throbber

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`METHOD FOR STABILIZING BIOLOGICAL SAMPLES FOR NUCLEIC ACID
`
`ANALYSIS
`
`RELATED APPLICATIONS
`
`5
`
`This application claims priority under 35 U.S.C. § I 19(e) to U.S. Provisional
`
`Application Serial No. 60/571,120, entitled "Method for Stabilizing a Biological Sample for a
`
`Nucleic Acid Integrity Assay" filed on May 14, 2004, the disclosure of which is herein
`
`incorporated by reference in its entirety.
`
`10
`
`15
`
`FIELD OF THE INVENTION
`
`The invention relates generally to assays to detect nucleic acid markers indicative of
`
`cancer and other diseases and more particularly to preparing nucleic acid-containing biological
`
`samples for use in these assays.
`
`BACKGROUND OF THE INVENTION
`
`Tissue and body fluid samples, including stools, contain shed cellular debris. In healthy
`
`patients, such debris is the result of apoptotic degradation as part of the normal cell cycle.
`
`Apoptosis reduces the integrity (intactness) of nucleic acids, proteins, and other cellular
`
`components in healthy individuals, so that only small fragments exist in the debris that results
`
`20
`
`from the apoptotic process (e.g., exfoliated cellular debris).
`
`In diseases in which cell cycle mechanisms are destroyed or impaired, cellular debris
`
`can include high-integrity cellular components, such as nucleic acids that have not been
`
`degraded by apoptosis. One class of disease in which cell cycle mechanisms are disrupted is
`
`cancer. An increased presence of high molecular weight nucleic acids in a biological sample
`
`25
`
`therefore can reveal the presence of cancer in a patient from whom the biological sample was
`
`obtained. Disease detection assays known as nucleic acid integrity analysis assays have been
`
`developed that are based on the increased levels of non-degraded nucleic acid in a cancerous
`
`tissue or body fluid as compared to the level of non-degraded nucleic acid in a non-cancerous
`
`tissue or body fluid.
`
`30
`
`Nucleic acids in patient samples tend to degrade after they have been removed from the
`
`patient. This degradation can diminish the effectiveness of a nucleic acid integrity assay that
`
`scores a sample as diseased (e.g., cancerous) based on the presence of intact nucleic acids; if
`
`Geneoscopy Exhibit 1009, Page 2
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`-2-
`
`the sample is excessively degraded, a sample that is actually positive may appear to be
`
`negative.
`
`SUMMARY OF THE INVENTION
`
`5
`
`The invention is based in part on the discovery of a method for stabilizing nucleic acids
`
`in tissue and body fluid samples so as to facilitate analysis of the samples in a nucleic acid
`
`integrity assay. It has been unexpectedly found that contacting a patient sample with a
`
`stabilization solution stabilizes the DNA so that intact nucleic acids indicative of diseased cells
`
`are more effectively detected in a nucleic acid integrity assay.
`
`10
`
`Accordingly, in one aspect, the invention provides a method for preparing a nucleic
`
`acid sample for a nucleic acid integrity analysis assay. In one aspect, the invention provides a
`
`method for preparing a nucleic acid sample for a nucleic acid integrity analysis assay. The
`
`method includes providing a patient sample that includes shed cells or cellular debris and a
`
`nucleic acid and contacting the patient sample with a stabilization solution under conditions
`
`15
`
`sufficient to stabilize the nucleic acid for nucleic acid integrity analysis. In one embodiment,
`
`the stabilization solution includes a buffer, a salt, and a chelating agent.
`
`In some embodiments, the conditions are sufficient to detect at least a three-fold
`
`genomic equivalent (GE) increase in a nucleic acid integrity analysis of a patient sample
`
`having adenoma or cancer as compared to the GE detected in a nucleic acid integrity analysis
`
`20
`
`of a sample from the patient that is not incubated with the stabilization solution. In one
`
`embodiment, the integrity analysis is performed by determining an amount of nucleic acid
`
`greater than about 200 bp in length using an assay that detects a nucleic acid (which can be a
`
`wild-type or mutant nucleic acid). The nucleic acid is present in a patient sample that includes
`
`shed cells or cellular debris. A patient is identified as having cancer or adenoma if the amount
`
`25
`
