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`FROM SPUTUM
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`Background of the Invention
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`The present invention relates to compositions and methods for preserving
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`nucleic acids at room temperature for extended periods of time and for simplifying
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`the isolation of nucleic acids.
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`DNA can be extracted from virtually every type of cell in the human body,
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`with the exception of red blood cells. The usual source of bodily samples for
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`extraction of DNA is venous blood, since the number of nucleated white blood
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`cells (principally neutrophils and lymphocytes) is relatively high and quite
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`consistent: the normal range is about 5 to 10 million white blood cells per milliliter
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`of blood. The DNA content of human cells is about 6 micrograms per million
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`cells, so 1 milliliter can theoretically yield from 30 to 60 micrograms of DNA.
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`However, there are about 5 billion red blood cells per milliliter of blood, which,
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`since they contain no DNA, must be removed to. obtain pure DNA. Furthermore,
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`the use of blood as a source of DNA has many other disadvantages. Collection of
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`blood is not a trivial procedure. Taking of venous blood requires trained
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`personnel. It is an invasive procedure, which frequently causes some distress and
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`pain to the donor. Precautions are needed to minimize exposure of personnel to
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`blood-borne pathogens. Once collected, the blood sample must be either frozen or
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`quickly transported to a laboratory for extraction of DNA. For these reasons,
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`venous blood is not the ideal source of DNA. A simpler procedure for obtaining
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`blood is to collect a few drops after a finger prick and blotting it onto a piece of
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`filter paper. Less training of personnel is required. Once dried, the DNA is quite
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`stable. The amount of DNA recovered is small but sufficient for many forensic
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`purposes. However, a finger prick is still an invasive procedure and heme derived
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`from hemoglobin in blood can inhibit some types of DNA analysis.
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`Swabbing the inside of the cheek with a brush (a buccal swab) is another
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`source of cells that contain DNA. It is much less invasive than taking of blood and
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`ANCESTRY EX. ‘I008
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`can be collected by individuals with less training than is required in the collection
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`of blood. Once collected, the time that useable DNA can be recovered can be
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`extended by either drying the swab or wiping onto filter paper and drying it.
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`However, as the inside of the mouth is not a sterile source (as compared to blood)
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`and microbes can degrade the quality of the DNA after a period of time. The
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`number of cells recovered by this procedure is not large and typically less than 1-2
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`niicrograms of DNA can be expected in the entire sample.
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`Saliva is a fairly clear, colorless fluid secreted principally by the major
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`salivary glands (parotid, submandibular, and sublingual). Its function is to
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`lubricate and cleanse the oral cavity, as well as to initiate the process of digestion.
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`The parotid gland primarily secretes serous (watery) saliva, while the other glands
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`secrete a mixture of serous and mucinous (sticky) saliva. Components of saliva
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`include albumin, globulin, mucins, and digestive enzymes. It has long been
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`known that cellular DNA is present in saliva and that this DNA is suitable for
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`forensic purposes. Forensic use is typically limited to victim or suspect
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`identification, using the tiny amounts of DNA from saliva that may recovered at a
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`crime scene or from the back of a postage stamp. The notion that saliva may be a
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`reliable source of genomic DNA and a rival to venous blood samples for this
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`purpose has been investigated more recently in a scientific publication (van Schie,
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`et al., J. Immunol. Methods 208:91-101, 1997). The authors used freshly collected
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`or frozen saliva samples and purified the DNA by a fairly complex extraction
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`procedure. Estimates of the quantity of DNA recovered were based upon light
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`absorption at 260 nm, a procedure known to be an unreliable method since other
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`common biological macromolecules, such as RNA, have essentially the same
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`ultraviolet light absorption spectrum. Nevertheless, these authors showed that
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`quality genomic DNA was indeed present by gel electrophoretic analysis and
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`polymerase chain reaction analysis for certain allelic polymorphisms. Another
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`communication (Terasaki, et al., Hum. Immunol. 591597-598, 1998) reported
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`similar results about the suitability of saliva as a source of DNA for HLA typing
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`by polymerase chain reaction analysis. Although the amount of DNA recovered
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`was reported, the method used to measure DNA was not. These authors provided
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`3 examples where saliva dried on filter paper yielded DNA suitable for analysis.
