Nucleotide analogs for nucleic acid labeling and detection
First Claim
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1. In the processof detecting the presence of a target polynucleotide, said process comprisingincorporating a deoxyribonucleotide, modified by the attachment thereto of a biotin, into a polynucleotide complementary to said target polynucleotide;
- hybridizing said complementary polynucleotide to said target polynucleotide; and
detecting the presence of said biotin;
the improvement comprising using, for said modified deoxyribonucleotide, compound of the form
space="preserve" listing-type="equation">(d)BTP--NH--L--X, whereTP represents triphosphate, and (D)BTP represents dATP, dCTP, dGTP, 3'"'"'-deoxy ATP, 3'"'"'-doexy CTP, 3'"'"'-deoxy GTP, 2'"'"',3'"'"'-dideoxy ATP, 2'"'"',3'"'"'-dideoxy CTP or 2'"'"',3'"'"'-dideoxy GTP;
L is bonded through the N of NH to the N6 position of B when B represents adenosine, to the N4 position of B when B represents cytidine, and to the N2 position of B when B represents guanosine;
L presents either(CH2)n, where 1≦
n≦
12,(CH2)n NH--CO-(CH2)m, where 2≦
n+m≦
24,(CH2)n O--CO--(CH2)m, where 2≦
n+m≦
24, or(CH2)n S--CO--(CH2)m, where 2≦
n+m≦
24; and
X represents ##STR1## where Y represents either NH, O, or S.
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Abstract
Nucleotide analogs, modified by the attachment at hydrogen bonding positions of linker groups, that is, the 6-position of adenine, 4-position of cytosine, and 2-position of guanine, are prepared. Such analogs, alone or with reporter groups attached, may be incorporated into DNA probes which effectively hybridize to target DNA.
369 Citations
12 Claims
-
1. In the processof detecting the presence of a target polynucleotide, said process comprising
incorporating a deoxyribonucleotide, modified by the attachment thereto of a biotin, into a polynucleotide complementary to said target polynucleotide; -
hybridizing said complementary polynucleotide to said target polynucleotide; and detecting the presence of said biotin; the improvement comprising using, for said modified deoxyribonucleotide, compound of the form
space="preserve" listing-type="equation">(d)BTP--NH--L--X,where TP represents triphosphate, and (D)BTP represents dATP, dCTP, dGTP, 3'"'"'-deoxy ATP, 3'"'"'-doexy CTP, 3'"'"'-deoxy GTP, 2'"'"',3'"'"'-dideoxy ATP, 2'"'"',3'"'"'-dideoxy CTP or 2'"'"',3'"'"'-dideoxy GTP; L is bonded through the N of NH to the N6 position of B when B represents adenosine, to the N4 position of B when B represents cytidine, and to the N2 position of B when B represents guanosine; L presents either (CH2)n, where 1≦
n≦
12,(CH2)n NH--CO-(CH2)m, where 2≦
n+m≦
24,(CH2)n O--CO--(CH2)m, where 2≦
n+m≦
24, or(CH2)n S--CO--(CH2)m, where 2≦
n+m≦
24; andX represents ##STR1## where Y represents either NH, O, or S. - View Dependent Claims (2, 3, 4)
-
- 5. In the process of detecting the presence of a target polynucleotide, said process including
incorporating a deoxyribonucleotide into a polynucleotide complementary to said target polynucleotide, and hybridizing said complementary polynucleotide to said target polynucleotide, the improvement comprising, using, for said deoxyribonucleotide, a compound of the form - space="preserve" listing-type="equation">(d)BTP--NH--M,
where TP represents triphosphate, and (d)BTP, represents dATP, dCTP, dGTP, 3'"'"'-deoxy ATP, 3'"'"'-deoxy CTP, 3'"'"'-deoxy GTP, 2'"'"',3'"'"'-dideoxy ATP, 2'"'"',3'"'"'-deoxy CTP or 2'"'"',3'"'"'-didexoy GTP; M is bonded through the N of NH to the N6 position of B when B represents adenosine, to the N4 position of B when B represents cytidine, and to the N2 position of B when B represents guanosine; and M represents a compound of the form L-X, where X represents NH2, OH or SH, and L represents either (CH2)n for an n such that 1≦
n≦
12, or (CH2)n NHCO(CH2)m for an n and m such that 2≦
n+m≦
24.- View Dependent Claims (6, 7, 8)
- 9. A compound having the structure
- space="preserve" listing-type="equation">(d)BTP--NH--L--NH--X
wherein (a) TP represents triphosphate, and (d)BTP represents either 2'"'"'-deoxy BTP, 3'"'"'-deoxy BTP, or 2'"'"',3'"'"'-dideoxy BTP, and B represents adenosine, cytidine or guanosine; (b) when B represents adenosine or guanosine, L is bonded through NH to the N6 position of adenosine or the N2 position of guanosine, and L represents either (CH2)n for an n such that 1≦
n≦
12, (CH2)n NHCO(CH2)m for an n and m such that 2≦
n+m≦
24, CH2 CONH(CH2)5 for 1≦
n≦
12, or CH2 CONH(CH2)n NHCO(CH2)5 for 1≦
n≦
12;(c) when B represents cytidine, L is bonded through NH to the N4 position of B and L represents either (CH2)n for an n such that 1≦
n≦
12 or (CH2)n NHCO(CH2)m for an n and m such that 2≦
n+m≦
24, and(d) X represents biotin. - View Dependent Claims (10, 11)
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12. A compound having the structure, ##STR2## wherein (a) TP represents triphosphate, and (d)R represents 2'"'"'-deoxyribose, 3'"'"'-deoxyribose or 2'"'"', 3'"'"'-dideoxyribose;
- and either
(b) B represents either adenine or cytosine and L represents either (CH2)n where 1≦
n≦
12, or (CH2)n NHCO(CH2)m, where 2≦
n+m≦
24, or(c) B represents adeine and L represents either CH2 CONH(CH2)n for 1≦
n≦
12 or CH2 CONH(CH2)n NHCO(CH2)5 for 1≦
n≦
12;and wherein, when B represents adenine, L is covalently bonded through NH to the N6 psition of B, and when B represents cytosine, L is covalently bonded through NH to the N4 position of B.
- and either
Specification