`of nucleic acid is greater than an amount of nucleic acid expected to be present in a sample
`
`obtained from a patient who does not have cancer or adenoma.
`
`The patient sample can be obtained from a patient that is, e.g., a vertebrate, including a
`
`mammal, a reptile, or an amphibian. A mammal can be, e.g., a human, a non-human primate
`
`(such as a gorilla or monkey, including a chimpanzee), a rodent (such as a mouse, rat, guinea
`
`30
`
`pig, or gerbil) dog, cat, horse, pig, goat, sheep, or cow. The patient sample can be any body
`
`tissue or fluid that is suspected of containing DNA from a diseased cell (such as a precancerous
`
`or cancerous cell). In one embodiment, the patient sample is a stool sample.
`
`Geneoscopy Exhibit 1009, Page 3
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`- 3 -
`
`In some embodiments, the patient sample can be obtained as part of a screen for, e.g., a
`
`disease or disease-associated condition that impairs, or could lead to impairment of, the proper
`
`function of the gastrointestinal system. Gastrointestinal diseases can include, e.g., those
`
`associated with the stomach, small intestine, and/or colon. The disease or condition can include
`
`5
`
`cancers or precancerous conditions such as an adenoma. Other conditions include
`
`inflammatory bowel syndrome, inflammatory bowel disease, Crohn's disease, and others in
`
`which a genomic instability is thought to play a role.
`
`In some embodiments, the patient sample is frozen and thawed prior to incubation with
`
`stabilization solution. In other embodiments, the patient sample is not frozen prior to
`
`10
`
`incubation with the stabilization solution.
`
`In general, the stabilization solution is added to the patient sample at a ratio of about 1
`
`ml/gram of patient sample to about 20 ml/gram of patient sample. In some embodiments, the
`
`stabilization solution is provided at 1-15 ml/gram, 2-12 ml/gram, 3-11 ml/gram, or 4-7
`
`ml/gram. However, higher or lower ratios may be used. When the patient sample is a stool
`
`15
`
`sample, and the stabilization solution is 10 mM Tris-Cl, 1 mM EDTA, and 150 mMNaCL, a
`
`suitable ratio of stabilization solution to patient sample is 7 ml/gm.
`
`In some embodiments, the patient sample and stabilization solution are incubated at
`
`about 4 to 28 degrees Centigrade. In some embodiments the temperature is 17 to 27 degrees
`
`Centigrade, e.g., about 20 to 25 degrees Centigrade. However, the sample and stabilization
`
`20
`
`solution may be exposed to higher or lower temperatures (e.g., the sample and stabilization
`
`solution may be frozen). Also, a sample and buffer may be exposed to changing temperatures
`
`during transport and/or storage.
`
`In various embodiments, the patient sample and stabilization solution are incubated at
`
`least 6 hours, e.g., at least 12 hours, at least 24 hours, or at least 36 hours.
`
`25
`
`In some embodiments, the buffer in the stabilization solution is 0.5 mM to 25 mM Tris,
`
`e.g., 5 mM to 15 mM Tris, 8 mM to 13 mM Tris or about 10 mM Tris. However, other buffers
`
`and/or concentrations may be used.
`
`In some embodiments, the chelating agent in the stabilization solution is 0.01 to 2.5
`
`mM EDTA, e.g., 0.75-1.25 mM EDTA, or lmM EDTA. However, other chelating agents
`
`30
`
`and/or concentrations may be used.
`
`In some embodiments, the salt in the stabilization solution is 75 mM to 225 mM NaCl, e.g.,
`
`100 mM to 175 mM NaCl, or 150 mM NaCL However, other salts (e.g., KCl etc.) and/or
`
`concentrations may be used.
`
`Geneoscopy Exhibit 1009, Page 4
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`-4-
`
`In some embodiments, the stabilization solution is provided at pH 7.0 to 9.0, e.g., pH
`
`7.5 to 8.5, or pH 8.0. However, higher or lower pHs may be used.
`
`In some embodiments, the method further includes determining in the incubated patient
`
`sample an amount of nucleic acid greater than about 200 hp in length using an assay that
`
`5
`
`detects wild-type or mutant nucleic acid, and identifying the patient as having cancer or
`
`adenoma if the amount is greater than an amount of nucleic acid expected to be present in a
`
`sample obtained from a patient who does not have cancer or adenoma ( e.g., more than about
`
`1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 50 fold, or more greater than an amount expected
`
`in a normal individual). In one aspect, a DNA integrity assay may include interrogating a
`
`10
`
