Nothing Special   »   [go: up one dir, main page]

WO1998058081A1 - Methods for stool sample preparation - Google Patents

Methods for stool sample preparation Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
sample
dna
allele
stool
probe
Prior art date
Application number
PCT/US1998/011550
Other languages
French (fr)
Inventor
Anthony P. Shuber
Stanley N. Ladidus
Gail E. Radcliffe
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/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention provides methods for the preparation of stool samples to increase the yield of relevant DNA, and further provides methods for isolating and analyzing target DNA for characteristics indicative of colorectal cancer. Methods for screening patients for the presence of cancerous or pre-cancerous colorectal lesions are also provided.

Description

METHODS FOR STOOL SAMPLE PREPARATION
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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. Generally, such symptoms are present only when the disease is well established, and often after metastasis has occurred. Early detection of colorectal cancer therefore is important in order to significantly reduce its morbidity. Invasive diagnostic methods such as endoscopic examination allow for direct visual identification, removal, and biopsy of potentially cancerous growths. Endoscopy is expensive, uncomfortable, inherently risky, and therefore not a practical tool for screening populations to identify those with colorectal cancer. Non-invasive analysis of stool samples for characteristics indicative of the presence of colorectal cancer or precancer is a preferred alternative for early diagnosis, but no known diagnostic method is available which reliably achieves this goal.
Current non-invasive screening methods involve assaying stool samples for the presence of fecal occult blood or for elevated levels of carcinoembryonic antigen, both of which are suggestive of the presence of colorectal cancer. Additionally, recent developments in molecular biology provide methods of great potential for detecting the presence of a range of DNA mutations or alterations indicative of colorectal cancer.
The presence of such mutations can be detected in DNA found in stool samples during various stages of colorectal cancer. However, 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.
An additional problem encountered in preparation of stool sample for detection of colorectal cancer is the difficulty of extracting sufficient quantities of relevant DNA from the stool. Stool samples routinely contain cell debris, enzymes, bacteria (and associated nucleic acids), and various other compounds that can interfere with traditional DNA extraction procedures and reduce DNA yield. Furthermore, DNA in stool often appears digested or partially digested, which can reduce the efficiency of extraction methods. SUMMARY OF THE INVENTION
It has now been appreciated that the yield of nucleic acid from a stool sample is increased by providing an optimal ratio of solvent volume to stool mass in the sample. Accordingly, the invention provides stool sample preparation protocols for increasing sample nucleic acid yield. In a preferred embodiment, 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.
Also in a preferred embodiment, methods further comprise the step of extracting DNA from the homogenized sample mixture using sequence-specific nucleic acid probes. Particularly preferred are 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. 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, such as a buffer comprising a detergent and a proteinase and optionally a DNase inhibitor.
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. Finally, surgical resection of abnormal tissue may be done in order to prevent the spread of cancerous or precancerous tissue. Accordingly, 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. Moreover, 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.
DESCRIPTION OF THE DRAWINGS
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.
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.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides improved methods for extraction of nucleic acids from stool. According to methods of the invention, 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.
In preferred methods of the invention a stool sample obtained for analysis comprises at le'ast a cross-section of a whole stool. As provided in co-owned, co- pending U.S. patent application, Serial No. 08/699,678, incorporated by reference herein, 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. Once the stool sample is collected, it is homogenized in a physiologically acceptable solvent. A preferred means of homogenization employs agitation with glass beads. 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.
According to methods of the invention, solvent (buffer) 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.
In preferred methods of the invention, 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.
In a preferred embodiment of the invention, extracted nucleic acids are placed in 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.
