(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`I CORRECTED VERSION I
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`1111111111111111 IIIIII IIIII 11111111111111111111 IIIII 111111111111111 IIIII IIII IIIIIII IIII IIII IIII
`
`( 43) International Publication Date
`18 December 2003 (18.12.2003)
`
`PCT
`
`(10) International Publication Number
`WO 2003/104251 A2
`
`(51) International Patent Classification 7:
`
`C07H21/00
`
`(21) International Application Number:
`PCT/CA2003/000869
`
`(22) International Filing Date:
`
`6 June 2003 (06.06.2003)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/386,397
`60/386,398
`60/386,399
`
`7 June 2002 (07.06.2002) US
`7 June 2002 (07.06.2002) US
`7 June 2002 (07.06.2002) US
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NI, NO, NZ, OM, PH, PL, PT, RO, RU, SC, SD,
`SE, SG, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US,
`UZ, VC, VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, Fl, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, RO,
`SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`(71) Applicant (for all designated States except US): DNA
`GENOTEK INC. [CA/CA]; 190 Merlose Avenue, Suite
`200, Ottawa, Ontario Kl Y 4K7 (CA).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): BIRNBOIM, Chaim,
`H. [CA/CA]; 1552 Featherston Drive, Ottawa, Ontario
`KlH 6P2 (CA).
`
`(48) Date of publication of this corrected version:
`11 March 2004
`
`(15) Information about Correction:
`see PCT Gazette No. 11/2004 of 11 March 2004, Section II
`
`(74) Agents: ROBINSON, Christopher, J. et al.; Smart &
`Biggar, Box 11560 Vancouver Centre, Suite 200, 650 West
`Georgia Street, Vancouver, British Columbia V6B 4N8
`(CA).
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`---iiiiiiii
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`== --
`iiiiiiii ----
`
`!!!!!!!!
`iiiiiiii
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`ln
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`"'1'
`Q
`,-...I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`~ (54) Title: COMPOSITION AND METHODS FOR OBTAINING NUCLEIC ACIDS FROM SPUTUM
`Q
`~ (57) Abstract: The present invention relates to compositions and methods for preserving and extracting nucleic acids from saliva.
`The compositions include a chelating agent, a denaturing agent, buffers to maintain the pH of the composition within ranges desirable
`0 for DNA and/or RNA. The compositions may also include a reducing agent and/or antimicrobial agent. The invention extends to
`> methods of using the compositions of the invention to preserve and isolate nucleic acids from saliva as well as to containers for the
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`;;, compositions of the invention.
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`Page i
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`Spectrum Ex. 1003
`IPR Petition - USP 10,000,795
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`WO 2003/104251
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`PCT/CA2003/000869
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`COMPOSITIONS AND METHODS FOR OBTAINING NUCLEIC ACIDS
`
`FROM SPUTUM
`
`Background of the Invention
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`5
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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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`10
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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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`15 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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`20
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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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`25
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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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`30
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`source of cells that contain DNA. It is much less invasive than taking of blood and
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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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`5
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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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`micrograms 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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`10
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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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`15
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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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`20
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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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`25
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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. 59:597-598, 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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`30
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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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`5
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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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`10
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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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`15
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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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`20
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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 further
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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-borne infection is
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`25
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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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`30 mammalian cells has been partially addressed by Bimboim 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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`5
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`cells, where they can provide a barrier to particulate matter and 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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`MUC5B, one of four gel-forming mucins that exist as multimeric proteins with
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`molecular weights greater than 20-40 million daltons. MUC5B is a large
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`10
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`oligomeric mucin composed of disulphide-linked subunits.
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`It is known that reagents that reduce disulfides also reduce the viscosity of
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`mucin, such as that found in sputum or saliva. Reducing agents, in particular
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`sulfur-containing chemicals such as ~-mercaptoethanol and dithiothreitol, are
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`widely used in biochemistry. However, many biochemically relevant reducing
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`15
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`agents are capable of reacting in solution with dissolved oxygen. This is known
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`are autooxidation (also called autoxidation or auto-oxidation), where I-electron
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`reduction intermediates of oxygen are formed, viz., superoxide (02-"), hydrogen
`peroxide (H20 2) and hydroxyl radical (OH} In addition, transitional metal cations
`function as catalysts and 0 2-- has been demonstrated to be an intermediate.