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`With the increasing use of DNA-based analysis in forensics, law
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`enforcement, military, human medicine, veterinary medicine, and research, there is
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`a need for a product that would allow saliva to become a standard reliable source
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`of DNA from an individual (to replace blood, the current standard). In forensic,
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`military and mass disaster situations, for example, DNA samples are now routinely
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`taken from living persons thought to be relatives of unidentified victims of
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`accident or foul play, to aid in identification of the dead. Military personnel or
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`other individuals who expect to encounter hazardous situations where their lives
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`may be at risk may wish to store DNA samples prior to exposing themselves to
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`these hazards. In the law enforcement area, convicted felons in both Canada and
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`the United States are now required to provide DNA samples. DNA-based tests are
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`expected to increase in medicine, such as testing for cystic fibrosis, cytochrome
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`P450 isotypes, polymorphisms affecting susceptibility to infectious and
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`autoimmune diseases, HLA typing, paternity issues, to name but a few. In clinical
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`studies, an example would be to screen populations for colon cancer-predisposing
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`genes or family members of a breast cancer victim for breast cancer predisposing
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`genes. In all of these cases, there are significant advantages to providing a saliva
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`sample rather than providing a blood sample as a source of DNA. All donors
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`would prefer donating saliva rather than blood because of the discomfort, pain, or
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`apprehension associated with phlebotomy or pin-pricks. Saliva has a ftuther
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`advantage of not requiring specialized personnel thereby reducing cost where mass
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`sample collection is being carried out. The risk of blood—bcme infection is
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`likewise decreased.
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`In addition to the problem of developing a standard collection and
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`preservation method for DNA in saliva, there remains an ongoing need to improve
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`methods of overcoming problems specific to the recovery of nucleic acids from
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`saliva. The problem of extraction of high molecular weight DNA and RNA from
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`mammalian cells has been partially addressed by Birnboim in Methods of
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`Enzymology 216:154—160, 1993, but this work was not extended to the recovery of
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`nucleic acids from mucin-containing bodily fluids.
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`Multimeric proteins called mucins are high molecular weight glycosylated
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`proteins that form a major part of a protective biofilm on the surface of epithelial
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`cells, where they can provide a barrier to particulate matter mad bind
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`microorganisms. These glycoproteins contribute greatly to the viscoelastic nature
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`of saliva. The major high—molecular-weight mucin in salivary secretions is
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`MUCSB, one of four gel-forming mucins that exist as multimeric proteins with
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`molecular weights greater than 20-40 million daltons. MUCSB is a large
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`oligomeric mucin composed of disulphide-linked subunits.
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`It is lcnown that reagents that reduce disulfides also reduce the viscosity of
`mucin, such as that found in sputum or saliva. Reducing agents,
`particular
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`sulfilr-containing chemicals such as [3-mercaptoethanol and dithiothreitol, are
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`' widely used in biochemistry. However, many biochemically relevant reducing
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`agents are capable of reacting in solution with dissolved oxygen. This is lcnown
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`are autooxidation (also called autoxidation or auto-oxidation), where 1-electron
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`reduction intennediates of oxygen are fonned, viz., superoxide (Of), hydrogen
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`peroxide (H202) and hydroxyl radical (OH'). In addition, transitional metal cations
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`function as catalysts and 02" has been demonstrated to be an intennediate.
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`Unfortunately, reducing agents and reducing compositions of the prior art have a
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`relatively short shelf life, especially in basic solutions, and stock solutions that
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`contain reducing agents cannot be prepared and stored under ambient conditions
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`for an extended period time, usually not more than a day or two.
