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WO1998058081A1 - Methodes de preparation d'echantillons de selles - Google Patents

Methodes de preparation d'echantillons de selles Download PDF

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Publication number
WO1998058081A1
WO1998058081A1 PCT/US1998/011550 US9811550W WO9858081A1 WO 1998058081 A1 WO1998058081 A1 WO 1998058081A1 US 9811550 W US9811550 W US 9811550W WO 9858081 A1 WO9858081 A1 WO 9858081A1
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WO
WIPO (PCT)
Prior art keywords
sample
dna
allele
stool
probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1998/011550
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English (en)
Inventor
Anthony P. Shuber
Stanley N. Ladidus
Gail E. Radcliffe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Exact Sciences Corp
Original Assignee
Exact Laboratories Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Exact Laboratories Inc filed Critical Exact Laboratories Inc
Priority to AU78168/98A priority Critical patent/AU7816898A/en
Publication of WO1998058081A1 publication Critical patent/WO1998058081A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to methods for the early detection of colon cancer in patients, and more particularly to methods for preparing stool samples in order to increase the yield of nucleic acids.
  • Stool samples frequently must be prepared for medical diagnostic analysis. Stool samples may be analyzed for diagnosis of medical conditions ranging from parasitic, bacterial or viral infections to inflammatory bowel disease and colorectal cancer.
  • Colorectal cancer is a leading cause of death in Western society. However, if diagnosed early, it may be treated effectively by removal of the cancerous tissue. Colorectal cancers originate in the colorectal epithelium and typically are not extensively vascularized (and therefore not invasive) during the early stages of development. Colorectal cancer is thought to result from the clonal expansion of a single mutant cell in the epithelial lining of the colon or rectum. The transition to a highly vascularized, invasive and ultimately metastatic cancer which spreads throughout the body commonly takes ten years or longer. If the cancer is detected prior to invasion, surgical removal of the cancerous tissue is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool.
  • stool comprises cells and cellular debris from the patient, from microorganisms, and from food, resulting in a heterogeneous population of cells. This makes detection of small, specific subpopulations difficult to detect reliably.
  • the invention provides stool sample preparation protocols for increasing sample nucleic acid yield.
  • methods of the invention comprise homogenizing a representative stool sample in a solvent in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5:1. DNA may then extracted from the homogenized sample mixture.
  • Providing an optimal solvent volume to stool mass ratio increases the yield of nucleic acid obtained from the sample.
  • An especially-preferred ratio of solvent volume to stool mass is between about 10:1 and about 30:1 , more preferably from about 10:1 to about 20:1 , and most preferably 10:1.
  • a preferred solvent for preparing stool samples according to the invention is a physiologically-compatible buffer, such as a buffer comprising Tris-EDTA-NaCI.
  • a preferred buffer is a Tris-EDTA-NaCI buffer comprising about 50 to about 100 mM Tris, about 10 to about 20 mM EDTA, and about 5 to about 15 mM NaCl at about pH 9.0.
  • a particularly preferred buffer is 50 mM Tris, 16 mM EDTA and 10 mM NaCl at pH 9.0.
  • Another preferred solvent is guanidine isothiocyanate (GITC).
  • a preferred GITC buffer has a concentration of about 1 M to about 5 M.
  • a particularly preferred GITC buffer has a concentration of about 3 M.
  • methods further comprise the step of extracting DNA from the homogenized sample mixture using sequence-specific nucleic acid probes.
  • probes hybridizing to mammalian DNA.
  • Methods of the invention are useful to screen for the presence in a stool sample of nucleic acids indicative of colorectal cancer.
  • Such methods comprise obtaining a representative stool sample (i.e., at least a cross-section); homogenizing the sample in a solvent having a solvent volume to stool mass ratio of at least 5:1 ; extracting DNA from the sample; and analyzing the DNA for characteristics of colorectal cancer.
  • a representative stool sample i.e., at least a cross-section
  • homogenizing the sample in a solvent having a solvent volume to stool mass ratio of at least 5:1 extracting DNA from the sample; and analyzing the DNA for characteristics of colorectal cancer.
  • Various methods of analysis of DNA characteristics exist, such as those disclosed in co-owned, copending U.S. Patent application Serial No. 08/700,583, incorporated by reference herein.
  • Methods of the invention also comprise obtaining a representative (i.e., cross- sectional) sample of stool and homogenizing the stool in a buffer,
  • the methods of the invention are especially and most preferably useful for detecting DNA characteristics indicative of a subpopulation of transformed cells in a representative stool sample.