After separation of probe-target hybrids, the resultant DNA is washed repeatedly to remove inhibitors, including those commonly introduced via the capture probe technique. In the methods of the present invention, 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. Finally, the target DNA is eluted into a small volume of distilled water by heating.
Assays using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis or other nucleic acid analysis methods may be used to detect DNA characteristics indicative of a disorder, such as colorectal cancer or pre- cancer. Several particularly useful analytical techniques are described in co-pending applications Serial Number 08/700,583, 08/815,576 and [Attorney
Docket No. EXT-007], the disclosures of which are incorporated herein by reference.
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.
Toward this end, applicants have constructed a suitable exemplary capture probe directed to the kras nucleotide sequence. The capture probe, designated 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. As illustrated more fully below, 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), and 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.
The following examples provide further details of methods according to the invention. However, numerous additional aspects of the invention will become apparent upon consideration of the following examples.
Example 1
Stool Sample Preparation
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.
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
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.
Total DNA from each sample was resuspended in 100 ul of 100 mM Tris, 10 mM EDTA buffer and 10 ul aliquots were loaded onto a 4% agarose gel for electrophoresis at 125 V constant voltage for about one hour. The results are shown in Figure 2. As shown in Figure 2, the yield of total DNA increased as the ratio of solvent to mass increased from 3x to 10x.
Example 3
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.
The results are shown in Figure 3. As shown, a ratio of 10:1 produced the greatest yield of nucleic acids. Figure 3 also shows the effect of RNase treatment on the yield of DNA from each stool sample. As shown in the Figure, RNase treatment virtually eliminates RNA from the sample, but leaves DNA intact. The results indicate that optimal solvent volume to stool mass ratios greatly increase DNA yield from stool samples. Example 4
Sequence-Specific Capture of target DNA.
Once extracted from stool, specific nucleic acids are isolated using sequence- specific capture probes. Total DNA was extracted from a stool sample according to the methods described in Example 1. The pelletized DNA was resuspended in 1 ml of TE buffer. A 100 μl aliquot of this solution was removed to a new tube and 100 μl of 6M guanidine isothiocyanate (GITC) was added to a final concentration of 3M GITC. A vast excess of biotinylated kras capture probe CP1 was the added to the sample. The mixture was heated to 95°C for 5 minutes to denature the DNA, then cooled to 37°C for 5 minutes. Finally, probe and target DNA were allowed to hybridize for 30 minutes at room temperature. Streptavidin-coated magnetized beads (320 mg) (Dynal Corp.) were suspended in 400 μl distilled water and added to the mixture. After briefly mixing, the tube was maintained at room temperature for 30 minutes.
Once the affinity binding was completed, a magnetic field was applied to the sample to draw the magnetized isolation beads (both with and without hybridized complex out of the sample. The beads were then washed four (4) times in 1M GITC/0.1 % Igepal (Sigma, St. Louis, MO) solution for 15 minutes, followed by two (2) washes with wash buffer (TE with 1 M NaCl) for 15 minutes in order to isolate complexed streptavidin. Finally, 10 μl distilled water was added to the beads and heated at 95°C for 3 minutes to elute the DNA. Sequencing and/or gel electrophoresis enable confirmation of the capture of kras-specific DNA.
Accordingly, 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. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: SHUBER, Anthony P. LAPIDUS, Stanley N. RADCLIFFE, Gail E.
(ii) TITLE OF INVENTION: Methods for Stool Sample Preparation
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Testa, Hurwitz & Thibeault
(B) STREET: 125 High St.
(C) CITY: Boston
(D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02110
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MEYERS, Thomas C.
(B) REGISTRATION NUMBER: 36,989
(C) REFERENCE/DOCKET NUMBER: EXT-006PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 248-7000
(B) TELEFAX: (617) 248-7100
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GCCTGCTGAA AATGACTGAA TATAAACTTG TGGTAGT 37
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : CCTGCTGAAA ATGACTGAA 19
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : CATGAAAATG GTCAGAGAAA 20
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 307 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GTACTGGTGG AGTATTTGAT AGTGTATTAA CCTTATGTGT GACATGTTCT AATATAGTCA 60
CATTTTCATT ATTTTTATTA TAAGGCCTGC TGAAAATGAC TGAATATAAA CTTGTGGTAG 120
TTGGAGCTGG TGGCGTAGGC AAGAGTGCCT TGACGATACA GCTAATTCAG AATCATTTTG 180
TGGACGAATA TGATCCAACA ATAGAGGTAA ATCTTGTTTT AATATGCATA TTACTGGTGC 240
AGGACCATTC TTTGATACAG ATAAAGGTTT CTCTGACCAT TTTCATGTAC AGAAGTCCTT 300
GCTAAGA 307