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`Therefore, in addition to a need for a means to collect sputum or saliva, and
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`subsequently preserving the nucleic acids contained therein by contacting them
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`with a stabilizing composition, there is a need for the inclusion of a stable reducing
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`agent into the composition, such that nucleic acids can be conveniently recovered
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`from it, especially after extended periods of time in the presence of oxygen at
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`neutral or mildly alkaline pH.
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`
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`Summary of the Invention
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`The present inventor has developed a composition, which, when mixed with
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`a mucin-containing bodily fluid, preserves the nucleic acids at room temperature
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`under ambient conditions for extended periods of time. There is no requirement
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`for freezing of the samples before nucleic acid recovery and purification. The
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`properties of this composition are that it (a) chemically stabilizes nucleic acids, (b)
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`inhibits nucleases that may be present in the saliva, and (c) is compatible with
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`proteolytic enzymes and other reagents used to purify/amplify oligo- or
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`polynucleotides. A fourth and novel property of this composition is that it
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`contains an agent that rapidly reduces the viscous properties of mucin, greatly
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`facilitating the extraction of nucleic acids contained within.
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`Accordingly, a first aspect of the invention features a composition for
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`preserving nucleic acids that includes a chelating agent, and a denaturing agent,
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`where the pH of the composition is greater than 5.0. In one embodiment, the
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`composition is an aqueous solution.
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`In another embodiment, the composition also includes a reducing agent. For
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`example, it can include one or more of the following: ascorbic acid, dithionite,
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`erythiorbate, N—acetylcysteine, cysteine, glutathione, dithiothreitol, 2-
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`mercaptoethanol, dierythritol, a resin-supported thiol, a resin-supported phosphine,
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`vitamin E, and trolox, or salts thereof. Desirably, the reducing agent is ascorbic
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`acid, erythiorbate, N-acetylcysteine, dithiothreitol, or 2—mercaptoethanol, and most
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`desirably, the reducing agent is ascorbic acid. In another embodiment, the
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`composition does not contain ascorbic acid. In yet another embodiment, the
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`concentration of the reducing agent in the composition is greater than or equal to
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`50 millimolar.
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`Antioxidant free-radical scavengers are also desirable reducing agents for
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`the composition of the present invention. Examples include antioxidant vitamins,
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`antioxidant hormones, antioxidant enzymes, thiols, and phenols.
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`Desirably, the reducing agent retains reducing activity for at least 46 days in
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`the presence of one or more of the following: oxygen, ambient air, ambient light,
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`and alkaline pH.
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`The chelating agent of the composition can be selected from the group
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`consisting of: ethylenediamine tetraacetic acid (EDTA), cyclohexane
`diaminetetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA),
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`tetraazacyclododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic
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`acid (TETA), and desferrioximine, or chelator analogs thereof. Desirably, the
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`chelating agent is cyclohexane diaminetetraacetate (CDTA), diethylenetriamine
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`pentaacetic acid (DTPA), tetraazacyclododccanetetraacetic acid (DOTA), or
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`desferrioximine, and most desirably, the chelating agent is cyclohexane
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`diaminetetraacetate (CDTA).
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`In another embodiment, the chelating agent of the composition inhibits
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`metal redox cycling. By "inhibits metal redox cycling" is meant the inhibition of
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`metal-based oxidation/reduction cycles that produce reactive oxygen fi'ee-radical
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`species. Examples of redox ion pairs involved in such cycles include Fe”/Fe“,
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`Cu”/Cu”, and various oxidation states of molybdenum, vanadium, nickel, and
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`cobalt. Chelators that bind one or both ions of a redox ion pair can inhibit the
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`production of reactive oxygen species such as, for example, hydroxyl radical
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`(HO-), hydroperoxyl radical (I-I00-), superoxide radical (Of), nitric oxide radical
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`(N0-), or peroxynitrite radical (ON02'-).
