA
polymerase I derived from E. coli, the Klenow fragment of DNA
polymerase derived from E. coli, T4 DNA polymerase, Taq DNA
polymerase, T. litoralis DNA polymerase, Tth DNA polymerase and Pfu
DNA polymerase.
[0029] The term "primer", as used herein, refers to an
oligonucleotide acting as a starting point from which the synthesis
begins in the presence of a DNA template, reagents for
polymerization and so on. Although a primer is preferably
single-stranded, double-stranded primers may also be used. When
double-stranded primers are used, it is desirable to convert them
into their single-stranded forms before use in an amplification
reaction. A primer may be synthesized using well known methods, or
may be isolated from an organism.
[0030] The term "saliva", as used herein, refers to the secretion,
or combination of secretions, from any of the salivary glands,
including the parotid, submaxillary, and sublingual glands,
optionally mixed with the secretions from the numerous small
labial, buccal, and palatal glands that line the mouth.
[0031] The term "sputum", as used herein, refers to 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.
[0032] The term "stable", as used herein to describe nucleic acid
within a saliva sample, refers to the ability of that nucleic acid
to support PCR amplification into a detectable product. For
example, nucleic acid within a sample of saliva is said to remain
stable for at least fourteen days if a PCR product is obtainable at
least fourteen days after being mixed with the aqueous composition
of the present invention.
[0033] The term "subject", as used herein, refers to an animal or
human. Desirably, the subject is a mammal that can produce saliva
for the purposes of nucleic acid detection. Most desirably, the
subject is human.
Composition
[0034] The composition of the present invention is for extracting
nucleic acid from a bodily fluid and maintaining the nucleic acid
contained therein stable for at least fourteen days at room
temperature. In a preferred embodiment, the bodily fluid is saliva.
The composition of the present invention further permits direct use
of the extracted nucleic acid in an amplification reaction without
further processing of the nucleic acid-containing composition. In
particular, the components of the composition are at a
concentration sufficiently low to permit nucleic acid amplification
when a portion that constitutes at least 2% of the total reaction
volume is added to an amplification reaction. The term "processing"
as used herein refers to mechanical or chemical steps used to
isolate or purify nucleic acid from a storage composition.
[0035] Selection of the specific components of the composition is
made based on various criteria, including, for example, cost,
availability, downstream application, and safety. As would be
readily appreciated by a worker skilled in the art, the
concentration of the components is sufficiently high to stabilize
the bodily fluid-derived nucleic acid for at least fourteen days at
room temperature, while not interfering with the direct use of a
portion of the nucleic acid-containing composition in an
amplification reaction. For example, when a sample of saliva is
mixed 1:1 (v/v) with the composition of the present invention, no
inhibition of nucleic acid amplification is observed when the
composition consists of 100 mM NaOH in water and an amount of the
composition/bodily fluid is added to the amplification reaction
such that it constitutes from 20% of the total reaction volume
(i.e., the composition of the present invention constitutes from 2%
to 20% of the total volume).
[0036] A major cause of nucleic acid instability in biological
samples is the presence of deoxyribonucleases and ribonucleases.
Deoxyribonucleases and ribonucleases are enzymes that break down
DNA or RNA, respectively. Their main source in the digestive tract
is secretions of the pancreas, although lower levels may be present
in saliva and in cells of the salivary gland and buccal mucosa. In
addition, microorganisms resident in the mouth or from recently
ingested foods may release deoxyribonucleases or ribonucleases.
Over time, the nucleic acid within a sample of saliva stored in
water would be expected to degrade or break down into smaller
fragments.
[0037] Desirably, the composition provides inhibition of nucleases,
including deoxyribonucleases, and chemical stabilization of nucleic
acids. Nuclease inhibition is achieved through the use of
denaturing agents, proteases and/or heating. Chemical stabilization
of the nucleic acids in saliva sample is achieved through the use
of pH buffers to maintain an appropriate pH, and/or 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.