  • the DNA characteristics may be, for example, mutations, including point mutations, deletions, additions, translocations, substitutions, and loss of heterozygosity.
  • Methods of the invention may further comprise a visual examination of the colon.
  • surgical resection of abnormal tissue may be done in order to prevent the spread of cancerous or precancerous tissue.
  • methods of the invention provide means for screening for the presence of a cancerous or precancerous subpopulation of cells in a heterogeneous sample, such as a stool sample.
  • Methods of the invention reduce morbidity and mortality associated with lesions of the colonic epithelium.
  • methods of the invention comprise more accurate and convenient screening methods than are currently available in the art, because such methods take advantage of the increased yield of relevant DNA. Further aspects and advantages of the invention are contained in the following detailed description thereof.
  • Figure 1 is a representation of a partial nucleotide sequence of the kras gene (base pairs 6282-6571 ) and the positions of capture probe CP1 , PCR primer A1 , and PCR primer B1 , in relation to the kras nucleotide sequence.
  • Figure 2 is an image produced using a Stratagene Eagle Eye II Still Video System (Stratagene, La Jolla, CA), of the results of a gel electrophoresis run with the uncut DNA extracted as described in Example 2.
  • Stratagene Eagle Eye II Still Video System Stratagene, La Jolla, CA
  • Figure 3 is an image produced using a Stratagene Eagle Eye II Still Video System (Stratagene, La Jolla, CA), of the results of a gel electrophoresis run with the DNA extracted as described in Example 3.
  • Stratagene Eagle Eye II Still Video System Stratagene, La Jolla, CA
  • the invention provides improved methods for extraction of nucleic acids from stool.
  • the yield of nucleic acids extracted from stool is increased by homogenizing the stool in a buffer at optimal ratio of buffer volume to stool mass.
  • Improved nucleic acid yields allow nucleic acid analysis of stool samples to be conducted more efficiently with less stool volume.
  • a stool sample obtained for analysis comprises at le ' ast a cross-section of a whole stool.
  • cells and cellular debris from the colonic epithelium is deposited onto and into stool in a longitudinal streak.
  • Obtaining at least a cross-section of a stool ensures that a representative sampling of colonic epithelial cells and cellular debris is analyzed.
  • a preferred solvent is a physiologically-compatible buffer comprising, for example, 1 M Tris, 0.5M EDTA, 5M NaCl and water to a final concentration of 500mM Tris, 16mM EDTA and 10mM NaCl at pH 9.
  • the buffer acts as a solvent to disperse the solid stool sample during homogenization. Applicants have discovered that increasing the volume of solvent in relation to solid mass of the sample results in increased yields of DNA.
  • solvent is added to the solid sample in a solvent volume to solid mass ratio of at least about 5:1.
  • the solvent volume to solid mass ratio is preferably in the range of about 10:1 to about 30:1 , and more preferably in the range of about 10: 1 to about 20: 1. Most preferably, the solvent volume to solid mass ratio is about 10:1.
  • total DNA is isolated from stool homogenate.
  • the homogenized mixture is centrifuged to form a pellet made up of cell debris and stool matter, and a supernatant containing nucleic acid and associated proteins, lipids, etc.
  • the supernatant is treated with a detergent, such as 20% SDS, and enzymes capable of degrading protein (e.g., Proteinase K).
  • the supernatant is then Phenol-Chloroform extracted.
  • the resulting purified nucleic acids are then precipitated by means known in the art. A variety of techniques in the art can then be employed to manipulate the resulting nucleic acids, including further purification or isolation of specific nucleic acids.
  • extracted nucleic acids are placed in a physiologically compatible buffer, such as guanidine isothiocyanate (GITC).
  • a physiologically compatible buffer such as guanidine isothiocyanate (GITC).
  • Capture probes are then added to the mixture to hybridize to target DNA in order to facilitate selective removal of target DNA from the sample.
  • Sequence specific capture of target DNA can be accomplished by initially denaturing sample DNA to form single-stranded DNA. Then, a sufficient quantity of sequence specific oligonucleotide probe that is complementary to at least a portion of a target polynucleotide (e.g., a sequence in or near the p53 allele) is added. The probe sequence (labeled with biotin) is allowed to hybridize to the complementary target DNA sequence.
  • Beads coated with avidin or streptavidin are then added and attach to the biotinylated hybrids by affinity-binding. The beads may be magnetized to facilitate isolation.
  • the resultant DNA is washed repeatedly to remove inhibitors, including those commonly introduced via the capture probe technique.
  • washes are preferably carried out approximately four times with 1 M GITC and 0.1 % detergent, such as Igepal (Sigma).