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`The nucleic acid to be preserved by the composition can be DNA or RNA,
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`including mRNA or viral RNA.
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`The pH of the composition can between from about 5.0 and about 11.0,
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`desirably from about 6.5 to about 7.5, and most desirably, about 7.0. For the
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`preservation of DNA, a pH from about 7.0 to about 10.0 can be used. Depending
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`on other components of the compositions, desirable pHs are about 7.5, about 8.0,
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`or a pH range from about 8.0 to about 9.0. A buffer, such as I-IEPES, TRIS, or
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`carbonate buffer can be added to the composition to maintain the pH in a constant
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`range. For the preservation of RNA, a pH from about 5.0 to about 7.0, desirably
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`from about 6.5 to about 6.8 can be used. Again, a buffer, such as BES, can be
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`used to maintain the pH in a constant range.
`The denaturing agent of the composition can be selected from the group
`consisting of: urea, dodecyl sulfate, guanidinium chloride, guanidinium
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`thiocyanate, perchlorate, and an alcohol. Desirably, the denaturing agent is urea,
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`dodecyl sulfate, or an alcohol, wherein the alcohol is 10% - 60% of the total
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`composition volume. The alcohols can be methanol, ethanol, n-propanol,
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`isopropanol, n-butanol, trifluoroethanol, phenol, or 2,6-di-tert-butyl-4-
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`methylphenol.
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`In another embodiment, the composition includes an antimicrobial agent.
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`By "antimicrobial agent" is meant a substance or group of substances which
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`reduces the rate of growth of an organism compared to the rate of growth of the
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`organism in their absence. A reduction in the rate of growth of an organism may
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`be by at least 5%, more desirably, by at least 10%, even more desirably, by at least
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`20%, 50%, or 75%, and most desirably, by 90% or more. The definition also
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`extends to substances which affect the viability, virulence, or pathogenicity of an
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`organism. An antimicrobial agent can be natural (e.g., derived from bacteria),
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`synthetic, or recombinant. An antimicrobial agent can be bacteriostatic,
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`bactericidal or both. An antimicrobial agent is bacteriostatic if it inhibits cell
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`division without affecting the viability of the inhibited cell. An antimicrobial agent
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`is bactericidal if it causes cell death. Cell death is commonly detected by the
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`absence of cell growth in liquid growth medium (e.g., absence of turbidity) or on a
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`solid surface (e.g., absence of colony formation on agar). Those of skill in the art
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`know that a substance or group of substances which is bacteriostatic at a given
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`concentration may be bactericidal at a higher concentration. Certain bacteriostatic
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`substances are not bactericidal at any concentration. Desirably, the composition of
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`the invention includes an alcohol as an antimicrobial agent, and most desirably the
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`composition includes ethanol.
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`In another embodiment, the composition also includes an inhibitor of
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`ribonuclease. Desirable inhibitors are selected from the group consisting of:
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`heparin, heparan sulfate, oligo(vinylsu1fonic acid), poly(vinylsulfonic acid),
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`20
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`3D
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`oligo(vinyIphosphonic acid), and poly(vinylsulfi.uic acid), or salts thereof. The
`inclusion of an inhibitor ofribonuclease in the composition ofthe invention is
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`particularly desirable when the nucleic acid to be preserved is RNA, desirably
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`mRNA, or when the nucleic acid to be preserved is from a virus or a bacterium.
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`A second aspect of the invention features a method of reducing the
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`viscosity of a mucin—containing bodily fluid or tissue by reducing disulfide bonds
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`inherent to mucin, wherein the bodily fluid or tissue is mixed with a composition
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`of the invention that includes a reducing agent. In one embodiment, the bodily
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`fluid is sputum, desirably saliva. By "sputum" is meant that mucoid matter
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`contained in or discharged from the nasal or buccal cavity of an animal, including
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`saliva and discharges from the respiratory passages, including the lungs. In
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`another embodiment, the method includes the recovery of a nucleic acid.