Claims

CLAIMSWhat is claimed is:
1. A method for extracting DNA from stool, comprising:
homogenizing a stool sample in a solvent for DNA in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5:1 ; and
extracting DNA from said homogenized sample mixture.
2. The method of claim 1 wherein the solvent volume to stool mass ratio is from about 10:1 to about 30:1.
3. The method of claim 2 wherein the solvent volume to stool mass ratio is about 10:1 to about 20:1.
4. The method of claim 2 wherein the solvent volume to stool mass ratio is about 10:1.
5. The method of claim 1 wherein the solvent comprises a physiologically compatible buffer.
6. The method of claim 5 wherein the buffer comprises Tris-EDTA-NaCI.
7. The method of claim 6 wherein the Tris-EDTA-NaCI buffer comprises a final concentration of about 50mM Tris, about 16 mM EDTA and about 10mM NaCl at about pH 9.0.
8. The method of claim 1 wherein the solvent comprises guanidine isothiocyanate buffer.
9. The method of claim 8 wherein the guanidine isothiocyanate buffer comprises a final concentration of from about 1 to about 5 M.
10. The method of claim 9 wherein the guanidine isothiocyanate buffer comprises a final concentration of about 3 M.
11. The method of claim 1 wherein said extracting step further comprises contacting said DNA with a sequence-specific capture probe.
12. The method of claim 1 wherein said solvent comprises a detergent and a proteinase.
13. The method of claim 1 wherein said DNA is human DNA.
14. A method of screening for the presence of a colorectal cancerous or pre- cancerous lesion in a patient, the method comprising the steps of:
obtaining a sample comprising at least a cross-sectional portion of a stool voided by the patient;
homogenizing the 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 ;
extracting target human DNA from said homogenized sample mixture; and
analyzing the extracted target human DNA for DNA characteristics indicative of the presence of said colorectal cancerous or pre-cancerous lesion.
15. The method of claim 14 wherein said analyzing step comprises amplifying the DNA with a polymerase chain reaction.
16. The method of claim 14 wherein said DNA characteristics comprise a loss of heterozygosity encompassing a polymorphic locus.
17. The method of claim 14 wherein said DNA characteristic is a mutation.
18. The method of claim 17 wherein said mutation is selected from the group consisting of loss of heterozygosity and microsatellite instability.
19. The method of claim 14 wherein said DNA characteristics comprise a deletion in a tumor suppressor allele.
20. The method of claim 14 wherein said analyzing step comprises determining whether a difference exists in said sample between a number X of a first allele known or suspected to be mutated in a subpopulation of cells in the sample and a number Y of a second allele that is known or suspected not to be mutated in a subpopulation of cells in the sample, the presence of a statistically-significant difference being indicative of a mutation in a subpopulation of cells in the sample and the potential presence of a cancerous or precancerous lesion.
21. The method of claim 14 wherein said analyzing step comprises determining whether a difference exists between a number of a target tumor suppressor allele in the sample and a number of a non-cancer-associated reference allele in the sample, the presence of a statistically-significant difference being indicative of a deletion of the target tumor suppressor allele in a subpopulation of cells in the sample and the potential presence of a cancerous or precancerous lesion.
22. The method of claim 14 wherein said analyzing step further comprises the steps of:
a) detecting an amount of a maternal allele at a polymorphic locus in the sample;
b) detecting an amount of a paternal allele at the polymorphic locus in the sample; and
c) determining whether a difference exists between the amounts of maternal and paternal allele,
the presence of a statistically-significant difference being indicative of a deletion at the polymorphic locus in a subpopulation of cells in the sample and the potential presence of a lesion.
23. The method of claim 22 wherein said polymorphic locus is a single base polymorphism and is heterozygous between said maternal and paternal alleles.
24. The method of claim 22 wherein said detecting steps comprise,
a) hybridizing probe to a portion of said polymorphic locus on both maternal and paternal alleles that is immediately adjacent to said single-base polymorphism;