`sample for the presence of long DNA :fragments ( e.g., longer than 200 nucleotides, longer than
`
`500 nucleotides, longer than 1,000 nucleotides, etc.) at two or more different loci. In one
`
`embodiment, a patient is identified as having cancer or adenoma if two or more loci (e.g., 3, 4,
`
`or more loci) are positive for the presence of abnormally high levels of long DNA.
`
`In one embodiment, the invention provides a method for preparing a nucleic acid
`
`15
`
`sample for a nucleic acid integrity and/or multiple mutation analysis assays for diagnosing a
`
`carcinoma or adenoma. The method may include providing a patient stool sample that
`
`includes shed cells or cellular debris and a nucleic acid, and incubating the patient sample with
`
`a stabilization solution under conditions sufficient to stabilize the nucleic acid for nucleic acid
`
`integrity and/or multiple mutation analysis. The stabilization solution may be about pH 7.5 to
`
`20
`
`about pH 8.5 and may include 0.5 mM to 25 mM Tris, 0.01 to 2.5 mM EDTA and 100 mM to
`
`200 mM NaCl. For example, the stabilization solution can be 10 mM Tris-Cl pH 8.0, 1 mM
`
`EDTA, and 150mM NaCl. In one embodiment, the conditions are sufficient to detect at least a
`
`three-fold genomic equivalent (GE) increase in a nucleic acid integrity analysis of a patient
`
`sample having adenoma or cancer as compared to the GE detected in a nucleic acid integrity
`
`25
`
`analysis of a sample from the patient that is not incubated with the stabilization solution.
`
`Aspects of the invention may be used for transporting or storing biological samples
`
`after they are obtained and before they are processed for analysis. For example, methods of the
`
`invention may be used to stabilize nucleic acid in biological samples (e.g., stool samples) for
`
`about 12 hours, about 24 hours, about 36 hours, or longer (e.g., 4 days, 5, days, 6 days, 1 week,
`
`30
`
`or longer), even in the absence ofrefrigeration or freezing.
`
`Aspects of the invention may be particularly useful for detecting indicia of adenomas,
`
`early stage cancers, and/or other diseases that may be characterized by very low frequencies of
`
`mutant nucleic acid in a sample.
`
`Geneoscopy Exhibit 1009, Page 5
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`- 5 -
`
`Aspects of the invention may be particularly useful for preserving biological samples
`
`for nucleic acid integrity assays or for multi-panel screens that include one or more nucleic
`
`acid integrity assays. Aspects of the invention may be particularly useful for detecting
`
`mutations in samples that contain very little human DNA (e.g., DIA negative samples).
`
`5 According to the invention, the amount of human DNA recovered from stool from different
`
`subjects may vary and it may be particularly important to preserve nucleic acids in samples
`
`that contain low amounts of human DNA.
`
`Accordingly, aspects of the invention may be useful for screening a population to
`
`identify individuals with indicia of disease, and for avoiding or reducing the number of false
`
`10
`
`negatives (subjects with a disease but who are identified as normal or healthy) in such screens.
`
`In one embodiment, average-risk individuals may screened for one or more indicia of a
`
`sporadic disease (e.g., adenoma, cancer, etc.). In one embodiment, high-risk individuals may
`
`be screened for a sporadic disease or a disease that may be inherited. In one embodiment, a
`
`screen may be performed on a population of individuals regardless of their risk profile.
`
`15
`
`Unless otherwise defined, all technical and scientific terms used herein have the same
`
`meaning as commonly understood by one of ordinary skill in the art to which this invention
`
`belongs. Although methods and materials similar or equivalent to those described herein can be
`
`used in the practice or testing of the invention, suitable methods and materials are described
`
`below. All publications, patent applications, patents, and other references mentioned herein are
`
`20
`
`incorporated by reference in their entirety. In the case of conflict, the present Specification,
`
`including definitions, will control. In addition, the materials, methods, and examples are
`
`illustrative only and not intended to be limiting.
`
`Other features and advantages of the invention will be apparent from the following
`
`detailed description and claims.
`
`25
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1 is a Table (Table 2) showing the results of DNA Integrity Assay (DIA)
