Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-24
2003-04-22
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S025400, C536S025410, C536S025420, C536S023740
Reexamination Certificate
active
06551777
ABSTRACT:
TECHNICAL FIELD
The invention provides methods for deoxyribonucleic acid (“DNA”) extraction from a biological sample. More particularly, the invention relates to methods for high yield DNA extraction from a heterogeneous biological sample by inhibiting DNA degradation.
BACKGROUND OF THE INVENTION
DNA is a relatively stable molecule that is routinely isolated from biological samples. Recently, many diseases involving instabilities (e.g., mutations) in genomic DNA have been characterized. Also, many pathogens have been identified by the presence or absence of a particular DNA in a biological sample. Many diseases, such as cancer, are optimally detected early in their progression. In order for early detection to be effective, relatively low levels of DNA which are indicative of cancer must be detected against a high background of other DNA (e.g., normal human DNA, bacterial DNA, etc.). This type of detection is technically difficult and typically results in low sensitivity of detection. Moreover, in certain complex specimens, including stool, what little species-specific DNA exists, is rapidly degraded, making efficient sequence-specific detection even more difficult. Thus, a need exists for methods to retain integrity of DNA in a sample, especially in samples in which the DNA to be detected is in low proportion relative to other DNA in the sample and is degraded quickly.
SUMMARY OF THE INVENTION
The present invention provides methods for preserving the integrity of DNA in a sample. In a preferred embodiment, methods of the invention prevent enzyme-mediated DNA degradation. Preservation of DNA integrity facilitates isolation and detection of DNA.
Methods of the invention are especially useful for extracting or detecting DNA in a biological specimen, especially one that contains low levels of relevant DNA. A good example of a specimen that contains lower-levels of relevant DNA is stool. Typical human stool contains only small amounts of intact human DNA. Most of the human DNA in stool from a healthy individual is presumably from exfoliated epithelial cells, and has undergone apoptotic degradation. As the forming stool passes through the colon, colonic epithelial cells are sloughed onto the stool as part of the cellular turnover that occurs in the colon. Stool also contains sloughed cells from other luminal sources (e.g., lung, stomach, esophagus, etc.) Sloughed cells typically have undergone or are undergoing apoptosis, leaving cellular DNA in small fragments. Enzymes, such as deoxyribonuclease (“DNase”) and Micrococcal nuclease contribute to the degradation of any intact human DNA that remains. Prior art methods, while using DNase inhibitors, have failed to achieve significant yields of intact, species-specific DNA from stool. Therefore, such methods failed to consider optimization of inhibition of DNA degradation. Methods of the invention are based on the realization that optimal inhibition of DNA degrading enzyme(s) effectively preserves DNA, especially large, diagnostically-relevant DNA fragments that are present in a sample.
In one aspect, the invention comprises inhibiting nucleic acid degradation in a sample and optionally extracting a target DNA with, for example, a phenol-chloroform extraction. Preferably, the inhibition of nucleic acid degradation is sufficient to produce a critical number of molecules of analyzable DNA. In one embodiment, methods of the invention comprise inhibiting an enzyme capable of DNA degradation in a stool sample. In a preferred embodiment, methods of the invention comprise exposing a stool sample to an ion chelator, such as a divalent ion chelator. Ion chelators, in certain embodiments inhibit DNase. Examples of preferred inhibitors include ethylenediaminetetraacetic acid (“EDTA”). Additional preferred methods of the invention comprise exposing a stool sample to a Micrococcal nuclease inhibitor, such as EGTA, also a divalent ion chelator. Inhibitors of DNA degradation may be used either alone or in combination to achieve optimal levels of DNA preservation.
Methods of the invention are practiced using any inhibitor of DNA degradation. The amount of inhibitor varies depending on the inhibitor that is used. However, an inhibitor must be used in an amount that preserves significant levels of DNA in the sample for subsequent analysis. Methods for determining sufficient levels of DNA are presented below. Such methods allow the skilled artisan to practice the invention with specificity regardless of the inhibitor used. According to preferred methods, an amount of inhibitor is used that preserves sufficient DNA in the sample for detection of a target DNA within a desired level of statistical confidence. Using methods described herein, the skilled artisan can determine an appropriate amount of any inhibitor for use in methods of the invention. The use of various specific inhibitors is exemplified below.
In another preferred embodiment, methods of the invention comprise obtaining a representative (circumfrential or cross-sectional) stool sample, exposing the sample or a portion thereof to a DNase inhibitor, and isolating DNA from the sample. One preferred DNase inhibitor is EDTA. Preferred amounts of EDTA are from about 0.042 g per gram of stool to about 0.782 g per gram of stool and especially from about 0.250 g per gram of stool to about 0.521 g per gram of stool. DNA may be extracted, for example, by a phenol-chloroform extraction. After extraction, the DNA may be analyzed by methods known in the art. For example, U.S. Pat. No. 5,830,665 and U.S. Pat. No. 5,670,325, which are incorporated by reference herein, disclose methods for analyzing DNA which has been extracted from a stool sample.
Methods of the invention are useful in any sample in which inhibition of DNA degradation is desired. For example, methods of the invention are especially effective in samples comprising exfoliated cells, especially exfoliated epithelial cells. The DNA contained in such samples typically degrades rapidly, making analysis of a particular DNA, especially one that exists in low proportion within the sample, difficult. For example, such samples include stool, sputum, urine, pus, and collostrum. Methods of the invention include inhibiting DNA degradation in such samples, thus preserving a sufficient amount of DNA for specific, sensitive detection. Any of the features described above, such as DNA degradation inhibitors or amounts of inhibitors that are used, can be useful in samples containing exfoliated cells.
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Huntress, Jr. Frederick A.
Moore James K.
Shuber Anthony P.
Exact Sciences Corporation
Katcheves Konstantina
Testa Hurwitz & Thibeault LLP
Yucel Remy
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