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`A third aspect of the invention features a method of preserving a nucleic
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`acid contained in sputum that includes the steps of obtaining sputum from a
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`subject, and contacting the sputum with a composition of the invention, thus
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`preserving the nucleic acid.
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`In one embodiment, when the nucleic acid is DNA, the DNA is stable for
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`more than 14 days, desirably more than 30 days, and more desirably more than 60
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`days. In another embodiment, when the nucleic acid is DNA and the composition
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`does not contain ascorbic acid, the DNA is stable for more than 60 days, and
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`desirably more than 360 days.
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`A fourth aspect of the invention features a method of recovering a nucleic
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`acid from sputum that includes the steps of: i) obtaining sputum from a subject, ii)
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`contacting the sputum with a composition of the invention to form a mixture, iii)
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`contacting the mixture with a protease, and iv) recovering the nucleic acid from
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`the mixture. Desirably, the protease is proteinase K or pronase.
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`In one embodiment of any of the second, third, or fourth aspects, the
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`sputum is saliva. In another embodiment, the sputum is from a mammal, desirably
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`a human. In yet another embodiment, the nucleic acid is DNA or RNA. If the
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`nucleic acid is RNA, desirably it is mRNA or viral RNA. The nucleic acid can be
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`from a source foreign to the subject fiom which the sputum sample is taken. For
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`
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`example, the nucleic acid can be from a bacterium or a virus that is residing in the
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`buccal, nasal, or respiratory passages of the subject.
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`In a fifth aspect, the invention features a method of preserving and/or
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`recovering a nucleic acid from a bodily fluid that includes, placing the bodily fluid
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`into a first region of a container, placing a composition of the invention into a
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`second region of the container, which is separated from the first region by a
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`barrier, closing the container, and disturbing the integrity of the barrier such that
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`the composition and the bodily fluid are brought into contact.
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`In one embodiment, the disestablishment of the barrier is coupled to the
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`closing of the container when a lid is placed on it. In one example, the barrier is
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`punctured. In a desirable example, the barrier is in the form of a pivoting sealing
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`disc. In this example, attachment of the lid to the container forces the disc to pivot
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`from its original position of spanning the space between the first region and the
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`second region to a position in which both regions are exposed to each other,
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`thereby fonning a mixture between a composition of the invention and the bodily
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`fluid is allowed. Desirably, the bodily fluid is sputum, and most desirably, saliva.
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`In a sixth aspect, the invention features a device for preserving andlor
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`isolating a nucleic acid obtained from a biological sample. The device includes: a
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`container that has a first region for collecting a biological sample and a second
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`region containing a composition for preserving a nucleic acid, a barrier between
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`the first region the second region that keeps the biological sample and the
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`composition separate, a means for closing the container, and a means for
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`disturbing the integrity of the barrier such that the composition is capable of
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`contacting the biological sample. The first region can have an opening of from 2.0
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`to 7.0 cm, desirably from 2.5 to 3.5 cm, and most desirably 3.0 cm. Desirably, the
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`biological sample is sputum, and most desirably, saliva.
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`In one embodiment of the sixth aspect, the nucleic acid-preserving
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`composition is a composition of the present invention. In another embodiment, the
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`means for closing the container is coupled to the means for disturbing the integrity
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`of the banier. In yet another embodiment, the means for closing the container is
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`an airtight lid.
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`In a seventh aspect, the invention features a method of manufacturing a
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`device for preserving a nucleic acid in a biological sample that includes: providing
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`a container that has a first region and a second region, with the first region suitable
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`for containing a composition of the invention and the second region having an
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`opening suitable for the application of a biological sample;
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`placing the composition into the first region; and applying a barrier to the
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`container between the first region and the second region, with the barrier being
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`impermeable to the composition and capable of being disestablished.