`
`analysis performed on stool samples under different experimental conditions.
`
`Figure 2 is a Table (Table 5) showing the results of DNA Integrity Assay (DIA)
`
`30
`
`analysis performed on stool samples under different experimental conditions.
`
`Geneoscopy Exhibit 1009, Page 6
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`- 6 -
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`It has been unexpectedly found that a DNA integrity signal can be stabilized and/or
`
`enhanced by mixing, or incubating, a patient sample known to or suspect of containing DNA
`
`indicative of a disease with a stabilization solution prior to performing the DNA integrity
`
`5
`
`assay. The stabilization solution typically includes one or more buffers, and/or chelating
`
`agents, and/or salts. Aspects of the invention are particularly useful for nucleic acid integrity
`
`assays. However, mutation detection (for example, in a multiple mutation assay) and
`
`hypermethylation analysis also can benefit from aspects of the invention.
`
`According to the invention, an important challenge for colon cancer detection from
`
`10
`
`stool is to preserve the integrity of human DNA in the hostile stool environment, in order to
`
`recover, amplify, and interrogate the DNA for known cancer related abnormalities. Nucleases
`
`that are active in stool have the potential to rapidly degrade DNA, including the minor human
`
`DNA component, and measures may be taken to minimize their negative impact. Typically,
`
`clinical samples are frozen as quickly as possible after collection. However, in order to use
`
`15
`
`fecal DNA tests in population screens, it should be expected that there will be some variability
`
`in the time between sample collection and shipping to testing labs, and furthermore, some
`
`variability in the temperature at which stool samples are transported. In order to eliminate any
`
`variables in sample handling that might have an impact on assay performance we have run
`
`controlled sample incubation experiments and looked at how different markers in a multi-
`
`20
`
`target assay are affected.
`
`Markers may be chosen that yield an acceptable clinical sensitivity for the intended
`
`application such as screening a population for indicia of a disease. In addition, for stool
`
`sample analysis, mutation detection methods should offer sufficient analytical sensitivity since
`
`the human DNA recovered from stool is highly heterogeneous. Normal cells are sloughed into
`
`25
`
`the colonic lumen along with the mutant cells. Therefore, in one embodiment, analytical
`
`methods should detect as little as 1 % (or less) mutant DNA in the presence of excess wild-type
`
`DNA. Also, certain sample preparation methodologies may be used for maximum recovery of
`
`human DNA from samples. The vast majority of DNA recovered from stool often is bacterial
`
`in origin, with the human DNA component representing only a small minority. Certain
`
`30
`
`purification methodologies can efficiently select for the rare human component, and since the
`
`mutant copies (when they exist) represent only a small percentage of the total human DNA
`
`from stool it may be important to maximize the recovery of human DNA in order to maximize
`
`the probability of amplifying mutant copies in the PCR reactions. In one embodiment, gel
`
`Geneoscopy Exhibit 1009, Page 7
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`-7-
`
`electrophoresis methods for capturing human DNA may be used. However, according to the
`
`invention, it may be particularly important to preserve san1ple DNA for purification, especially
`
`when looking for early indicia of diseases ( e.g., indicia of adenomas or early stage cancers that
`
`may be present in less than about 1 %, or about 0.1 % or less of human genomes isolated from a
`
`5
`
`stool sample). A common method to insure that DNA remains stable is to freeze stool samples
`
`as quickly as possible after collection, or to receive samples in centralized testing labs as
`
`quickly as possible. However, in order to provide the option of decentralized sample analysis
`
`and still retain maximum sample integrity, it is desirable to use a more robust and standardized
`
`sample handling method.
`
`10
`
`In one aspect, the invention provides methods for stabilizing biological samples (e.g.,
`
`stool samples) by adding a stabilization solution to a sample as soon as possible after the