  • the initial washes are then preferably followed by two washes with a standard wash buffer (such as Tris-EDTA-NaCI) to remove the GITC from the mix, since GITC is a known inhibitor of DNA polymerases, including those associated with PCR.
  • a standard wash buffer such as Tris-EDTA-NaCI
  • PCR polymerase chain reaction
  • RFLP restriction fragment length polymorphism
  • Methods of the present invention are particularly useful for isolation and analysis of nucleic acids that encompass genes that have mutations implicated in colorectal cancer, such as kras.
  • the kras gene has a length of more than 30 kbp and codes for a 189 amino acid protein characterized as a low-molecular weight GTP-binding protein. The gene acquires malignant properties by single point mutations, the most common of which occurs at the 12th amino acid.
  • Several studies have confirmed that approximately 40% of primary colorectal adenocarcinoma cells in humans contain a mutated form of the kras gene. Accordingly, the kras gene is a particularly suitable target for the methods of colorectal cancer detection of the present invention.
  • CP1 has the following sequence: 5' GCC TGC TGA AAA TGA CTG AAT ATA AAC TTG TGG TAG T 3' (SEQ, ID NO: 1 ), and is preferably biotinylated at the 5' end in order to facilitate isolation.
  • CP1 is effective in the sequence specific capture of kras DNA.
  • Suitable PCR primers for the analysis of extracted kras DNA sequence have also been determined.
  • Primer A1 has the sequence: 5' C CTG CTG AAA ATG ACT GAA 3' (SEQ ID NO: 2)
  • Primer B1 has the sequence: 5' CAT GAA AAT GGT CAG AGA AA 3' (SEQ ID NO: 3).
  • the PCR primers A1 and B1 as well as capture probe CP1 , are depicted in Figure 1 , showing their relation to the kras nucleotide sequence, base pairs 6282-6571 (SEQ ID NO: 4).
  • One skilled in the art can construct other suitable capture probes and PCR primers for kras or other target genes or nucleotide sequences, using techniques well known in the art.
  • Voided stool was collected from a patient and a cross-sectional portion of the stool was removed for use as a sample. After determining the mass of the sample, an approximately 10x volume of Tris-EDTA-NaCI lysis buffer was added to the solid sample in a test tube. The final concentration of the buffer was 500mM Tris, 16mM EDTA and 10mM NaCl, at a pH of about 9.0. Four 10mm glass balls were placed in the tube and the tube and contents were homogenized in an Exactor II shaker for 15 minutes. The homogenized mixture was then allowed to stand 5 minutes at room temperature.
  • the tube was then centrifuged for 5 minutes at 10,000 rpm in a Sorvall Centrifuge, and the supernatant was transferred to a clean test tube. A 20% SDS solution was added to the tube to a final concentration of 0.5%. Proteinase K was also added to the tube to a final concentration of 500mg/ml. The tube was then incubated overnight at 37°C.
  • Example 2 After incubation, the contents of the tube were extracted with an equal volume of phenol/chloroform and centrifuged at 3500 rpm for 3 minutes. The aqueous layer was then transferred to a new tube and extracted three (3) times with equal volumes of chloroform and centrifuged at 3500 rpm for 3 minutes. The aqueous layer was then transferred to a new tube and 0.1x volume of 3M NaOAc was added to the aqueous portion, which was then extracted with an equal volume of isopropanol, and centrifuged for 5 minutes at 12,000 rpm. The supernatant was discarded, and the pellet was washed with 10ml of 70% ethanol, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded and the pellet containing isolated DNA was dried by inverting the tube.
  • Example 2 Example 2
  • a comparative analysis of solvent volume to mass ratios was conducted. Three separate stool samples were prepared as described above. A first sample, designated SS88-3x, was homogenized in buffer at a volume to mass ratio of 3:1. A second sample, designated SS88-5x, was homogenized at a ratio of 5:1 ; and a third sample, designated SS88-1 Ox, was homogenized at a ratio of 10: 1.
  • a second set of four equivalent samples was prepared from a single stool sample. Each of the four samples was of equal mass, and was homogenized as described in Example 1 at a solvent volume to stool mass ratio of 5: 1 , 10: 1 , 20: 1 , and 30:1 , respectively. After homogenization each sample was subdivided into 8 aliquots, 4 treated with RNase, and 4 untrreated. Total DNA was then isolated as described above and analyzed on agarose gels.
  • methods of the invention produce increased yields of DNA from stool, thereby allowing more efficient sequence-specific capture of target nucleic acid.
  • Methods of the invention provide improvements in the ability to detect disease-related nucleic acid mutations present in stool.
  • the skilled artisan will find additional applications and embodiments of the invention useful upon inspection of the foregoing description of the invention. Therefore, the invention is limited only by the scope of the appended claims.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Pathology (AREA)
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  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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Abstract