b) exposing said sample to a mixture of detectably-labeled dideoxy nucleoside triphosphates under conditions which allow appropriate binding of said dideoxy nucleoside triphosphates to said single- base polymorphism;
c) washing the sample; and
d) counting an amount of each detectably-labeled dideoxy nucleoside triphosphate remaining for the sample.
25. The method of claim 24 wherein said detectable label is selected from the group consisting of radioisotopes, fluorescent compounds, and particles.
26. The method of claim 14 wherein said analyzing step comprises a method for detecting heterozygosity at a single-nucleotide polymorphic locus, comprising the steps of:
a) hybridizing probes to a sequence immediately adjacent to a single- base polymorphism;
b) exposing the sample to a plurality of different labeled dideoxy nucleotides
c) washing the sample;
d) determining which of said dideoxy nucleotides are incorporated into said probes; and e) detecting heterozygosity at the single-nucleotide polymorphic site as the detection of two dideoxy nucleotides having been incorporated into the probe.
27. The method of claim 14 wherein said analyzing step comprises:
(a) exposing the sample to a plurality of a first oligonucleotide probe and to a plurality of a second oligonucleotide probe under hybridization conditions, thereby to hybridize
(1 ) said first oligonucleotide probes to copies of a first polynucleotide segment characteristic of wild-type cells of the organism, and
(2) said second oligonucleotide probes to copies of a second polynucleotide segment characteristic of a wild-type genomic region suspected to be deleted or mutated in colorectal cancer cells;
(b) detecting a first number of duplexes formed between said first probe and said first segment and a second number of duplexes formed between said second probe and said second segment; and
(c) determining whether there is a difference between the number of duplexes formed between said first probe and said first segment and the number of duplexes formed between said second probe and said second segment,
the presence of a statistically-significant difference being indicative of the presence in said sample of a colorectal cancer or precancerous lesion.
28. The method of claim 27 wherein said first and second oligonucleotide probes each are coupled to a distinct detectable label.
29. The method of claim 27 wherein
said first oligonucleotide probes are attached to a first particle in a ratio of one first oligonucleotide probe to one particle and said second oligonucleotide probes are attached to a second particle detectably distinct from said first particle in a ratio of one second oligonucleotide probe to one second particle, wherein
said detecting step comprises separating hybridized from unhybridized first and second oligonucleotide probes and subsequently passing hybridized first and second oligonucleotide probes through a detector to determine said first and second numbers.
30. The method of claim 29 wherein said first and second particles are of detectably different sizes.
31. The method of claim 29 wherein said first and second particles are of detectably different colors.
32. The method of claim 27 further comprising, prior to step a) the steps of converting double-stranded DNA in said sample to single-stranded DNA and removing complement to said first and second polynucleotide segments.
33. The method of claim 32 wherein said removing step comprises hybridizing said complement to a nucleic acid probe attached to a magnetic particle and subsequently removing said magnetic particle from the sample.
34. The method of claim 14 wherein said analyzing step comprises a method for detecting a nucleic acid sequence change in a target allele in the sample, comprising the steps of:
(a) determining
(i) an amount of wild-type target allele in the sample, and
(ii) an amount of a reference allele in the sample; and
(b) detecting a nucleic acid sequence change in the target allele in the sample, a statistically significant difference in the amount wild-type target allele and the amount of reference allele obtained in said determining step being indicative of a nucleic acid sequence change.
35. The method according to claim 34 wherein said determining step comprises exposing said sample to a first oligonucleotide probe capable of hybridizing with a portion of said wild-type allele and to a second oligonucleotide probe capable of hybridizing to a portion of said reference allele, and removing from said sample any unhybridized first or second oligonucleotide probe.
PCT/US1998/011550 1997-06-16 1998-06-04 Methods for stool sample preparation WO1998058081A1 (en)