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`20 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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`25
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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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`30
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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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`5
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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 w1th
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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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`10
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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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`15
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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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`20
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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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`25
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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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`5
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`consisting of: ethylenediamine tetraacetic acid (EDTA), cyclohexane
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`diaminetetraacetate (CDT A), 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 (CDT A), diethylenetriamine
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`10
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`pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), or
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`desferrioximine, and most desirably, the chelating agent is cyclohexane
`
`diaminetetraacetate ( CDT A).
`
`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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`15 metal-based oxidation/reduction cycles that produce reactive oxygen free-radical
`species. Examples of redox ion pairs involved in such cycles include Fe2+ /Fe3+,
`Cu1+/Cu2+, and various oxidation states of molybdenum, vanadium, nickel, and
`cobalt. Chelators that bind one or both ions of a redox ion pair can inhibit the
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`production of reactive oxygen species such as, for example, hydroxyl radical
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`20
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`(HO·), hydroperoxyl radical (HOO·), superoxide radical (02-- ), nitric oxide radical
`(NO·), or peroxynitrite radical (ONO2- · ).
`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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`25
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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 HEPES, 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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`30
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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.
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`The denaturing agent of the composition can be selected from the group
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`consisting of: urea, dodecyl sulfate, guanidinium chloride, guanidinium
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`5
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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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`10
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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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`15
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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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`20
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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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`25
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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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`30
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`ribonuclease. Desirable inhibitors are selected from the group consisting of:
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`heparin, heparan sulfate, oligo(vinylsulfonic acid), poly(vinylsulfonic acid),
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`oligo(vinylphosphonic acid), and poly(vinylsulfuric acid), or salts thereof. The
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`inclusion of an inhibitor of ribonuclease in the composition of the invention is
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`particularly desirable when the nucleic acid to be preserved is RNA, desirably
`
`mRNA, or when the nucleic acid to be preserved is from a virus or a bacterium.
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`5
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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
`
`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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`10
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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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`15
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`subject, and contacting the sputum with a composition of the invention, thus
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`preserving the nucleic acid.
`
`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
`
`days. In another embodiment, when the nucleic acid is DNA and the composition
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`20
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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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`25
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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 Kor pronase.
`
`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
`
`a human. In yet another embodiment, the nucleic acid is DNA or RNA. If the
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`30
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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 from which the sputum sample is taken. For
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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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`5
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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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`10
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`closing of the container when a lid is placed on it. In one example, the barrier is
`
`punctured. In a desirable example, the barrier is in the form of a pivoting sealing
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`disc. In this example, attachment of the lid to the container forces the disc to pivot
`
`from its original position of spanning the space between the first region and the
`
`second region to a position in which both regions are exposed to each other,
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`15
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`thereby forming a mixture between a composition of the invention and the bodily
`
`fluid is allowed. Desirably, the bodily fluid is sputum, and most desirably, saliva.
`
`In a sixth aspect, the invention features a device for preserving and/or
`
`isolating a nucleic acid obtained from a biological sample. The device includes: a
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`container that has a first region for collecting a biological sample and a second
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`20
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`region containing a composition for preserving a nucleic acid, a barrier between
`
`the first region the second region that keeps the biological sample and the
`
`composition separate, a means for closing the container, and a means for
`
`disturbing the integrity of the barrier such that the composition is capable of
`
`contacting the biological sample. The first region can have an opening of from 2.0
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`25
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`to 7.0 cm, desirably from 2.5 to 3.5 cm, and most desirably 3.0 cm. Desirably, the
`
`biological sample is sputum, and most desirably, saliva.
`
`In one embodiment of the sixth aspect, the nucleic acid-preserving
`
`composition is a composition of the present invention. In another embodiment, the
`
`means for closing the container is coupled to the means for disturbing the integrity
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`30
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`of the barrier. In yet another embodiment, the means for closing the container is
`
`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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`5
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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.
`
`In an embodiment of either the sixth or seventh aspect, the barrier can be a
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`10
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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
`
`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 free-radical scavenger" is meant a substance that reduces a
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`reactive oxygen free radical species. Such free radicals include, for example,
`
`hydroxyl radical (HO·), hydroperoxyl radical (HOO·), superoxide radical (02-·),
`nitric oxide radical (NO·), or peroxynitrite radical (ONO2-· ).