`
`sample is obtained. Methods of the invention do not require refrigeration or freezing. Aspects
`
`of the invention are based, in part, on the surprising discovery that nucleic acids in certain
`
`biological samples are stable at room temperature for hours, and even days ( e.g., 1 day, 2 days,
`
`15
`
`3 days, or longer). However, in certain embodiments, samples with stabilization solution may
`
`be refrigerated or frozen. Aspects of the invention are particularly useful for preserving
`
`samples for nucleic acid integrity analysis. However, methods of the invention may be used to
`
`preserve samples for other assays including mutation detection and/or hypermethylation
`
`assays. In certain embodiments, methods of the invention are used to preserve a sample for
`
`20
`
`analysis using a nucleic acid integrity assay along with a mutation detection assay ( e.g., a
`
`multiple mutation panel assay), a hypermethylation assay, or both.
`
`Nucleic acid integrity assays are known in the art and are described in, e.g., US Patent
`
`Application No. 20040043467, US Patent Application No. 20040014104, US Patent No.
`
`6,143,529, and Boynton et al., Clin. Chem. 49:1058-65, 2003. Nucleic acid integrity assays
`
`25
`
`are based on higher levels of intact nucleic acid that appear in debris from cells that lyse non(cid:173)
`
`apoptoTically. Healthy patient generally produces cellular debris through normal apoptotic
`
`degradation, resulting in relatively small fragments of cellular components in tissue and body
`
`fluid samples, especially luminal samples. Patients having a disease generally produce cells
`
`and cellular debris, a proportion of which has avoided normal cell cycle regulation, resulting in
`
`30
`
`relatively large cellular components. As a result, the disease status of a patient is determined by
`
`analysis of patient cellular components produced in specimens obtained from the patient. The
`
`presence of such fragments is a general diagnostic screen for disease.
`
`Geneoscopy Exhibit 1009, Page 8
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`- 8 -
`
`Nucleic acids in patient samples tend to degrade after they have been removed from the
`
`patient. This degradation can diminish the effectiveness of a nucleic acid integrity assay that
`
`scores a sample as diseased ( e.g., cancerous) based on the presence of intact nucleic acids; if
`
`the sample is excessively degraded, a sample that is actually positive may appear to be
`
`5
`
`negative. While not wishing to be bound by theory, it is postulated that the stabilization buffer
`
`of the invention inhibits the nucleases that degrade the nucleic acids present in the diseased
`
`patient samples.
`
`In some aspects of the invention, the addition of a stabilization solution to a biological
`
`sample may be used to preserve nucleic acid molecules containing one or more mutations that
`
`10 may be detected in a multiple mutation analysis ( e.g., an analysis that involves interrogating a
`
`sample for the presence of a mutation at one or more loci, for example at about 2, about 3,
`
`about 4, about 5, about 10, about 15, about 20, about 25, or more loci). In some embodiments,
`
`the addition of a stabilization solution may be used to preserve nucleic acid for a methylation
`
`specific analysis to detect the presence of hyper-methylated nucleic acid molecules at one or
`
`15 more loci that may be indicative of cancer, adenoma, or other disease. In some embodiments,
`
`the addition of a stabilization solution may be used to preserve nucleic acid for a combination
`
`of a nucleic acid integrity assay and/or a multiple mutation analysis and/or a methylation
`
`detection assay. Assays may be performed under conditions to detect a small amount of
`
`mutant nucleic acid in a heterogeneous sample containing an excess of non-mutant nucleic acid
`
`20
`
`( e.g., where the mutant nucleic acid represents less than 10%, less than 5%, less than 1 %, or
`
`about O .1 % or less of the nucleic acid at a particular locus). In some embodiments, a digital
`
`assay may be performed on the preserved nucleic acid in order to detect rare genetic events. In
`
`some aspects, stabilization methods of the invention may preserve more than 80%, 85%, 90%,
`
`91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the nucleic acids indicative of a
`
`25
`
`disease ( e.g., long nucleic acid :fragments, nucleic acid molecules containing one or more
`
