Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-15
2001-03-20
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024310, C536S024300, C536S023500, C435S091200
Reexamination Certificate
active
06203992
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to oncology and, in particular, it relates to oligonucleotides for detecting carcinoma in a test sample.
BACKGROUND OF THE INVENTION
Studies have suggested that the presence of epithelial cells in the hematopoietic system indicates the spread of cancer from a localized area to other parts of the body (also known as metastisis). This discovery is important since metastisis is diagnostic of certain stages of cancer, and decisions concerning the proper treatment of a cancer patient are largely dependent upon properly characterizing the stage of the disease. In particular, treatment of patients having localized cancer can be vastly different from treatment of patients in metastatic stages of cancer.
Early efforts to detect the spread of cancer by detecting epithelial cells in the hematopoietic system included immunocytological assay procedures. Unfortunately, these methods are largely inaccurate because antibodies used in these assays, and ostensibly specific for epithelial cells, demonstrate crossreactivity for cells normally found in the hematopoietic system. Hence, “normal hematopoietic cells” are sometimes detected in the absence of metastatic cells and therefore, false positive results can be obtained according to these assay procedures. Additionally, immunocytological assays lack sensitivity and can produce false negative results when low levels of epithelial cells are actually present in the hematopoietic system. Accordingly, early stages of metastatic cancer can be misdiagnosed using immunocytological asays.
With the advent of nucleic acid amplification reactions such as the polymerase chain reaction (PCR), epithelial cells present in the hematopoietic system can be detected at the nucleic acid level instead of at the protein level. Hence, problems associated with crossreactive antibodies are avoided. Additionally, it is well known that nucleic acid amplification reactions are significantly more sensitive than more conventional antibody based assay methods. Amplification based assays for detecting epithelial cells in the blood stream have therefore provided significant advantages over immunocytological assay methods for detecting early stages of metastatic cancer.
PCR based assays employed to detect epithelial cells in the hematopoietic system have been reported in the literature. Most of these assays target a nucleic acid sequence encoding cytokeratin 19 (CK19), a protein found on the surface of epithelial cells. However, psuedogenes (comprising a nucleic acid sequence that closely mimics the gene for CK19) are present in the human genome. Thus, one challenge facing those developing amplification assays to detect a CK19 target sequence is to design assays that amplify and detect a sequence from the CK19 gene but not the closely related pseudogene.
Additionally, it is well known that amplification primer sequences can be selected based upon computer comparisons of closely related sequences. Theoretically, sequences selected in this manner effectively should produce copies of the selected target sequence when employed according to nucleic acid amplification principles. Notwithstanding the theoretical efficacy of sequences selected in the above manner, it is often times true that such sequences do not produce acceptable amounts of amplification product. Unfortunately, this phenomenon is not understood. Accordingly, while primers initially can be screened using computer programs efficacy cannot be adequately determined until such primers are employed in practice.
A further challenge faces those designing PCR assays that use microparticle capture based detection procedures for detecting amplification products. Specifically, amplified target sequences detected with the assistance of microparticles must be sufficiently short so that amplification product captured on the microparticle does not interfere with the capture of additional amplification product. Accordingly, those choosing to detect amplification products with the assistance of a microparticle are faced with an added restriction in terms of selection of a suitable target sequence. In particular, suitable target sequences are constrained to sequences that are relatively short.
There is therefore a need in the art for a method and sequences that can be employed according to nucleic acid amplification principles to detect a CK 19 target sequence using microparticle based detection techniques.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid sequences that can be used to specifically and sensitively detect a CK 19 target sequence. In particular, primers sequences employed in the present invention are designated SEQ ID NO 2 and SEQ ID NO 3. Sequences identified herein as SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7, SEQ ID NO 10, are employed as probes for detecting the amplification product produced by SEQ. ID. NOs. 2 and 3. Combinations of the above sequences can be provided in kits along with other reagents for performing an amplification reaction to detect a CK 19 target sequence in peripheral blood.
The CK 19 target sequence, designated herein as SEQ. ID. NO. 1, can be amplified by forming a reaction mixture comprising nucleic acid amplification reagents, a test sample containing a CK 19 target sequence, and a primer set containing SEQ ID NOs. 2 and 3. Following amplification, the amplified target sequence can be detected. For example, any probe or any combination of the probes designated SEQ ID NOs. 4, 5, 6, and 7 can be employed to hybridize to the amplified target sequence to form a probe/amplification product hybrid which can then be detected using microparticle capture techniques. Hence, the primers or probes can be labeled to capture and detect the amplified target sequence and therefore indicate the presence of the target sequence in the test sample.
DETAILED DESCRIPTION OF THE INVENTION
As previously mentioned, the present invention provides reagents, methods, and kits for amplifying and detecting a CK-19 target sequence in a test sample. In particular, SEQ. ID. Nos. 2 and 3 can be employed as amplification primers to amplify the CK 19 target sequence designated herein as SEQ. ID. NO. 1. It was discovered that these primers specifically and sensitively produce an amplification product that is amenable to microparticle capture and detection techniques. Probe sequences, having SEQ. ID. Nos. 4 through 7 can be employed to insure specificity and detect the amplification product.
The primer and probe sequences disclosed herein, may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or nucleic acid analogs such as uncharged nucleic acid analogs including but not limited to peptide nucleic acids (PNAs) which are disclosed in International Patent Application WO 92/20702 or morpholino analogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506, and 5,142,047 all of which are herein incorporated by reference. Such sequences can routinely be synthesized using a variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, Calif.); DuPont, (Wilmington, Del.); or Milligen, (Bedford, Mass.). Similarly, and when desirable, the sequences can be labeled using methodologies well known in the art such as described in U.S. Pat. Nos. 5,464,746; 5,424,414; and 4,948,882 all of which are herein incorporated by reference. It will be understood, however, that the sequences employed as primers should at least comprise DNA at the 3′ end of the sequence and preferably are completely comprised of DNA.
A “target sequence” as used herein means a nucleic acid sequence that is detected, amplified, both amplified and detected or otherwise is complementary to one of the sequences herein provided. While the term target sequence is sometimes referred to as single stranded, those skilled in the art will recognize that the target sequence may actually be double stranded.
The term “test
Granados Edward N.
Scheffel Christi Powell
Abbott Laboratories
Einsmann Juliet C
Fredman Jeffrey
Yasger Paul D.
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