Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-18
2002-11-05
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06475738
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods of detecting cancer, precancer, or other diseases or disorders using nucleic acid markers.
BACKGROUND OF THE INVENTION
Numerous diseases are associated with disruptions in genomic stability. For example, sickle cell anemia, phenylketonuria, hemophilia, cystic fibrosis, and various cancers have been associated with one or more genetic mutation(s). Cancer is thought to arise from a multi-step process that typically involves multiple genetic mutations leading to uncontrolled cell growth. Many cancers are curable if detected early in their development. For example, colorectal cancers typically originate in the colonic epithelium, and are not extensively vascularized (and therefore not invasive) during early stages of development. The transition to a highly-vascularized, invasive and ultimately metastatic cancer commonly takes ten years or longer. If the presence of cancer is detected prior to extensive vascularization, surgical removal typically is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and bloody stool. Generally, such symptoms are present only when the disease is well established, and often after metastasis has occurred. Similarly, with the exception of the Pap smear for detection of pre-malignant cervical lesions, diagnostic screening methods for other types of cancer are best at detecting established disease. Increased knowledge of the molecular basis for disease has lead to a proliferation of screening assays capable of detecting disease-associated nucleic acid mutations.
A variety of detection methods have been developed which exploit sequence variations in DNA using enzymatic and chemical cleavage techniques. A commonly-used screen for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of Southern blots, as reported by Botstein et al.,
Am. J Hum. Genet.,
32: 314-331 (1980) and White et al.,
Sci. Am.,
258: 40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of the DNA. Thus, a difference in restriction fragment lengths is indicative of the presence of a mutation in the recognition sequence. A problem with this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect a mutation outside of the recognition sequence and which, consequently, does not affect cleavage with a restriction endonuclease. One study reported that only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA were detected using RFLP mapping. Jeffreys,
Cell,
18: 1-18 (1979).
Single-base mutations have been detected by differential hybridization techniques using allele-specific oligonucleotide probes. Saiki et al.,
Proc. Natl. Acad. Sci.,
86: 6230-6234 (1989). Mutations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Disadvantages of this approach for mutation analysis include the requirement for optimization of hybridization for each probe, and the limitations imposed by the nature of the mismatch and the local sequence on the degree of discrimination of the probes. In practice, tests based only on parameters of nucleic acid hybridization function poorly when the sequence complexity of the test sample is high (e.g., in a heterogeneous biological sample). This is due partly to the small thermodynamic differences in hybrid stability generated by single nucleotide changes. Therefore, nucleic acid hybridization is generally combined with some other selection or enrichment procedure for analytical and diagnostic purposes.
Recently, a number of genetic mutations, including alterations in the BAT-26 segment of the MSH2 mismatch repair gene, the p53 gene, the Kras oncogene, and the APC tumor suppressor gene have been associated with the multi-step pathway leading to cancer. The BAT-26 segment contains a long poly-A tract. In certain cancers, a characteristic 5 base pair deletion occurs in the poly-A tract. Detection of that deletion may provide diagnostic information. For example, it has been suggested that mutations in those genes might be a basis for molecular screening assays for the early stages of certain types of cancer. See e.g., Sidransky, et al.,
Science,
256: 102-105 (1992). Attempts have been made to identify and use nucleic acid markers that are indicative of cancer. However, even when such markers are found, using them to screen patient samples, especially heterogeneous samples, has proven unsuccessful either due to an inability to obtain sufficient sample material, or due to the low sensitivity that results from measuring only a single marker. For example, simply obtaining adequate human DNA from one type of heterogeneous sample (stool) has proven difficult. See Villa, et al.,
Gastroenterol.,
110: 1346-1353 (1996) (reporting that only 44.7% of all stool specimens, and only 32.6% of stools from healthy individuals produced sufficient DNA for mutation analysis). Other reports in which adequate DNA has been obtained have reported low sensitivity in identifying a patient's disease status based upon a single cancer-associated mutation. See Eguchi, et al.,
Cancer,
77: 1707-1710 (1996) (using a p53 mutation as a marker for cancer).
Therefore, there is a need in the art for high-sensitivity, high-specificity assays for the detection of molecular indicia of cancer, pre-cancer, and other diseases or disorders, especially in heterogeneous samples. Accordingly, the invention provides methods for detecting deletions in genomic regions, such as BAT-26 and others, which may be associated with disease.
SUMMARY OF THE INVENTION
Methods of the invention provide assays for identification of a mutation in a genomic region suspected to be indicative of disease. In general, methods of the invention comprise annealing a primer upstream of a region in which, for example, a deletion is suspected to occur, extending the primer through the region, terminating extension at a known end-point, and comparing the length and/or weight of the extended primer with that of an extended primer from the corresponding wild-type (non-affected) region or a molecular weight standard (either known or run in parallel). Also according to the invention, assays described herein are combined with invasive detection methods to increase sensitivity of detection.
Methods of the invention further provide for the determination of whether a target point mutation is present at a genetic locus of interest. In one embodiment, the invention comprises contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of a genetic locus, extending the primer in the presence of a labeled nucleotide that is complementary to a target nucleotide suspected to be present at the target position. The primer is further extended in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from the target nucleotide, but is not complementary to the target nucleotide, thereby generating an extension product. The presence of a labeled nucleotide in the extension product is indicative of the presence of the target point mutation at the genetic locus.
In addition, methods of the invention provide for the identification of a target single nucleotide polymorphic variant present at a genetic locus of interest. In one embodiment, the method comprises contacting a nucleic acid in a biological sample with a primer, extending the primer in the presence of at least a first and a second differentially labeled nucleotide, the first labeled nucleotide being complementary to a first nucleotide suspected to be present at said target position, the second labeled nucleotide being complementary to a second nucleotide alternatively suspected to be present at the target position. The primer is further extended in t
Pierceall William
Shuber Anthony P.
Chunduru Suryaprabha
Exact Sciences Corporation
Fredman Jeffrey
Testa Hurwitz & Thibeault
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