La présente invention concerne des méthodes de préparation d'échantillons de selles en vue d'augmenter la production d'ADN approprié; l'invention concerne également des méthodes d'isolation et d'analyse d'ADN cible présentant des caractéristiques indiquant la présence de cancer du côlon et du rectum. L'invention concerne en outre des méthodes de détection chez des patients de lésions cancéreuses ou pré-cancéreuses du côlon et du rectum.
PCT/US1998/011550 1997-06-16 1998-06-04 Methodes de preparation d'echantillons de selles Ceased WO1998058081A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU78168/98A AU7816898A (en) 1997-06-16 1998-06-04 Methods for stool sample preparation

Applications Claiming Priority (2)

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US87663897A 1997-06-16 1997-06-16
US08/876,638 1997-06-16

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WO1998058081A1 true WO1998058081A1 (fr) 1998-12-23

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050640A1 (fr) * 1999-02-25 2000-08-31 Exact Laboratories, Inc. Methodes de conservation de l'integrite de l'adn
WO2000061808A3 (fr) * 1999-04-09 2001-08-02 Exact Lab Inc Procedes de detection d'acides nucleiques revelateurs de cancer
US6280947B1 (en) 1999-08-11 2001-08-28 Exact Sciences Corporation Methods for detecting nucleotide insertion or deletion using primer extension
US6428964B1 (en) 2001-03-15 2002-08-06 Exact Sciences Corporation Method for alteration detection
WO2001042504A3 (fr) * 1999-12-07 2002-08-29 Penn State Res Found Detection d'acide nucleique extracellulaire associe aux tumeurs dans le plasma et le serum sanguins
US6511805B1 (en) 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
WO2000031303A3 (fr) * 1998-11-23 2003-07-03 Exact Lab Inc Techniques de preparation d'echantillons de selles
US6849403B1 (en) 1999-09-08 2005-02-01 Exact Sciences Corporation Apparatus and method for drug screening
US6919174B1 (en) 1999-12-07 2005-07-19 Exact Sciences Corporation Methods for disease detection
EP1599489A4 (fr) * 2002-02-13 2007-01-03 Medimolecular Pty Ltd Procede permettant d'isoler des acides nucleiques a partir d'echantillons de selles
US7368233B2 (en) 1999-12-07 2008-05-06 Exact Sciences Corporation Methods of screening for lung neoplasm based on stool samples containing a nucleic acid marker indicative of a neoplasm
US7790395B2 (en) 1996-03-15 2010-09-07 The Penn State Research Foundation Evaluation of diseases and conditions associated with translocated genes using plasma or serum DNA
US7833757B2 (en) * 2003-09-23 2010-11-16 Universitael Posidam Method for conducting non-invasive early detection of colon cancer and/or of colon cancer precursor cells
US8048629B2 (en) 1996-03-15 2011-11-01 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum

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WO1997009600A2 (fr) * 1995-09-06 1997-03-13 Medical Research Council Procede d'isolement de cellules