[0038] The action of deoxyribonucleases and ribonucleases can be
inhibited by denaturing agents that destroy the complex structures
of these enzymes (proteins). Hence, denaturing agents can be
included in the composition of the present invention. A
non-limiting example of a suitable denaturing agent is sodium
dodecyl sulfate (SDS).
[0039] Low specificity proteases, such as proteinase K, are
frequently used to digest proteins. In one embodiment of the
present invention, a protease is added to the composition before,
after or at the time of mixing with saliva. Since the proteases are
themselves proteins, their action can be inhibited by denaturing
agents. Thus, a balance must be struck between the concentration of
the 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.
[0040] The method of the present invention optionally includes the
step of heating the saliva-containing mixture. Heating of saliva
samples (in the range 45 to 90.degree. C.) can act as a
`reversible` denaturing agent, particularly when used in
combination with denaturing agents. That is, it has been found that
a lower concentration of a chemical denaturing agent may be used if
combined with the denaturing action of heat. Optionally, a heating
step is included in the method of the present invention prior to
the amplification step in order to improve the release of DNA and
improve the yield of the amplification reaction. It should be
appreciated that this optional heating step does not constitute a
"processing" step for isolation or purification of the nucleic acid
with the sample.
[0041] 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, one embodiment
of the present invention provides a composition comprising one or
more chelators that can form complexes with metal ions to prevent
them from binding to DNA, remove metal ions that have already bound
to DNA, or bind metal ions strongly enough to inhibit their redox
cycling, and hence, the formation of reactive oxygen species. The
amount or concentration of chelator will depend upon the strength
of the chelator, which would need to be determined empirically.
Cyclohexanediamine tetraacetate (CDTA) is a commonly used chelator.
In a specific example, 20 mM CDTA is used; however, the skilled
worker would appreciate and readily determine other concentrations
of CDTA that would be appropriate.
[0042] The chemical backbone and the purine bases of DNA are
understood to be most stable at slightly alkaline pH, with an
optimal stability generally recognized as being within a pH range
of about 7-11, and desirably at a pH of about 8. Below a pH of
about 6, depurination (i.e., spontaneous loss of purine bases from
the deoxyriboside phosphate backbone) can occur. Above a pH of
about 10, spontaneous loss of amino groups from cytosine 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, in
one embodiment of the present invention, the pH of the composition
is maintained by including a buffering agent, desirably those that
best control the pH within the range of about 5 to 11. One
non-limiting example of a suitable buffering agent in the pH range
6.5 to 9.5 is Tris hydrochloride.
[0043] In accordance with one embodiment of the present invention,
the composition comprises a denaturing agent and optionally
proteinase K, a chelator and a pH buffer to maintain the pH within
the range of 7-11.
[0044] In accordance with an alternative embodiment of the present
invention, the composition does not contain a denaturing agent, a
proteinase K, a chelator or a pH buffer. Rather, inhibition of
nucleases and extraction and storage of DNA is achieved by
maintaining a basic pH.
[0045] Surprisingly, it has been found that a mixture of a bodily
fluid, such as saliva, with an aqueous composition containing a
basic agent, for example, an alkali metal hydroxide, a soluble
alkaline earth metal hydroxide, an alkali metal oxide or an organic
base can release DNA from the bodily fluid such that the released
DNA can function as a template in a PCR reaction. While not wishing
to be bound by theory, it is believed the composition achieves
denaturation of proteins, inhibition of nucleases and extraction
and storage of DNA by maintaining a basic pH. In selecting the
appropriate base it is understood that a base containing a metal
that is reactive with DNA, or that would interfere with the DNA
polymerase employed in the amplification reaction, would not be
suitable for use in the composition of the present invention.
[0046] In accordance with a specific embodiment of the present
invention, the composition for use in extracting and storing DNA
from saliva is a sodium hydroxide (NaOH) solution containing from
50 mM to 400 mM.