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)

Application Number Priority Date Filing Date Title
US87663897A 1997-06-16 1997-06-16
US08/876,638 1997-06-16

Publications (1)

Publication Number Publication Date
WO1998058081A1 true WO1998058081A1 (en) 1998-12-23

Family

ID=25368233

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/011550 WO1998058081A1 (en) 1997-06-16 1998-06-04 Methods for stool sample preparation

Country Status (2)

Country Link
AU (1) AU7816898A (en)
WO (1) WO1998058081A1 (en)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000031303A2 (en) * 1998-11-23 2000-06-02 Exact Laboratories, Inc. Methods for stool sample preparation
WO2000050640A1 (en) * 1999-02-25 2000-08-31 Exact Laboratories, Inc. Methods for preserving dna integrity
WO2000061808A2 (en) * 1999-04-09 2000-10-19 Exact Laboratories, Inc. Methods for detecting nucleic acids indicative of cancer
WO2001042504A2 (en) * 1999-12-07 2001-06-14 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
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
US6511805B1 (en) 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
EP1599489A1 (en) * 2002-02-13 2005-11-30 Medimolecular Pty. Ltd. Method of isolating nucleic acids from stool samples
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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19530132A1 (en) * 1995-08-16 1997-02-20 Max Planck Gesellschaft Process for the purification, stabilization or isolation of nucleic acids from biological materials
WO1997009600A2 (en) * 1995-09-06 1997-03-13 Medical Research Council Method of isolating cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19530132A1 (en) * 1995-08-16 1997-02-20 Max Planck Gesellschaft Process for the purification, stabilization or isolation of nucleic acids from biological materials
WO1997009600A2 (en) * 1995-09-06 1997-03-13 Medical Research Council Method of isolating cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
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 *
SIDRANSKY ET AL.: "Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors", SCIENCE, vol. 256, 1992, pages 102 - 105, XP000605488 *

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US6511805B1 (en) 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
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
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
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
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
WO2000031303A3 (en) * 1998-11-23 2003-07-03 Exact Lab Inc Methods for stool sample preparation
WO2000031303A2 (en) * 1998-11-23 2000-06-02 Exact Laboratories, Inc. Methods for stool sample preparation
AU752817B2 (en) * 1999-02-25 2002-10-03 Exact Sciences Corporation Methods for preserving DNA integrity
JP2002537777A (en) * 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 Methods for preserving DNA integrity
JP2011015701A (en) * 1999-02-25 2011-01-27 Exact Sciences Corp Method for preserving dna integrity
WO2000050640A1 (en) * 1999-02-25 2000-08-31 Exact Laboratories, Inc. Methods for preserving dna integrity
WO2000061808A3 (en) * 1999-04-09 2001-08-02 Exact Lab Inc Methods for detecting nucleic acids indicative of cancer
WO2000061808A2 (en) * 1999-04-09 2000-10-19 Exact Laboratories, Inc. Methods for detecting nucleic acids indicative of cancer
US6280947B1 (en) 1999-08-11 2001-08-28 Exact Sciences Corporation Methods for detecting nucleotide insertion or deletion using primer extension
WO2001042504A3 (en) * 1999-12-07 2002-08-29 Penn State Res Found Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
WO2001042504A2 (en) * 1999-12-07 2001-06-14 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
US6428964B1 (en) 2001-03-15 2002-08-06 Exact Sciences Corporation Method for alteration detection
EP1599489A4 (en) * 2002-02-13 2007-01-03 Medimolecular Pty Ltd Method of isolating nucleic acids from stool samples
EP1599489A1 (en) * 2002-02-13 2005-11-30 Medimolecular Pty. Ltd. Method of isolating nucleic acids from stool samples
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
US11198899B2 (en) 2014-03-07 2021-12-14 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples

Also Published As

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

Similar Documents

Publication Publication Date Title
US6268136B1 (en) Methods for stool sample preparation
US6406857B1 (en) Methods for stool sample preparation
EP1169479B1 (en) Methods for detecting nucleic acids indicative of cancer
JP3633932B2 (en) Method for detecting mammalian nucleic acid isolated from stool sample and reagent for detection thereof
US6632610B2 (en) Methods of identification and isolation of polynucleotides containing nucleic acid differences
US20080145852A1 (en) Methods and compositions for detecting adenoma
EP1185693A2 (en) Methods for improving sensitivity and specificity of screening assays
EP0817968A1 (en) Methods for detecting colon cancer from stool samples
WO1998058081A1 (en) Methods for stool sample preparation
AU767833B2 (en) Methods of detecting colorectal disease
CA3100912A1 (en) Tumor marker col4a1,methylation detection reagent, kit and use thereof
US20210189461A1 (en) Tumor marker, methylation detection reagent, kit and use thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1999504508

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA