Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-12-12
2004-04-27
Horlick, Kenneth R. (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024310, C536S024330
Reexamination Certificate
active
06727354
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of diagnostic assays and assays for identifying patients at risk for development of glaucoma.
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Glaucoma is the second most common cause of blindness in the United States. It is estimated that some two million Americans have glaucoma, with half of those suffering unaware of the presence of the disease. Primary open-angle glaucoma (“POAG”) is the most common form of glaucoma, accounting for some 60 to 70% of all glaucomas.
POAG is characterized by obstruction of the normal aqueous outflow of fluids through the trabecular meshwork, canal of Schlemm, intrascleral channels, and episcleral and conjunctival veins. In open-angle glaucoma, this obstruction exists despite an angle that appears open. Generally, a patient that has not been otherwise diagnosed as having glaucoma first becomes aware of the disease due to losses in the visual field. By this point, the degree of optic nerve atrophy resulting from the disease may be quite severe, and is irreversible. Thus, early diagnosis and treatment play a key role in patient management.
Several risk factors have been identified as being related to POAG, including elevated intraocular pressure (“IOP,” 50% of patients present with an IOP of <22 mm Hg), increased age (POAG is 6× more common in persons >60 years of age), a family history of the disease (a 15× increased chance of developing glaucoma), race (African Americans are at an increased risk for more serious disease), diabetes, hypertension, myopia, and the use of corticosteroids.
While elevated IOP is a primary risk factor for development of POAG, about ⅙ of patients exhibit an IOP within the normal range. Additionally, there is currently no reliable method for predicting which patients presenting with elevated IOP will progress to POAG. Recently, the relationship of the trabecular meshwork-inducible glucocorticoid response (“TIGR”) gene and protein have been studied for possible associations with glaucoma and related diseases. See, e.g., U.S. Pat. Nos. 5,861,497, 5,916,778, 5,925,748, and 6,248,867; Morissette et al., Nat. Genet. 19: 319-21 (1998); Brezin et al., Am. J. Med. Genet. 76: 438-45 (1998); Shimizu et al., Am. J. Ophthalmol. 130: 165-77 (2000); Nguyen et al., J. Biol. Chem. 273: 6341-50 (1998); and Lindblad-Toh et al., Nat. Genet. 24: 381-6 (2000). Each publication and patent in the foregoing section is hereby incorporated by reference in its entirety, including all tables, figures, and claims.
SUMMARY OF THE INVENTION
The present invention is drawn to methods and compositions for the screening of samples for one or more TIGR polymorphisms. The sample can be a biological sample, such as a sample from a subject. The invention can be used to determine which of a plurality of TIGR polymorphisms are present in the genome of a subject. Preferably, a plurality of different samples are assayed, each in its own individual reaction mixture, and/or several different polymorphisms in the TIGR gene are assayed in that single reaction mixture. Thus, a plurality of samples may be simultaneously assayed for several different TIGR polymorphisms in a single cycle (batch run) of the assay.
In a first aspect, the invention provides methods of testing for the presence of one or more polymorphisms of a TIGR gene, in one or more samples comprising TIGR nucleic acids, by generating a labeled nucleic acid that provides a means of identifying a particular polymorphism, thus distinguishing that polymorphism from other polymorphisms that might be present in the same gene. The particular polymorphism may be identified, for example, by determining both the length of the labeled nucleic acid and the identity of a distinctively labeled nucleotide incorporated at an end of the nucleic acid.
In preferred embodiments, these methods comprise one or more of the following steps: (a) preparing a reaction mixture that contains (i) sample nucleic acid suspected of containing a TIGR nucleic acid sequence, (ii) a nucleic acid polymerase, (iii) one or more extension primers, wherein the extension primers comprise nucleotide sequences that terminate at positions located one nucleotide 3′ from the positions of one or more preselected polymorphism(s) of interest, and (iv) one or more labeled dideoxynulceotide triphosphates, or ddNTPs; (b) incubating the reaction mixture under conditions such that extension primers that hybridize to the TIGR nucleic acids are labeled by addition of one of the ddNTPs comprising a label to the 3′-end of the detection primer, in order to generate one or more labeled oligonucleotides; and (c) detecting a signal from the labeled oligonucleotides. The presence of a specific polymorphism can be identified by the presence of a distinctive signal at a position in the sequence of the extended nucleic acid.
In certain embodiments, TIGR nucleic acid obtained from a sample is amplified to provide an amount of TIGR nucleic acid sufficient for primer extension to determine the presence or absence of one or more polymorphic forms of TIGR in the original sample. While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (“PCR”), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods.
The phrase “TIGR nucleic acid” refers to any nucleic acid containing sequences directly associated with production of the TIGR protein, including TIGR genomic DNA, TIGR-encoding hnRNA, mature TIGR-encoding mRNA, or amplification products thereof. Any TIGR nucleic acid may be the subject of the methods described herein. In certain embodiments for example, a TIGR RNA may be reverse transcribed into DNA, and the DNA subjected to the analysis methods described hereinafter. In preferred embodiments, the methods are applied to TIGR gene sequences.
The phrases “TIGR gene sequences,” “TIGR genomic DNA,” and “TIGR gene” as used herein refer to the nucleic acid unit present in the genome of an animal, preferably a human, encoding the TIGR protein, and includes both the TIGR coding sequence and the upstream enhancer and promoter regions operably associated with the TIGR coding sequence in the genome.
The term “biological sample” as used herein refers to a sample obtained from a biological source, e.g., an organism, cell culture, tissue sample, etc. A biological sample can, by way of non-limiting example, consist of or comprise blood, sera, urine, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and/or chorionic villi. Convenient biological samples may be obtained by, for example, scraping cells from the surface of the buccal cavity.
The term “subject” as used herein refers to any eukaryotic organism. Preferred subjects are fungi, invertebrates, insects, arachnids, fish, amphibians, reptiles, birds, marsupials and mammals. A mammal can be a cat, dog, cow, pig, horse, ox, elephant, simian. Most preferred subjects are humans. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “animals” includes prenatal forms of animals, such as fetuses.
As used herein, a “plurality of samples” refers to at least two. Preferably, a plurality refers to a relatively large number of samples. A plurality of samples is from about 5 to about 500 samples, preferably about 25 to about 200 samples, most preferably from about 50 to about 200 samples. Samples that are processed in a single batch run of the method of the invention are usually prepared in plates having 24, 48, 96, 144, or 192 wells. The term “samples” includes samples per se as well as controls, st
Horlick Kenneth R.
Quest Diagnostics Investments Inc.
Warburg Richard J.
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