`specific mutations, and/or hyper-methylated nucleic acid molecules).
`
`In general any body organ, tissue, or fluid known to or suspected of containing nucleic
`
`acid that can be characterized in a nucleic acid integrity analysis, multiple mutation assay, or
`
`methylation study may be used. Suitable patient samples include those likely to contain
`
`30
`
`sloughed cellular debris. Such specimens include, but are not limited to, stool, blood serum or
`
`plasma, sputum, pus, colostrum, and others. In diseases, such as cancer, in which genomic
`
`instabilities or abnormalities have interfered with normal cell cycle regulation, specimens such
`
`as those identified above contain relatively intact fragments of cellular components.
`
`Geneoscopy Exhibit 1009, Page 9
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`- 9 -
`
`The stabilization solution can be applied to a biological sample that is isolated directly
`
`from a patient, i.e., a freshly isolated biological sample. Alternatively, the method can be used
`
`on a biological sample that has been frozen (e.g., at -20 °C or -80 °C).
`
`In aspects of the invention, stabilization solution may be added to a biological sample
`
`5
`
`at any suitable ratio of sample to buffer. Ratios may be determined as a weight to volume
`
`(w/v) ratio. In some embodiments, the ratio may be about 1:1, about 1:2, about 1:3, about 1:4,
`
`about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9 or about 1: 10 (w/v) sample to stabilization
`
`solution. However, higher or lower ratios may be used.
`
`In aspects of the invention, a biological sample may be weighed before a buffer is
`
`10
`
`added. In some embodiments, a sample (e.g., a stool sample) may weigh about 5 g, about 10 g,
`
`about 15 g, about 20 g, about 25 g, about 30 g, about 35 g, about 40 g, about 45 g, about 50 g,
`
`about 55 g, about 60 g, about 65 g, about 70 g, about 75 g, about 80 g, about 85 g, about 90 g,
`
`about 95 g, about 100 g, or more. It should be appreciated that a large amount of sample ( e.g.,
`
`over 25 g, 30 g, 50 g, 100 g, or more of stool) may be required in order to detect conditions
`
`15
`
`such as adenomas or early stage cancers where a very small amount of mutant or abnormal
`
`nucleic acid may be present in a sample. According to the invention, it may be particularly
`
`important to immediately stabilize biological samples with a stabilization solution when the
`
`samples are to be interrogated for indicia of adenoma or early stage cancer. However, it also
`
`may be useful to stabilize biological samples for detecting indicia of later stage diseases.
`
`20
`
`In general, a stabilization solution may include one or more buffers and/or one or more
`
`chelating agents and/or one or more salts, or any combination of two or more thereof. The
`
`choices of buffer, chelating agent, and salt can be determined by the artisan. The suitability of
`
`a particular stabilization solution can be determined by comparing a nucleic acid integrity
`
`assay on samples that have been incubated with the stabilization solution to a parallel
`
`25
`
`biological sample that has not been incubated with the stabilization solution. A suitable
`
`stabilization solution is a solution that shows a significant average fold increase in genome
`
`equivalents (GE) in a nucleic acid integrity assay compared to a GE determination made on
`
`parallel samples that have not been treated with the stabilization solution. Methods of
`
`calculating genome equivalents (GE) are known in the art (see, e.g., e.g., US Patent
`
`30 Application No. 20040043467, US Patent Application No. 20040014104, US Patent No.
`
`6,143,529, and Boynton et al., Clin. Chem. 49:1058-65, 2003) and are illustrated in the
`
`Examples.
`
`Geneoscopy Exhibit 1009, Page 10
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`- 10-
`
`The temperature and pH optimum can also be determined empirically and optimized
`
`according to the combination of buffer, chelating agent and salt in the stabilization solution.
`
`While room temperature has been found to be a suitable temperature for incubating the patient
`
`sample and the stabilization solution, higher or lower temperatures ( e.g., 4 °C to 16 °C or 25
`
`5
`
`°C to about 37 °C) can also be used, provided they do not undermine the effectiveness of the
`
`stabilization solution. The mixed patient sample and stabilization solution is preferably
`
`subjected to a minimum of agitation. However, according to the invention, the addition of a
