Methods of using labeled nucleotides
DC CAFCFirst Claim
1. 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 C1'"'"' -position of the sugar moiety, provided that whenever B'"'"' or B" is purine or 7-deazapurine, the sugar moiety is attached at the N9 -position of the purine or 7-deazapurine, and whenever B'"'"' or B" is pyrimidine the sugar moiety is attached at the N1 -position of the pyrimidine;
wherein B represents 7-deazapurine or pyrimidine moiety covalently bonded to the C1'"'"' -position of the sugar moiety, provided that whenever B is 7-deazapurine, the sugar moiety is attached at the N9 -position of the 7-deazapurine, and whenever B is pyrimidine the sugar moiety is attached at the N1 -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.
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Abstract
Compounds having the structure: ##STR1## wherein B represents a purine, 7-deazapurine, or pyrimidine moiety covalently bonded to the C1'"'"' -position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached at the N9 -position of the purine or 7-deazapurine and when B is pyrimidine, it is attached at the N1 -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.
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Citations
42 Claims
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1. 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 C1'"'"' -position of the sugar moiety, provided that whenever B'"'"' or B" is purine or 7-deazapurine, the sugar moiety is attached at the N9 -position of the purine or 7-deazapurine, and whenever B'"'"' or B" is pyrimidine the sugar moiety is attached at the N1 -position of the pyrimidine;
wherein B represents 7-deazapurine or pyrimidine moiety covalently bonded to the C1'"'"' -position of the sugar moiety, provided that whenever B is 7-deazapurine, the sugar moiety is attached at the N9 -position of the 7-deazapurine, and whenever B is pyrimidine the sugar moiety is attached at the N1 -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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
- ##STR26## wherein each of B'"'"' and B" represents a purine, 7-deazapurine, or pyrimidine moiety covalently bonded to the C1'"'"' -position of the sugar moiety, provided that whenever B'"'"' or B" is purine or 7-deazapurine, the sugar moiety is attached at the N9 -position of the purine or 7-deazapurine, and whenever B'"'"' or B" is pyrimidine the sugar moiety is attached at the N1 -position of the pyrimidine;
Specification