US5328824A
Expired / Lapsed
US
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Methods of using labeled nucleotides
Assignee: Yale University
•
Filing: 1987-12-08
•
Granted: 1994-07-12
•
Expires: 2007-12-08
Abstract
Abstract Compounds having the structure: ##STR1## wherein B represents a purine, 7-deazapurine, or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached at the N 9 -position of the purine or 7-deazapurine and when B is pyrimidine, it is attached at the N 1 -position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex, or DNA-RNA hybrid; wherein the dotted line represents a chemical linkage joining B and A, provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and wherein each of x, y and z represents ##STR2## either directly, or when incorporated into oligo- and polynucleotides, provide probes which are widely useful. Applications include detection and localization of polynucleotide sequences in chromosomes, fixed cells, tissue sections, and cell extracts. Specific applications include chromosomal karyotyping, clinical diagnosis of nucleic acid-containing etiological agents, e.g. bacteria, viruses, or fungi, and diagnosis of genetic disorders.
Inventors
- 👤 David C. Ward
- 👤 Pennina R. Langer
- 👤 Alexander A. Waldrop, III
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 08:38
CPC Classifications
C; C07; C07H; C07H19/00; C07H19/02; C07H19/04; C07H19/16; C07H19/20; C07H21/00; C12; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12Q; C12Q1/00; C12Q1/02; C12Q1/18; C12Q1/68; C12Q1/6804; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 43
Claim 1
Independent Claim
291 words
A method of detecting the presence or absence of a nucleic acid in a sample which comprises the steps of (a) contacting under hybridizable conditions said sample with at least one compound comprising the structure: ##STR26## wherein each of B' and B" represents a purine, 7-deazapurine, or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that whenever B' or B" is purine or 7-deazapurine, the sugar moiety is attached at the N 9 -position of the purine or 7-deazapurine, and whenever B' or B" is pyrimidine the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein B represents 7-deazapurine or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that whenever B is 7-deazapurine, the sugar moiety is attached at the N 9 -position of the 7-deazapurine, and whenever B is pyrimidine the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or indirectly through a linkage group, said linkage group not interfering substantially with the characteristic ability of said compound to hybridize with said nucleic acid or of A to be detected; wherein if B is 7-deazapurine, A is attached to the 7-position thereof, and if B is pyrimidine, A is attached to the 5-position thereof; wherein m, n and p are integers, provided that m and p are not simultaneously 0 and provided further n is never 0; and wherein z represents H-- or HO--; and (b) detecting said compound or compounds so as to detect said nucleic acid.
Claim 2
Independent Claim
291 words
A method of detecting the presence or absence of a nucleic acid in a sample which comprises the steps of (a) contacting under hybridizable conditions said sample with at least one compound comprising the structure: ##STR26## wherein each of B' and B" represents a purine, 7-deazapurine, or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that whenever B' or B" is purine or 7-deazapurine, the sugar moiety is attached at the N 9 -position of the purine or 7-deazapurine, and whenever B' or B" is pyrimidine the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein B represents 7-deazapurine or pyrimidine moiety covalently bonded to the C 1' -position of the sugar moiety, provided that whenever B is 7-deazapurine, the sugar moiety is attached at the N 9 -position of the 7-deazapurine, and whenever B is pyrimidine the sugar moiety is attached at the N 1 -position of the pyrimidine; wherein A comprises at least three carbon atoms and represents at least one component of a signalling moiety capable of producing a detectable signal; wherein B and A are covalently attached directly or indirectly through a linkage group, said linkage group not interfering substantially with the characteristic ability of said compound to hybridize with said nucleic acid or of A to be detected; wherein if B is 7-deazapurine, A is attached to the 7-position thereof, and if B is pyrimidine, A is attached to the 5-position thereof; wherein m, n and p are integers, provided that m and p are not simultaneously 0 and provided further n is never 0; and wherein z represents H-- or HO--; and (b) detecting said compound or compounds so as to detect said nucleic acid.
Claim 3
Dependent Claim
12 words
The method of claim 1 wherein the moiety A is a ligand.
Claim 4
Dependent Claim
24 words
The method of claim 2 wherein the ligand is selected from the group consisting of a hapten, an antigen, a cofactor, biotin and iminobiotin.
Claim 5
Dependent Claim
22 words
The method of claim 2 wherein the ligand is selected from the group consisting of dinitrophenol, lipoic acid and an olefinic compound.
Claim 6
Dependent Claim
20 words
The method of claim 2 wherein the ligand is capable of forming a complex by binding with a detectable polypeptide.
Claim 7
Dependent Claim
29 words
The method of claim 5 wherein the detectable polypeptide is selected from the group consisting of an antibody, an enzyme capable of depositing insoluble reaction products, streptavidin and avidin.
Claim 8
Dependent Claim
33 words
The method of claim 5 wherein the sample is contacted with the detectable polypeptide after hybridization of the compound or compounds to said nucleic acid under suitable conditions as to form the complex.
Claim 9
Dependent Claim
18 words
The method of claim 5 wherein an indicator molecule is associated with or bound to the detectable polypeptide.
Claim 10
Dependent Claim
22 words
The method of claim 8 wherein the indicator molecule is fluorescent, electron dense, or an enzyme capable of depositing insoluble reaction products.
Claim 11
Dependent Claim
20 words
The method of claim 9 wherein the enzyme is selected from the group consisting of alkaline phosphatase, peroxidase and 6-galactosidase.
Claim 12
Dependent Claim
20 words
The method of claim 9 wherein the fluorescent indicator molecular is selected from the group consisting of fluorescein and rhodamine.
Claim 13
Dependent Claim
23 words
The method of claim 9 wherein the electron dense indicator molecule is selected from the group consisting of ferritin, hemocyanin and colloidal gold.