`
`stabilization solution with little or no agitation is surprisingly effective at preserving nucleic
`
`acids for subsequent analysis.
`
`10
`
`In one aspect of the invention, a stabilization solution may be particularly useful when
`
`samples are not refrigerated or frozen or when there is a risk that a sample may not be
`
`maintained at a sufficiently low temperature to preserve indicia of disease. For example, a
`
`stabilization solution may be particularly useful if a sample is obtained at a remote location and
`
`mailed or delivered to a testing center. However, stabilization solutions also may be useful to
`
`15
`
`preserve samples that are being processed on-site at a medical center.
`
`Buffers
`
`Suitable buffers include, e.g. tris(hydroxymethyl)aminomethane, sodium phosphate,
`
`sodium acetate, MOPS, and other buffering agents as long as a buffer has the capacity to resist
`
`a 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion. A
`
`20
`
`combination of buffering agents can be used, so long as the solution has the required buffering
`
`capacity. Methods for determining the buffering capacity of a solution are well known in the
`
`art.
`
`The comparison of buffering capacity is preferably carried out in the presence of the
`
`salt and chelating agent to be used in the stabilization solution, at the salt concentration to be
`
`25
`
`used, and with the solutions being compared at about the same temperature, preferably at a
`
`temperature within the range of about 15° C to about 25° C.
`
`Chelating Agents
`
`Table 1 provides a representative list of chelating agents that can be used in the
`
`stabilization solution. The list provided in Table 1 is not meant to be exhaustive. In some
`
`30
`
`embodiments, chelating agents are those which bind trace metal ions with a binding constant
`
`ranging from 101 to 10100; in some embodiments, chelating agents bind trace metal ions with
`
`Geneoscopy Exhibit 1009, Page 11
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`- 11 -
`
`a binding constant ranging from 1010 to 1080 ; in some embodiments, the chelators bind trace
`
`metal ions with a binding constant ranging from 1015 to 1060.
`
`Table 1. Examples of Chelating Agents
`
`ABB RE VIA TION
`
`EDT A free acid
`EDTA2Na
`EDTA 3Na
`
`CHELATORS
`
`FULL NAME
`
`Ethylenediamine-N ,N ,N' ,N' ,-tetraacetic acid
`Ethylenediamine-N,N,N',N',-tetraacetic acid, disodium salt, dihydrate
`
`Ethylenediamine-N,N,N',N',-tetraacetic acid, trisodium salt, trihydrate
`
`EDTA4Na
`
`IEthylenediamine-N,N,N',N'-tetraacetic acid, Tetrasodium salt, tetrahydrate
`
`IEDTA 2K
`
`IEthylenefisminr-N,N,N',N'-tetraacetic acid, dipotassium salt, dihydrate
`IEthylenefisminr-N,N,N',N'-tetraacetic acid, dipotassium salt, dihydrate
`
`IEDTA 2Li
`
`IEthylenediamine-N,N,N',N'-tetraacetic acid, dilithium salt, monhydrate
`
`IEDTA 2NH4
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, diammonium salt
`
`IEDTA 3K
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, Tripotassium salt, dihydrate
`
`Ba(ll)-EDTA
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, barium chelate
`
`Ca(II)-EDTA
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, calcium chelate
`
`Ce(III)-EDTA
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, cerium chelate
`
`Co(Il)-EDT A
`Cu( 11 )-EDT A
`
`Dy(Ill)-EDT A
`Eu(Ill)-EDTA
`Fe(III)-EDTA
`
`In(III)-EDTA
`La(lll)-EDTA
`
`Mg(II)-EDT A
`Mn( [1)-EDTA
`
`Ni(II)-EDTA
`Sm(IIl)-EDTA
`Sr(ll)-EDT A
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, cobalt chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, copper chelate
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, dysprosium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, europium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, iron chelate
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, indium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, lanthanum chelate
`
`Ethylenediamine-N,N,N',N'-tetraacetic acid, magnesium chelate
`Ethylenediamine-N,N,N',N'-tetraacetic acid, manganese chelate
`Ethylenediamine-N,N,N ,N -tetraacetic acid, nickel chelate
`
`IEthylenediamine-N,N,N' ,N'-tetraacetic acid, samarium chelate
`IEthylenediamine-N,N,N',N'-tetraacetic acid, strontium chelate
`
`Z:n(Il)-EDTA
`CyDTA
`
`IEthylenediamine-N,N,N',N'-tetraacetic acid, zinc chelate
`~rans- I ,2-Diaminocyclohexane-N ,N ,N' ,N'-tetraaceticacid,monohydrate
`
`Geneoscopy Exhibit 1009, Page 12
`
`