[0047] As noted above, in order to be used directly in an
amplification reaction, it is necessary to ensure that the
concentration of the various components of the composition of the
present invention are such that they do not inhibit the
amplification reaction. One example of a method for determining the
interference of a component on an amplification reaction is the
inclusion of graded concentrations of the composition within the
amplification reaction to determine the maximum amount of
composition that can be added to the reaction without inhibiting
the reaction. A second example of such a methodology includes first
combining the composition and the saliva, then including graded
volumes of the saliva/composition mixture within the PCR
amplification reaction to determine the maximum amount of the
saliva/composition mixture to be added to the reaction without
inhibiting the reaction.
[0048] Once a determination is made as to the amount of a component
that can be tolerated in a PCR, or other amplification reaction,
that information is used to calculate the amount to be included in
the composition of the present invention.
Method
[0049] In accordance with another aspect of the present invention,
there is provided a method for storing and amplifying a nucleic
acid sample derived from saliva. The method comprises the steps of
mixing a sample of saliva with the composition of the present
invention, subsequently mixing an aliquot of the resulting
saliva-containing mixture with an amplification reaction mixture
and amplifying the nucleic acid within the aliquot of the
saliva-containing mixture.
[0050] To collect saliva from a 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 toothbrush
without toothpaste. 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 that
there is more DNA in 2 ml of unstimulated saliva than in 2 ml of
stimulated saliva. After rinsing of the mouth and waiting about two
or three minutes for the mouth to clear of water, the donor may
spit a volume (for example, about 1 ml) of "unstimulated" saliva
into the receiving tube. If this proves to be difficult, saliva
flow can conveniently be stimulated with a few grains of table
sugar, or any other such saliva-stimulatory substance that does not
interfere with DNA stability or subsequent amplification.
[0051] The method of the present invention optionally includes the
step of heating the saliva-containing mixture. Heating of saliva
samples (in the range 45 to 90.degree. C.) can act as a
`reversible` denaturing agent, particularly when used in
combination with denaturing agents. That is, it has been found that
a lower concentration of a chemical denaturing agent may be used if
combined with the physical denaturing action of heat. Therefore, an
optional heating step is included in the method of the present
invention prior to the amplification step in order to improve the
release of DNA and improve the yield of the amplification
reaction.
[0052] Methods of the invention are conveniently practiced by
providing the compositions used in such method in the form of a
kit. Such a kit preferably contains appropriate solutions, enzymes,
salts or detergents (or equivalents thereof). At least one type of
positive standard may be provided and can include either nucleic
acid (DNA or RNA) template useful in the detection of a target gene
or DNA, or primers.
[0053] Optionally the kit includes a container, such as that
described in International PCT Application No. WO 03/104251, which
contains the composition of the present invention and that is
suitable for saliva collection.
[0054] To gain a better understanding of the invention described
herein, the following examples are set forth. It should be
understood that these examples are for illustrative purposes only.
Therefore, they should not limit the scope of this invention in any
way.
EXAMPLES
Example 1
Protocol for Obtaining Saliva Samples from Subjects Capable of
Following Instructions
[0055] The subject is instructed to wait for a period of 30-60
minutes before last eating. The subject will brush his teeth
without using toothpaste, if possible. The subject will rinse
his/her mouth with 50 ml of cool or tepid water. The subject will
be requested to wait for 2 minutes to allow the mouth to clear of
water, then spit saliva into the special collection tube until the
level of saliva reaches the 1 ml mark. Waiting after last eating
and rinsing the mouth is desirable but not essential. Collection of
saliva may take several minutes. If the subject finds that he/she
is unable to deliver sufficient saliva, he/she will be given a few
grains of table sugar to chew, and told not to be concerned if some
of the sugar is spit into the tube.
[0056] Where two or more samples are to be taken from a donor for
purposes of comparing two compositions, the donor is asked to
deliver small amounts of saliva alternating between two or more
tubes until each tube is filled to the 1 ml mark. This is necessary
because the composition of saliva can vary during the process of
spitting.
