Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-21
2002-06-18
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C436S063000, C436S064000
Reexamination Certificate
active
06406857
ABSTRACT:
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.
Use of the polymarase chain reaction (PCR) has made detection of nucleic acids more routine, but any PCR is limited by the amount of DNA present in a sample. A minimum amount of material must be present for specific analysis and this limitation becomes more relevant when one seeks to detect a nucleic acid that is present in a sample in small proportion relative to other nucleic acids in the sample, which is often the case when analyzing stool sample for detecting DNA characteristics of colorectal cancer. If a low-frequency mutant strand is not amplified in the first few rounds of PCR, any signal obtained from the mutant strand in later rounds will be obscured by background or by competing signal from amplification of ubiquitous wild-type strand.
An additional problem encountered 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, then enriching the homogenized sample for the target (human) DNA. The human DNA may then be analyzed for the characteristics of disease. 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-NaCl. A preferred buffer is a Tris-EDTA-NaCl 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 enriching the homogenized sample mixture for human DNA by, for example, using sequence-specific nucleic acid probes hybridizing to target human DNA.
In an alternative preferred embodiment, the methods of the invention comprise homogenizing, a stool sample in a physiologically-acceptable solvent for DNA in order to form a homogenized sample mixture having a solvent volume to stool mass ratio of at least 5:1; ensuring that the homogenized sample has at least a minimum number N of total DNA molecules to facilitate detection of a low-frequency target DNA molecule; and analyzing the target DNA for the characteristics of disease, preferably by amplifying the target DNA with a polymerase chain reaction.
In another embodiment, the present invention provides methods for analyzing DNA extracted from stool which comprise 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; enriching the homogenized sample for human DNA; ensuring that the enriched homogenized sample has at least a minimum number N of total DNA molecules to provide for detection of a low-frequency target DNA molecule; and analyzing the target DNA for DNA characteristics indicative of disease.
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; enriching the sample for target human DNA; 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 Ser. 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 repres
Lapidus Stanley N.
Radcliffe Gail E.
Shuber Anthony P.
Exact Sciences Corporation
Horlick Kenneth R.
Testa Hurwitz & Thibeault LLP
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