Claim 14
Dependent Claim
16 words
The method of claim 8 wherein the indicator molecule is covalently linked to the detectable polypeptide.
Claim 15
Dependent Claim
28 words
The method of claim 8 wherein the detectable polypeptide is indirectly detectable by specifically complexing the detectable polypeptide with a second polypeptide covalently linked to an indicator molecule.
Claim 16
Dependent Claim
33 words
The method of claim 14 wherein said detectable polypeptide is selected from the group consisting of avidin and streptavidin and the second polypeptide is selected from the group consisting of biotin and iminobiotin.
Claim 17
Dependent Claim
34 words
The method of claim 5 wherein at least one of said compounds is labeled with a first indicator molecule and at least one other of said compounds is labeled with a second indicator molecule.
Claim 18
Dependent Claim
46 words
The method of claim 16 wherein the compound labeled with the first indicator molecule is allowed to hybridize to the nucleic acid and is detected and then the compound labeled with the second indicator molecule is allowed to hybridize to the nucleic acid and is detected.
Claim 19
Dependent Claim
13 words
The method of claim 1 wherein the moiety A comprises an indicator molecule.
Claim 20
Dependent Claim
23 words
The method of claim 18 wherein said indicator molecule is fluorescent, electron dense, or is an enzyme capable of depositing insoluble reaction products.
Claim 21
Dependent Claim
20 words
The method of claim 19 wherein the enzyme is selected from the group consisting of alkaline phosphatase, peroxidase and β-galactosidase.
Claim 22
Dependent Claim
20 words
The method of claim 19 wherein the fluorescent indicator molecule is selected from the group consisting of fluorescein and rhodamine.
Claim 23
Dependent Claim
22 words
The method of claim 19 wherein the electron dense compound is selected from the group consisting of ferritin, hemocyanin and colloidal gold.
Claim 24
Dependent Claim
32 words
A method of claim 1 wherein said signalling moiety is capable of producing a detectable signal when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex or DNA-RNA hybrid.
Claim 25
Dependent Claim
22 words
The method of claim 1 wherein said detecting step (b) is carried out when the compound is hybridized to the nucleic acid.
Claim 26
Dependent Claim
15 words
The method of claim 1 wherein said nucleic acid is immobilized on a solid support.
Claim 27
Dependent Claim
15 words
The method of claim 1 wherein the moiety A comprises at least 5 carbon atoms.
Claim 28
Dependent Claim
11 words
The method of claim 1 wherein the moiety A is non-aromatic.
Claim 29
Dependent Claim
18 words
The method of claim 1 wherein B is selected from the group consisting of uracil, cytosine, deazaadenine, deazaguanine.
Claim 30
Dependent Claim
19 words
The method of claim 1 wherein the linkage group comprises an olefinic bond at the α-position relative to B.
Claim 31
Dependent Claim
15 words
The method of claim 29 wherein the linkage group comprises the moiety --CH═CH--CH 2 --NH--.
Claim 32
Dependent Claim
13 words
The method of claim 29 wherein the linkage group comprises the moiety ##STR27##
Claim 33
Dependent Claim
15 words
The method of claim 1 wherein said nucleic acid is derived from a living organism.
Claim 34
Dependent Claim
19 words
The method of claim 32 wherein said living organism is selected from the group consisting of prokaryotes and eukaryotes.
Claim 35
Dependent Claim
25 words
The method of claim 1 wherein said sample is suspected of containing an etiological agent and said nucleic acid is associated with said etiological agent.
Claim 36
Dependent Claim
29 words
The method of claim 34 wherein said sample is of human or animal origin and said etiological agent is selected from the group consisting of bacteria, viruses and fungi.
Claim 37
Dependent Claim
44 words
The method of claim 1 wherein said sample comprises a microorganism suspected of containing a nucleic acid which imparts resistance to an antibiotic and wherein said compound comprises a polynucleotide complementary to the nucleic acid of said microorganism which confers resistance to said antibiotic.
Claim 38
Dependent Claim
53 words
The method of claim 36 wherein when said microorganism is Streptococcus pyrogenes or Neisseria meningitidis, said antibiotic is penicillin, wherein when said microorganism is Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Streptococcus pyrogenes, or Neisseria gonorrhoeae, said antibiotic is a tetracycline, and wherein when said microorganism is Mycobacterium tuberculosis, said antibiotic is an aminoglycoside.
Claim 39
Dependent Claim
37 words
The method of claim 1 wherein said sample is suspected of containing a nucleic acid associated with a genetic disorder and wherein said compound comprises a polynucleotide complementary to the nucleic acid associated with said genetic disorder.
Claim 40
Dependent Claim
42 words
The method of claim 1 wherein said sample is suspected of containing a nucleic acid associated with or absent in thalassemia and wherein said compound comprises a polynucleotide complementary to the nucleic acid which is associated with or absent in thalassemic subjects.
Claim 41
Dependent Claim
33 words
The method of claim 1 for chromosomal karyotyping which comprises contacting said sample with a series of said compounds which are complementary to a series of known genetic nucleic acids located on chromosomes.
Claim 42
Dependent Claim
52 words
The method of claim 1 wherein said sample is suspected of containing a nucleic acid which codes for expression of a polypeptide associated with a tumor cell and wherein said compound comprises a polynucleotide complementary to the messenger ribonucleic acid transcribed from a deoxyribonucleic acid associated with the production of said polypeptide.
Claim 43
Dependent Claim
11 words
The method of claim 41 wherein said polypeptide is α-fetal protein.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1997-07-11 | 1998-01-10 | 1998-07-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-07-11 | 2002-01-10 | 2002-07-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-07-11 | 2006-01-10 | 2006-07-12 | $7700.00 | Window Closed / Lapsed |