`

`WO 2005/113769
`
`PCT/US2005/017046
`
`DREG
`
`DTPA-OH
`DTPA
`EDDA
`EDDP
`
`EDDPO
`
`EDTA-OH
`
`EDTPO
`
`IEGTA
`IHBED
`IHDTA
`HIDA
`
`IDA
`Methyl- EDTA
`
`NTA
`NTP
`NTPO
`0-Bistren
`
`TTHA
`
`- 12 -
`
`N,N-Bis(2-hydroxyethyl)glycine
`
`1,3-Diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid
`1,3-Di aminopropane-N,N,N',N'-tetraacetic acid
`
`Ethylenediamine-N,N'-diacetic acid
`Ethylenediamine-N,N'-dipropionic acid dihydrochioride
`
`Ethylenediamine-N,N'-bis(methylenephosphonic Acid), hemihydrate
`
`N-(2-Hydroxyethy l)ethy lenediamine-N ,N' ,N'-triacetic acid
`Ethylenediamine-N,N,N',N'-tetrakis(methylenephosponic acid)
`
`O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid
`IN,N-bis(2-hydroxybenzyl)ethylenediamine- N,N-diacetic acid
`1,6-H ex am ethyl enediamine-N,N,N',N'- tetraacetic acid
`~ -(2-Hydroxyethy l)irninodiacetic acid
`
`Jminodiacetic acid
`
`1,2-Diaminopropane-N, N ,N' ,N'-tetraacetic acid
`
`Nitrilotriacetic acid
`Nitrilotripropionic acid
`
`Nitrilotris(methylenephosphoric acid), trisodium salt
`7,19,30-Trioxa-1,4,10,13,16,22,27,33 -octaabicyclo [ 11, 11, 11] pentatriacontane,
`Triethylenetetramine-N,N,N',N",N"',N"'- hexaacetic acid
`
`Salts
`
`Candidate salts include, e.g, Nal, NaBr, NaCl, LiCl, KCl, KI, KBr, CsCl, GNHCl and
`
`GNSCN. In some embodiments, the salt is chaotropic and has an anion such as perchlorate,
`
`iodide, thiocyanate, acetate, trichloroacetate, hexafluorosilicate, tetrafluoroborate and the like.
`
`5 Cations for a chaotropic salt can include, e.g., the elements lithium, sodium, potassium,
`
`cesium, rubidium, guanidine and the like. More than one salt can be present in the buffered
`
`aqueous salt solution.
`
`Patient samples that have been treated with a stabilization solution can be subject to the
`
`nucleic acid integrity assay. For example the treated sample can be used in method that
`
`10
`
`includes determining an amount of nucleic acid greater than about 200 bp in length using an
`
`assay that detects wild-type or mutant nucleic acid, wherein said nucleic acid is present in a
`
`patient sample comprising shed cells or cellular debris; and identifying

This document is available on Docket Alarm but you must sign up to view it.


Or .

Accessing this document will incur an additional charge of $.

After purchase, you can access this document again without charge.

Accept $ Charge
throbber

Still Working On It

This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.

Give it another minute or two to complete, and then try the refresh button.

throbber

A few More Minutes ... Still Working

It can take up to 5 minutes for us to download a document if the court servers are running slowly.

Thank you for your continued patience.

This document could not be displayed.

We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.

Your account does not support viewing this document.

You need a Paid Account to view this document. Click here to change your account type.

Your account does not support viewing this document.

Set your membership status to view this document.

With a Docket Alarm membership, you'll get a whole lot more, including:

  • Up-to-date information for this case.
  • Email alerts whenever there is an update.
  • Full text search for other cases.
  • Get email alerts whenever a new case matches your search.

Become a Member

One Moment Please

The filing “” is large (MB) and is being downloaded.

Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.

Your document is on its way!

If you do not receive the document in five minutes, contact support at support@docketalarm.com.

Sealed Document

We are unable to display this document, it may be under a court ordered seal.

If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.


Access Government Site

We are redirecting you
to a mobile optimized page.





Document Unreadable or Corrupt

Refresh this Document
Go to the Docket

We are unable to display this document.

Refresh this Document
Go to the Docket