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`In an embodiment of either the sixth or seventh aspect, the barrier can be a
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`pivoting disc, where in a first position, the disc spans the compartment and
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`separates the first and second areas. Pivoting the disc to a second position (e.g., by
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`connecting a screw-on lid to a plunger mechanism which contacts the disc, causing
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`it to pivot when the lid is screwed on) disestablishes the barrier and allows the
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`biological sample contained in the first region to contact the composition that is
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`contained in the second region.
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`By “about” is meant +/- 10% of the stated value or a chemical or obvious
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`equivalent thereof.
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`By "alcohol" is meant a water-miscible organic compound containing a
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`hydroxyl group, including water—miscible mixtures of hydroxyl-containing organic
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`compounds.
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`By “antioxidant firee—radical scavenger” is meant a substance that reduces a
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`reactive oxygen free radical species. Such free radicals include, for example,
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`hydroxyl radical (HO-), hydroperoxyl radical (H00-), superoxide radical (Of-),
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`nitric oxide radical (NO), or peroxynitrite radical (ONO;'-).
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`By “aqueous solution” is meant a solution or suspension that contains 30%
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`or more water by volume.
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`By "bodily fluid" is meant a naturally occurring fluid from an animal, such
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`as saliva, serum, plasma, blood, urine, mucus, gastric juices, pancreatic juices,
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`semen, products of lactation or menstration, tears, or lymph.
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`By “biological sample” is meant any sample containing nucleic acids that
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`has been obtained from or deposited by an animal. Non-limiting examples include
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`slcin, hair, bodily fluids, fecal matter, and tissue.
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`By “chelator analog" is meant a derivative chelator compound with the
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`same backbone structure and having the same general properties as the parent
`chelator compound.
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`I
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`By “denaturing agent” is meant a substance that alters the natural state of
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`that to which it is added.
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`By "mucin" is meant any mucoprotein that raises the viscosity of the
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`medium surrounding the cells that secrete it.
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`By “mucoid” is meant any bodily fluid containing mucin
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`By "nucleic acid" is meant a chain of the nucleotides, including
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`deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically found in
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`chromosomes, mitochodria, ribosomes, bacteria, or viruses.
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`By “nucleic acid-preserving composition" is meant any composition of the
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`present invention, unless otherwise specified.
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`When referring to a nucleic acid, by “stable” is meant that at least about
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`50% of the initial amount of high molecular weight nucleic acid (DNA, RNA,
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`mRNA, or viral RNA) contained in a sample is still present after storing the
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`sample at ambient temperature (i.e., 20°C to 25°C) for the specified time period.
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`The amount of high molecular weight DNA in a sample can quantified by
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`densitometry analysis of the high molecular weight DNA band from an agarose gel
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`(see Figure 1 and Example 4).
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`By “resin-supported phosphine" is meant a polymer that contains a
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`multiplicity of covalently-bound phosphine groups.
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`By “resin-supported thiol” is meant is meant a polymer that contains a
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`multiplicity of covalently-bound sulflnydryl groups.
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`By “saliva” is meant the secretion, or combination of secretions, from any
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`of the salivary glands, including the parotid, submaxillary, and sublingual glands,
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`optionally mixed with the secretion from the buccal glands.
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`11
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`11
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`By “sputum" is meant that mucoid matter contained in or discharged from
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`the nasal or buccal cavity of a mammal, including saliva and discharges from the
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`respiratory passages, including the lungs.
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`By “subject” is meant any animal. Desirably, the subject is a mammal that
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`can produce saliva for the purposes of nucleic acid extraction. Most desirably, the
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`subject is a human.
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`Other features and advantages of the present invention will become
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`apparent from the following detailed description. It should be understood,
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`however, that the detailed description and the specific examples, while indicating
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`preferred embodiments of the invention are given by way of illustration only, since
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`various changes and modifications within the spirit and scope of the invention will
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`become apparent to those skilled in the art from this detailed description.
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`Brief Description of the Drawings
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`15
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`Figure 1 is an electrophoresis agarose analysis of DNA isolated from saliva
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`using the capacity of methods of one embodiment of the invention.