[0057] When the required amount of saliva is collected, it is mixed
with 1 ml of an aqueous composition. The precise way in which this
will be introduced will depend upon the container design. Once the
aqueous composition is introduced, the container is securely
capped. The DNA-containing sample can be maintained at room
temperature for at least fourteen days. A portion of the
DNA-containing sample in aqueous solution can be used as a DNA
template at any time up to fourteen days for direct addition into a
PCR reaction.
Example 2
Stability of DNA in Saliva and PCR Amplification of Saliva-Derived
DNA with a Heating Step
Method
[0058] Saliva was collected from a single donor, using the protocol
described in Example 1, and individual samples of the saliva were
mixed 1:1 with a solution consisting of 20 mM CDTA; 400 mM NaOAc
(sodium acetate); 200 mM Tris-HCl, 0.05% SDS (sodium dodecyl
sulfate); pH adjusted to 8.0. 20 .mu.L of Proteinase K (1 mg/mL
concentration) was then added to this mixture (10 .mu.g/mL final
concentration). The samples were vortexed, and then allowed to sit
at room temperature (approximately 24.degree. C.). At Day 1, Day
14, and Day 365, 2 .mu.l of the saliva/solution sample was added
directly to a standard 50 .mu.l PCR reaction following incubation
for 1 hour at 60.degree. C.
PCR Conditions
[0059] PCR reactions were performed in an Eppendorf
Mastercycler.TM. gradient PCR machine. The total reaction volume
was 50 .mu.L. Each reaction contained Invitrogen PCR Mastermix plus
Tris-HCl (pH 8.0, 10 mM final concentration), MgCl.sub.2 (2 mM
final concentration), each of the 4 dNTPs (400 .mu.M final
concentration), 10 pmoles of each PCR primer for a 560 by fragment
of the thymidylate synthase gene (Forward:
5'-ATGCTTAGTAGGCAATTCTG-3', Reverse: 5'-TTTGGTTGTCAGCAGAGG-3'), and
2 units of Taq DNA polymerase.
[0060] Thermocycling conditions consisted of: 1 cycle of 94.degree.
C. for 1 min; 30 cycles of 94.degree. C. for 30 sec, 55.degree. C.
for 60 sec, 72.degree. C. for 120 sec; 1 cycle of 72.degree. C. for
4 min.
Agarose Gel Electrophoresis
[0061] Eight-.mu.l from each PCR reaction was loaded onto a 1%
agarose gel. A 100-bp ladder was used as a marker. Following
electrophoresis, the agarose gel was stained with ethidium bromide
(1 .mu.g/ml, 15 min), and photographed using a UVP Digi Doc-it.TM.
System under transillumination at 300 nm.
SUMMARY
[0062] As can been seen in FIG. 1, DNA from saliva was stable when
mixed 1:1 with the composition and stored at room temperature for
1, 14, and 365 days.
Example 3
Stability of DNA in Saliva and PCR Amplification of Saliva-Derived
DNA
Method
[0063] Saliva was collected from a single donor, using the protocol
described in Example 1, and individual samples of the saliva were
mixed 1:1 with each of the following solutions: [0064] water [0065]
100 mM NaOH (sodium hydroxide) [0066] 100 mM KOH (potassium
hydroxide) [0067] 200 mM Na.sub.2CO.sub.3 (sodium carbonate)
[0068] The samples were vortexed and allowed to sit at room
temperature (approximately 24.degree. C.). At Day 1, Day 7, Day 14,
Day 21, and Day 365, 2 .mu.l of the saliva/solution sample was
added directly to a standard 50 .mu.l PCR reaction.
PCR Conditions
[0069] PCR reaction conditions were the same as in Example 2.
Thermocycling conditions were the same as in Example 2.
Agarose Gel Electrophoresis
[0070] Agarose gel electrophoresis was performed as described in
Example 2.
SUMMARY
[0071] As can been seen in FIG. 2, DNA from saliva was stable and
usable for PCR when mixed 1:1 with 100 mM NaOH, 100 mM KOH and 200
mM Na.sub.2CO.sub.3, and stored at room temperature for 1, 7, 14
21, and 365 days.