`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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`skin, 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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`5
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`same backbone structure and having the same general properties as the parent
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`chelator compound.
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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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`10 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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`15
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`By "nucleic acid-preserving composition" is meant any composition of the
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`present invention, unless otherwise specified.
`
`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,
`
`mRNA, or viral RNA) contained in a sample is still present after storing the
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`20
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`sample at ambient temperature (i.e., 20°C to 25°C) for the specified time period.
`
`The amount of high molecular weight DNA in a sample can quantified by
`
`densitometry analysis of the high molecular weight DNA band from an agarose gel
`
`(see Figure 1 and Example 4).
`
`By "resin-supported phosphine" is meant a polymer that contains a
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`25 multiplicity of covalently-bound phosphine groups.
`
`By "resin-supported thiol" is meant is meant a polymer that contains a
`
`multiplicity of covalently-bound sulfhydryl groups.
`
`By "saliva" is meant the secretion, or combination of secretions, from any
`
`of the salivary glands, including the parotid, submaxillary, and sublingual glands,
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`30
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`optionally mixed with the secretion from the buccal glands.
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`By "sputum" is meant that mucoid matter contained in or discharged from
`
`the nasal or buccal cavity of a mammal, including saliva and discharges from the
`
`respiratory passages, including the lungs.
`
`By "subject" is meant any animal. Desirably, the subject is a mammal that
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`5
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`can produce saliva for the purposes of nucleic acid extraction. Most desirably, the
`
`subject is a human.
`
`Other features and advantages of the present invention will become
`
`apparent from the following detailed description. It should be understood,
`
`however, that the detailed description and the specific examples, while indicating
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`10
`
`preferred embodiments of the invention are given by way of illustration only, since
`
`various changes and modifications within the spirit and scope of the invention will
`
`become apparent to those skilled in the art from this detailed description.
`
`Brief Description of the Drawings
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`15
`
`Figure 1 is an electrophoresis agarose analysis of DNA isolated from saliva
`
`using the capacity of methods of one embodiment of the invention.
`
`Figure 2 is a graph illustrating real time PCR of stimulated saliva DNA of
`
`Example 5.
`
`Figure 3 is a graph illustrating real time PCR of unstimulated saliva DNA
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`20
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`of Example 6.
`
`Figure 4 is an electrophoresis agarose analysis of the DNA in saliva
`
`samples mixed with compositions of the invention, the mixtures having been
`
`incubated for various times at various temperatures.
`
`Figure 5 shows structures of ( oxidized) ascorbate anion, (reduced)
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`25
`
`dehydroascorbic acid, and a free radical intermediate
`
`Figure 6 is a compilation of two spectrophotometric scans of sodium
`
`ascorbate (100 µM) in CB (1 mM CDTA, 10 mM BES, pH 7.4), prepared under
`
`aerobic conditions over 30 minutes at room temperature (scan 1) and 3 minutes
`
`after addition of a few crystals of MnC12.( scan 2), as per Example 8.
`
`30
`
`Figure 7 is a compilation of spectrophotometric scans, at the indicated
`
`times, of the 100 µM sodium ascorbate prepared in CB of Example 8. The
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`solution was exposed to ambient atmosphere and temperature between scans but
`
`was not contacted with MnCh (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-HCl,
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`5
`
`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
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`10
`
`addition of MnCh. Scan 2 was taken 2 minutes after the addition MnC}i. Scan 3
`
`was taken 8 minutes after the addition MnC}i. Scan 4 was taken 27 minutes after
`
`· the addition MnCh.
`
`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
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`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.
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`20
`
`Detailed Description
`
`The following standard abbreviations are used herein: DNA, deoxyribonucleic
`
`acid; RNA, ribonucleic acid; mRNA, messenger RNA; HEPES, 4-(2-
`
`hydroxyethyl)-1-piperazineethanesulfonic acid; BES, N,N-bis[2-hydroxyethyl]-2-
`
`25
`
`aminoethane-sulfonic acid; TRIS, tris(hydroxymethyl)aminomethane, COTA,
`
`cyclohexane diaminetetraacetate; DTPA, N,N-bis(2-
`
`(bis( carboxymethyl)amino )ethyl)glycine; DOTA, 1,4,7,10-
`
`tetraz