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`Figure 2 is a graph illustrating real time PCR of stimulated saliva DNA of
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`Example 5.
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`Figure 3 is a graph illustrating real time PCR of unstimulated saliva DNA
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`of Example 6.
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`Figure 4 is an electrophoresis agarose analysis of the DNA in saliva
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`samples mixed with compositions of the invention, the mixtures having been
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`incubated for various times at various temperatures.
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`Figure 5 shows structures of (oxidized) ascorbate anion, (reduced)
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`dehyclroascorbic acid, and a free radical intermediate
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`Figure 6 is a compilation of two spectrophotometric scans of sodium
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`ascorbate (100 |.LI\/I) in CB (1 mM CDTA, 10 mM BES, pH 7.4), prepared under
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`aerobic conditions over 30 minutes at room temperature (scan 1) and 3 minutes
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`after addition of a few crystals of MnCl2.(scan 2), as per Example 8.
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`30
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`Figure 7 is a compilation of spectrophotomenic scans, at the indicated
`
`times, of the 100 pM sodium ascorbate prepared in CB of Example 8. The
`
`12
`
`12
`
`
`
`solution was exposed to ambient atmosphere and temperature between scans but
`
`was not contacted wifli MnCl2 (see Example 9).
`
`Figure 8 is a graph of absorbances at 265 nm, obtained at the indicated
`times, of a solution of sodium ascorbate (250 mM) containing 30 mM Tris-HCI,
`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.
`
`Figure 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 MnC12. Scan 2 was taken 2 minutes after the addition MnCl;. Scan 3
`
`10
`
`was taken 8 minutes after the addition MnCI;. Scan 4 was taken 27 minutes after
`
`- the addition MnCl2.
`
`Figure 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
`
`15
`
`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 Figure 11).
`Figure 11 is a side view of the sample container of Figure 10, now showing
`
`sealing disc 7.
`
`20
`
`25
`
`30
`
`Detailed Description
`
`The following standard abbreviations are used herein: DNA, deoxyribonucleic
`
`acid; RNA, ribonucleic acid; mR'NA, messenger RNA; HEPES, 4-(2-
`hydroxyethyl)-1-piperazineethanesulfonic acid; BES, N,N-bis[2-hydroxyethyl]-2»
`aminoethane-sulfonic acid; TRIS, tris(hydroxymethy1)aminomethane, CDTA,
`
`cyclohexane diaminetetraacetate; DTPA, N,N-bis(2-
`
`(bis(carboxyrnethyl)amino)ethyl)glycine; DOTA, 1,4,7,10-
`
`tetrazacyclododecanetetraacetic acid; and TETA, 1,4,8,11-
`
`tetraazacyclotetradecanetetraacetic acid.
`
`13
`
`13
`
`
`
`Compositions of the Invention
`
`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
`
`10
`
`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 ofthe invention. The compositions of the invention can also include
`
`one or more reducing agents, which can reduce sample viscosity, thereby making
`
`15
`
`nucleic acid recovery an easier process.
`
`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,
`
`20
`
`more desirably within a pH range of about 7 .5 to about 10.0, and most desirably,
`
`within a pH of about 7.0.
`
`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
`
`30
`
`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
`
`14
`
`14
`
`
`
`the pH within the range of about 5 to about 1 1. Examples of pH buffers with
`
`desirable properties include, but not limited to, TRIS hydrochloride, I-IEPES and
`
`BBS.
`
`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
`
`10
`
`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, TBTA,
`
`15
`
`and desferioximine, or chelator analogs thereof. The amount or concentration of
`
`chelator will depend upon the strength of the chclator, 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.
`
`20
`
`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.
`
`25
`
`Pancreatic deoxyribonuclease is known to require divalent metal ions such as
`
`Mg(II), Mn(lI) and/or Ca(lI) for enzymatic activity. The strong chelators
`
`described above, in addition to providing chemical stability to