Example 4
Determination of the Concentration of Sodium Hydroxide that is
Non-Inhibitory in a PCR Reaction
Method
[0072] A series of 9 PCR reaction tubes was set up, with each tube
having a different final concentration of NaOH. The final
concentration of NaOH ranged from 0 to 14 mM, as indicated in FIG.
3. This is equivalent to a starting concentration of 50 to 400 mM
(i.e. 1 ml of 400 mM NaOH plus 1 ml of saliva; then 2 .mu.l of this
mixture is used in a 50 .mu.l PCR reaction). To isolate the effect
of NaOH, no saliva was added to the reaction tubes.
PCR Conditions
[0073] In addition to the NaOH, each reaction contained KCl (50 mM
final concentration), Tris-HCl (30 mM final concentration, pH 8.4),
MgCl.sub.2 (2 mM final concentration), each of the 4 dNTPs (400
.mu.M final concentration), 10 pmoles of each PCR primer for a 560
by fragment of the thymidylate synthase gene (Forward:
5'-ATGCTTAGTAGGCAATTCTG-3', Reverse: 5'-TTTGGTTGTCAGCAGAGG-3'), and
2 units of Taq DNA polymerase. 50 ng of purified control human DNA
(from saliva, collected and purified using Oragene.TM.) was added
as the template. PCR reactions were performed in an Eppendorf
Mastercycler.TM. gradient PCR machine. The total reaction volume
was 50 .mu.l. The thermal cycling conditions were the same as in
Example 2.
Agarose Gel Electrophoresis
[0074] Agarose gel analysis was performed as described in Example
2.
SUMMARY
[0075] As can be seen in FIG. 3, 8 mM NaOH was the highest final
concentration of NaOH that could be used in a 50 .mu.l conventional
PCR reaction without strongly inhibiting the reaction. This is
equivalent to a starting concentration of 400 mM NaOH that would
then be diluted 1:1 with a saliva sample before being used for
PCR.
Example 5
Determination of the Volume of a NaOH/saliva Mixture that is
Non-Inhibitory in a PCR Reaction
Method
[0076] One-ml of saliva was collected from a single donor and mixed
with an equal volume of 100 mM sodium hydroxide solution. The
sample was vortexed and stored at room temperature. After 7 days of
storage at room temperature, aliquots were removed and added to a
standard 50 .mu.l PCR reaction. The aliquots ranged in volume from
1 to 20 .mu.l.
PCR Conditions
[0077] The PCR conditions were the same as in Example 2. The total
reaction volume was kept constant at 50 .mu.l. The thermal cycling
conditions were the same as in Example 2.
Agarose Gel Electrophoresis
[0078] Agarose gel analysis was performed as described in Example
2.
SUMMARY
[0079] As can be seen in FIG. 4, up to 10 .mu.L of NaOH/saliva
mixture could be added to a 50 .mu.l conventional PCR reaction
without inhibiting the reaction.
Example 6
Inhibition of a PCR Reaction by Oragene.TM.
[0080] Oragene.TM. (DNA Genotek Inc.) is a saliva collection device
that contains an aqueous solution that stabilizes the DNA in saliva
at room temperature.
Method
[0081] 100 mM sodium hydroxide, 100 mM potassium hydroxide, 200 mM
sodium carbonate, or Oragene.TM. DNA-preserving solutions were
diluted 1:1 with distilled water and 2 .mu.l of each diluted
composition was added to a standard PCR reaction.
PCR Conditions
[0082] The PCR conditions were the same as in Example 2. The
thermal cycling conditions were the same as in Example 2. Fifty-ng
of purified DNA was added to each reaction as the template.
Agarose Gel Electrophoresis
[0083] Agarose gel analysis was performed as described in Example
2.
SUMMARY
[0084] As can be seen in FIG. 5, Oragene.TM. DNA-preserving
solution is inhibitory to PCR. In contrast, this example
demonstrates that the sodium hydroxide, potassium hydroxide, and
sodium carbonate compositions can be added directly to a PCR
reaction without inhibiting the reaction.
