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Reagents and methods useful for detecting diseases of the gastrointestinal tract

a technology of gastrointestinal tract and reagents, applied in the field of detecting diseases of the gastrointestinal tract, can solve the problems of few gi tract cancer detections, high sensitivity, and high cost, and achieve the effect of maintaining the integrity of the specimen and avoiding denaturation or irreversible adsorption of the sampl

Inactive Publication Date: 2002-09-12
BILLING MEDEL PATRICIA A +8
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0013] The present invention further provides a method of detecting a target CS193 polynucleotide in a test sample suspected of containing target CS193 polynucleotides, which comprises (a) contacting the test sample with at least one CS193 oligonucleotide as a sense primer and at least one CS193 oligonucleotide as an anti-sense primer, and amplifying same to obtain a first stage reaction product; (b) contacting the first stage reaction product with at least one other CS193 oligonucleotide to obtain a second stage reaction product, with the proviso that the other CS193 oligonucleotide is located 3' to the CS193 oligonucleotides utilized in step (a) and is complementary to the first stage reaction product; and (c) detecting the second stage reaction product as an indication of the presence of a target CS193 polynucleotide in the test sample. The CS193 oligonucleotides selected as reagents in the method have at least 50% identity to a sequence selected from the group consisting of SEQUENCE ID NOS 1-18, and fragments or complements thereof. Amplification may be performed by the polymerase chain reaction. The test sample can be reacted either directly or indirectly with a solid phase

Problems solved by technology

In addition to its high incidence, GI tract cancers can be extremely lethal; for example, greater than 97% of pancreatic cancer patients will die of the disease.
However, only few GI tract cancers are detected at this stage.
Although FOBT is noninvasive, simple and inexpensive, its sensitivity is low; for example, sensitivity for detecting colorectal cancer was only 26% in one study.
Further, although flexible sigmoidoscopy is highly sensitive for detecting early cancer and precursor polyps, it is invasive, costly, and too technically demanding to be used for routine screening.
These procedures are invasive and costly.
Moreover, an erroneous diagnosis can result from any of these procedures due to technical reasons, the subjective interpretation of results, or lack of sensitivity of the procedure.
Inaccurate staging can result in poor therapeutic decisions and is a major clinical problem in colorectal cancer.
These monitoring techniques, however, have failed to provide an accurate and effective means to monitor the progress of these patients.
Currently, no universally acceptable marker(s) exist(s) for the early detection of pancreatic, stomach, and esophageal cancers.

Method used

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  • Reagents and methods useful for detecting diseases of the gastrointestinal tract
  • Reagents and methods useful for detecting diseases of the gastrointestinal tract
  • Reagents and methods useful for detecting diseases of the gastrointestinal tract

Examples

Experimental program
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Effect test

example 1

Identification of Gastrointestinal Tract Tissue Library CS193 Gene-Specific Clones

[0191] A. Library Comparison of Expressed Sequence Tags (ESTs) or Transcript Images. Partial sequences of cDNA clone inserts, so-called "expressed sequence tags" (ESTs), were derived from cDNA libraries made from GI tract tumor tissues, GI tract non-tumor tissues and numerous other tissues, both tumor and non-tumor and entered into a database (LIFESEQ.TM. database, available from Incyte Pharmaceuticals, Palo Alto, Calif.) as gene transcript images. See International Publication No. WO 95 / 20681. (A transcript image is a listing of the number of EST's for each of the represented genes in a given tissue library. ESTs sharing regions of mutual sequence overlap are classified into clusters. A cluster is assigned a cone number from a representative 5' EST. Often, a cluster of interest can be extended by comparing its consensus sequence with sequences of other EST's which did not meet the criteria for automat...

example 2

Sequencing of CS193 EST-Specific Clones

[0193] The full-length DNA sequences of clones 774134 and 774419 of the CS193 gene contig were determined using dideoxy termination sequencing with dye terminators following known methods (F. Sanger et al., PNAS U.S.A. 74:5463 (1977). These full-length sequences are referred to herein as clones 7741341H (SEQUENCE ID NO 16) and 7744191H (SEQUENCE ID 17), respectively.

[0194] Because the pINCY vector (available from Incyte Pharmaceuticals, Inc., Palo Alto, Calif.) contains universal priming sites just adjacent to the 3' and 5' ligation junctions of the inserts, approximately 300 bases of the insert were sequenced in both directions using two universal primers (SEQUENCE ID NO 21 and SEQUENCE ID NO 22, available from New England Biolabs, Beverly, Mass., and Applied Biosystems Inc, Foster City, Calif.). The sequencing reactions were run on a polyacrylamide denaturing gel, and the sequences were determined by an Applied Biosystems 377 Sequencer (avail...

example 3

Nucleic Acid

[0195] A. RNA Extraction from Tissue. Total RNA is isolated from GI tract tissues and from non-GI tract tissues. Various methods are utilized, including but not limited to the lithium chloride / urea technique, known in the art and described by Kato et al. (J. Virol. 61:2182-2191, 1987), and TRIzol.TM. (Gibco-BRL, Grand Island, N.Y.).

[0196] Briefly, tissue is placed in a sterile conical tube on ice and 10-15 volumes of 3 M LiCl, 6 M urea, 5 mM EDTA, 0.1 M P-mercaptoethanol, 50 mM Tris-HCl (pH 7.5) are added. The tissue is homogenized with a Polytrone homogenizer (Brinkman Instruments, Inc., Westbury, N.Y.) for 30-50 sec on ice. The solution is transferred to a 15 ml plastic centrifuge tube and placed overnight at -20.degree. C. The tube is centrifuged for 90 min at 9,000.times.g at 0-4.degree. C. and the supernatant is immediately decanted. Ten ml of 3 M LiCl are added and the tube is vortexed for 5 sec. The tube is centrifuged for 45 min at 11,000.times.g at 0-4.degree. C...

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Abstract

A set of contiguous and partially overlapping cDNA sequences and polypeptides encoded thereby, designated as CS193 and transcribed from GI tract tissue, are described. These sequences are useful for the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the GI tract, such as GI tract cancer. Also provided are antibodies which specifically bind to CS193-encoded polypeptide or protein, and agonists or inhibitors which prevent action of the tissue-specific CS193 polypeptide, which molecules are useful for the therapeutic treatment of GI tract diseases, tumors or metastases.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001] This application is a continuation-in-part of U.S. application Ser. No. 08 / 828,856, filed Mar. 31, 1997, from which priority is claimed pursuant to 35 U.S.C. .sctn.120 and which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002] The invention relates generally to detecting diseases of the gastrointestinal tract organs, and more particularly, relates to reagents such as polynucleotide sequences and the polypeptide sequences encoded thereby, as well as methods which utilize these sequences, which are useful for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining predisposition to diseases and conditions of the GI tract such as cancer.[0003] The organs of the GI tract include the esophagus, stomach, small and large intestines, rectum and pancreas. Of the approximately 225,900 new cases of GI tract cancer projected for the United States during 1996, 131,200 wil...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12Q1/68
CPCC07K16/3046C12Q1/6886C12Q2600/106C12Q2600/136C07K2317/34
Inventor BILLING-MEDEL, PATRICIA A.COHEN, MAURICECOLPITTS, TRACEY L.FRIEDMAN, PAULA N.HAYDEN, MARK A.KLASS, MICHAEL R.ROBERTS-RAPP, LISARUSSELL, JOHN C.STROUPE, STEPHEN D.
Owner BILLING MEDEL PATRICIA A