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WO1997009600A2 (fr) * 1995-09-06 1997-03-13 Medical Research Council Procede d'isolement de cellules

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CALDAS C ET AL: "DETECTION OF K-RAS MUTATIONS IN THE STOOL OF PATIENTS WITH PANCREATIC ADENOCARCINOMA AND PANCREATIC DUCTAL HYPERPLASIA", CANCER RESEARCH, vol. 54, 1 July 1994 (1994-07-01), pages 3568 - 3573, XP002050203 *
NOLLAU P ET AL: "Isolation of DNA from stool and bodily fluids for PCR amplification.", BIOTECHNIQUES, (1996 MAY) 20 (5) 784-8. JOURNAL CODE: AN3. ISSN: 0736-6205., United States, XP002084081 *
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Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7790395B2 (en) 1996-03-15 2010-09-07 The Penn State Research Foundation Evaluation of diseases and conditions associated with translocated genes using plasma or serum DNA
US6511805B1 (en) 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
US8048629B2 (en) 1996-03-15 2011-11-01 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
US7935484B2 (en) 1996-03-15 2011-05-03 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assay
US7935487B2 (en) 1996-03-15 2011-05-03 The Penn State Research Foundation Method of detecting tumor-associated DNA in plasma or serum from humans without cancer
US8361726B2 (en) 1996-03-15 2013-01-29 The Penn State Research Foundation Method of detecting tumor-associated DNA in plasma or serum with a premalignant solid tumor
WO2000031303A3 (fr) * 1998-11-23 2003-07-03 Exact Lab Inc Techniques de preparation d'echantillons de selles
AU752817B2 (en) * 1999-02-25 2002-10-03 Exact Sciences Corporation Methods for preserving DNA integrity
WO2000050640A1 (fr) * 1999-02-25 2000-08-31 Exact Laboratories, Inc. Methodes de conservation de l'integrite de l'adn
JP2011015701A (ja) * 1999-02-25 2011-01-27 Exact Sciences Corp Ｄｎａの完全性を保存するための方法
JP2002537777A (ja) * 1999-02-25 2002-11-12 エグザクト サイエンシーズ コーポレイション Ｄｎａの完全性を保存するための方法
US6964846B1 (en) 1999-04-09 2005-11-15 Exact Sciences Corporation Methods for detecting nucleic acids indicative of cancer
WO2000061808A3 (fr) * 1999-04-09 2001-08-02 Exact Lab Inc Procedes de detection d'acides nucleiques revelateurs de cancer
US6280947B1 (en) 1999-08-11 2001-08-28 Exact Sciences Corporation Methods for detecting nucleotide insertion or deletion using primer extension
US6849403B1 (en) 1999-09-08 2005-02-01 Exact Sciences Corporation Apparatus and method for drug screening
WO2001042504A3 (fr) * 1999-12-07 2002-08-29 Penn State Res Found Detection d'acide nucleique extracellulaire associe aux tumeurs dans le plasma et le serum sanguins
US6919174B1 (en) 1999-12-07 2005-07-19 Exact Sciences Corporation Methods for disease detection
US7368233B2 (en) 1999-12-07 2008-05-06 Exact Sciences Corporation Methods of screening for lung neoplasm based on stool samples containing a nucleic acid marker indicative of a neoplasm
US6428964B1 (en) 2001-03-15 2002-08-06 Exact Sciences Corporation Method for alteration detection
EP1599489A4 (fr) * 2002-02-13 2007-01-03 Medimolecular Pty Ltd Procede permettant d'isoler des acides nucleiques a partir d'echantillons de selles
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum
US7833757B2 (en) * 2003-09-23 2010-11-16 Universitael Posidam Method for conducting non-invasive early detection of colon cancer and/or of colon cancer precursor cells
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US11536632B2 (en) 2011-06-19 2022-12-27 DNA Genotek, Inc. Biological collection system
US11549870B2 (en) 2011-06-19 2023-01-10 DNA Genotek, Inc. Cell preserving solution
US11592368B2 (en) 2011-06-19 2023-02-28 DNA Genotek, Inc. Method for collecting and preserving a biological sample
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US11198899B2 (en) 2014-03-07 2021-12-14 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples

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Publication number Publication date
AU7816898A (en) 1999-01-04

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