Example 7
Improved Extraction of DNA from Saliva
[0085] French et al. (French et al. (2002) Molecular and Cellular
Probes. 16: 319-326) diluted fresh whole saliva 1:1 with water and
used this mixture for quantitative real-time PCR. Diluted saliva
samples were either used immediately or stored at 4.degree. C. (2
to 3 days) or -20.degree. C. The calculated concentration of DNA
from the sample available for real-time PCR was found to be between
0.1 ng/.mu.l and 3.5 ng/.mu.l, varying between samples obtained
from different individuals.
[0086] The examples in Tables I, II and III show the compositions
of present invention make available significantly more DNA from
saliva than a mixture of saliva and water.
Method
[0087] One-ml of saliva was collected from a single donor and mixed
with 1 ml of 100 mM sodium hydroxide, 100 mM potassium hydroxide,
200 mM sodium carbonate, or distilled water. The mixtures were
stored at room temperature. At Day 1 (Table I), Day 14 (Table II),
and Day 365 (Table III), 1 .mu.l of each mixture was added directly
to a real-time PCR reaction as described below.
[0088] Additionally, 1 ml of saliva from the same donor was
collected and mixed with a solution of 20 mM CDTA; 400 mM NaOAc
(sodium acetate); 200 mM Tris-HCl, 0.05% SDS; pH adjusted to 8.0.
Following saliva collection and prior to storate, 20 .mu.l of
Proteinase K (1 mg/ml concentration) was added to the mixture (10
.mu.g/mL final concentration). Samples were stored at room
temperature. At Day 1, Day 14, and Day 365, 1 .mu.l of this mixture
was added directly to a real-time PCR reaction as described below
after incubation at 60.degree. C. for 1 hour.
Real-Time PCR Conditions
[0089] Real-time PCR reactions were performed in a Rotor-Gene.TM.
3000 real-time thermal cycler (Corbett Research). The total
reaction volume was 25 .mu.l.
[0090] Each reaction contained KCl (50 mM final concentration),
Tris HCl (20 mM, pH 8.4), MgCl.sub.2 (3 mM), each of the 4 dNTPs
(400 .mu.M); 5 pmoles of each primer to generate a 143 by fragment
of the human thymidylate synthase gene: (Forward primer:
5'-GCCCTCTGCCAGTTCTA-3', Reverse primer: 5'-TTCAGGCCCGTGATGT-3'), 2
units of Taq DNA polymerase, SYBR Green I dye (Molecular Probes),
1:25,000 final concentration.
[0091] Thermocycling conditions consisted of: 1 cycle of 96.degree.
C. for 5 min; 40 cycles of 95.degree. C. for 20 sec, 55.degree. C.
for 20 sec, 72.degree. C. for 30 sec. Melting curve analysis
conditions consisted of: 72-97.degree. C.; 45 sec at 72.degree. C.,
then 5 sec per degree Celsius to 97.degree. C.
[0092] A standard curve was constructed using purified DNA of known
concentration.
Results
[0093] The DNA concentration of the input sample was automatically
calculated using the Rotor Gene.TM. 3000 software with reference to
the standard curve.
TABLE-US-00006 TABLE I Real-time PCR results from Day 1 Threshold
crossing Calculated DNA concentration of point from real-time input
sample based on Ct value Composition PCR (Ct) (ng/.mu.l) Control
DNA, 30 ng/.mu.L 21.06 26.80 Control DNA, 15 ng/.mu.L 22.14 14.43
Control DNA, 7.5 ng/.mu.L 23.21 7.84 Control DNA, 3.75 ng/.mu.L
24.06 4.84 Control DNA, 1.90 ng/.mu.L 25.83 1.76 Control DNA, 1.00
ng/.mu.L 26.67 1.09 Control DNA, 0.50 ng/.mu.L 28.31 0.43 No
template 0 Water + saliva 34.6 0.01 CDTA/SDS/NaOAC/Tris + 23.33
7.32 saliva 100 mM sodium hydroxide + 23.54 6.51 saliva 100 mM
potassium hydroxide + 24.96 2.89 saliva 200 mM sodium carbonate +
25.19 2.54 saliva
TABLE-US-00007 TABLE II Real-time PCR results from Day 14 Threshold
crossing Calculated DNA concentration of point from real-time input
sample based on Ct value Composition PCR (Ct) (ng/.mu.l) Control
DNA, 30 ng/.mu.L 20.56 25.88 Control DNA, 15 ng/.mu.L 21.2 17.71
Control DNA, 7.5 ng/.mu.L 22.44 8.50 Control DNA, 3.75 ng/.mu.L
23.97 3.45 Control DNA, 1.90 ng/.mu.L 25.07 1.80 Control DNA, 1.00
ng/.mu.L 26.19 0.92 Control DNA, 0.50 ng/.mu.L 27.11 0.54 No
template 0 Water + saliva 31.81 0.03 CDTA/SDS/NaOAC/Tris + 23.62
4.23 saliva 100 mM sodium hydroxide + 23.01 6.06 saliva 100 mM
potassium hydroxide + 24.12 3.15 saliva 200 mM sodium carbonate +
24.02 3.35 saliva
TABLE-US-00008 TABLE III Real-time PCR results from Day 365
Threshold crossing Calculated DNA concentration of point from
real-time input sample based on Ct value Composition PCR (Ct)
(ng/.mu.l) Control DNA, 30 ng/.mu.L 21.21 27.91 Control DNA, 15
ng/.mu.L 22.11 16.32 Control DNA, 7.5 ng/.mu.L 23.43 7.44 Control
DNA, 3.75 ng/.mu.L 24.68 3.52 Control DNA, 1.9 ng/.mu.L 25.5 2.15
Control DNA, 1.0 ng/.mu.L 26.81 0.99 Control DNA, 0.5 ng/.mu.L
28.04 0.47 No template 33.99 0.01 Water + saliva 0
CDTA/SDS/NaOAc/Tris + 23.51 7.07 saliva 100 mM sodium hydroxide +
26.06 1.54 saliva 100 mM potassium hydroxide + 26.48 1.20 saliva
200 mM sodium carbonate + 25.35 2.35 saliva
SUMMARY
[0094] The purpose of this study was to demonstrate the improved
performance of the compositions of the present invention for
storing DNA in comparison to the use of water alone for storing
DNA. It should be appreciated that the real-time PCR technique used
in this study is merely semi-quantitative and, due to the inherent
degree of error in this technique, the calculated DNA concentration
values may not be the actual DNA concentration. However, as would
be readily appreciated by a worker skilled in the art, this method
does provide an accurate comparison between the samples tested
[0095] As can be seen in Tables I, II, and III, there is negligible
DNA detectable by real-time PCR when saliva is stored in water for
1, 14, or 365 days. In contrast, the concentrations of DNA in the
various compositions of the present invention, range from 2.54 to
7.32 ng/.mu.L at Day 1 (Table I), 3.15 to 6.06 ng/.mu.L at Day 14
(Table II), and 1.20 to 7.07 ng/.mu.L at Day 365 (Table III).
[0096] All publications, patents and patent applications mentioned
in this Specification are indicative of the level of skill of those
skilled in the art to which this invention pertains and are herein
incorporated by reference to the same extent as if each individual
publication, patent, or patent applications was specifically and
individually indicated to be incorporated by reference.
[0097] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be obvious to one skilled in
the art are intended to be included within the scope of the
following claims.
Sequence CWU 1
1
4120DNAArtificial SequenceSynthetic Construct 1atgcttagta
ggcaattctg 20218DNAArtificial SequenceSynthetic Construct
2tttggttgtc agcagagg 18317DNAArtificial SequenceSynthetic Construct
3gccctctgcc agttcta 17416DNAArtificial SequenceSynthetic Construct
4ttcaggcccg tgatgt 16
* * * * *