Fluorescent probes for DNA detection by hybridization with improved sensitivity and low background
First Claim
Patent Images
1. An oligonucleotide-probe comprising an oligonucleotide portion having a 3′
- -end and a 5′
-end, a minor groove binder moiety attached to at least one of said nucleotide units through a linking group which covalently binds the minor groove binder moiety to the oligonucleotide, and a fluorophore and quencher, said probe having the formula;
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Abstract
Minor groove binder-oligonucleotide probes are provided along with methods for their use wherein the probes have an attached fluorophore which, in an unhybridized form exhibits very low background signal.
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Citations
34 Claims
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1. An oligonucleotide-probe comprising an oligonucleotide portion having a 3′
- -end and a 5′
-end, a minor groove binder moiety attached to at least one of said nucleotide units through a linking group which covalently binds the minor groove binder moiety to the oligonucleotide, and a fluorophore and quencher, said probe having the formula;
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
- -end and a 5′
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10. A method for continuous monitoring of polynucleotide amplification, comprising:
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(a) combining a sample containing a target sequence, with one or more oligonucleotide primers complementary to regions of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a formula;
wherein MB is a minor groove binder, Q is a quencher, ODN is an oligonucleotide or modified oligonucleotide having a sequence that is complementary to a portion of said target sequence being amplified, K is a bond or a linking group, Fl is a fluorophore, W is a trivalent linking group that provides sufficient spacing between the MB, F and ODN components such that;
i) MB binds in the minor groove when said oligonucleotide conjugate hybridizes to said target sequence;
ii) in an unhybridized form, the fluorescence of Fl in said oligonucleotide conjugate is less than 10% of unquenched fluorescence; and
iii) when said oligonucleotide conjugate hybridizes to said target sequence, the fluorescence of Fl is at least 50% of its unquenched fluorescence;
to provide a mixture;
(b) incubating said mixture under conditions favorable for amplification of said polynucleotide; and
(c) continuously monitoring said amplification by monitoring the fluorescence produced upon conjugate hybridization to amplified target. - View Dependent Claims (11, 12, 13, 14, 15, 16)
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17. A method for monitoring gene expression comprising:
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(a) providing an array of oligonucleotide probes of different sequences, (b) incubating a population of polynucleotides with the array under hybridization conditions, and (c) determining to which of the oligonucleotide probes in the array the population hybridizes;
wherein one or more of the oligonucleotide probes is an oligonucleotide conjugate having the formula;
wherein MB is a minor groove binder, Q is a quencher, ODN is an oligonucleotide or modified oligonucleotide having a sequence that is complementary to a portion of said target sequence being amplified, K is a bond or a linking group, Fl is a fluorophore, W is a trivalent linking group that provides sufficient spacing between the MB, F and ODN components such that;
i) MB binds in the minor groove when said oligonucleotide conjugate hybridizes to said target sequence;
ii) in an unhybridized form, the fluorescence of Fl in said oligonucleotide conjugate is less than 10% of unquenched fluorescence; and
iii) when said oligonucleotide conjugate hybridizes to said target sequence, the fluorescence of Fl is at least 50% of its unquenched fluorescence. - View Dependent Claims (18, 19, 20)
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21. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising:
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(a) separately incubating each of at least two polynucleotides with an oligonucleotide conjugate having the formula;
wherein MB is a minor groove binder, Q is a quencher, ODN is an oligonucleotide or modified oligonucleotide having a sequence that is complementary to a portion of said target sequence being amplified, K is a bond or a linking group, Fl is a fluorophore, W is a trivalent linking group that provides sufficient spacing between the MB, F and ODN components such that;
i) MB binds in the minor groove when said oligonucleotide conjugate hybridizes to said target sequence;
ii) in an unhybridized form, the fluorescence of Fl in said oligonucleotide conjugate is less than 10% of unquenched fluorescence; and
iii) when said oligonucleotide conjugate hybridizes to said target sequence, the fluorescence of Fl is at least 50% of its unquenched fluorescence, said conjugate having a defined sequence under hybridization conditions, wherein one of the polynucleotides has a target sequence that is perfectly complementary to said oligonucleotide conjugate and at least one other of the polynucleotides has a target sequence having a single-nucleotide mismatch with the oligonucleotide conjugate; and
(b) determining the hybridization strength between each of the polynucleotides and the oligonucleotide conjugate. - View Dependent Claims (22, 23)
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24. A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence, the method comprising:
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(a) contacting the mixture of polynucleotides with an oligonucleotide conjugate having the formula;
wherein MB is a minor groove binder, Q is a quencher, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group, Fl is a fluorophore and W is a trivalent linking group that provides sufficient spacing between the MB, F and ODN components such that;
i) MB binds in the minor groove when said oligonucleotide conjugate hybridizes to said target sequence;
ii) in an unhybridized form, the fluorescence of Fl in said oligonucleotide conjugate is less than 10% of unquenched fluorescence; and
iii) when said oligonucleotide conjugate hybridizes to said target sequence, the fluorescence of Fl is at least 50% of its unquenched fluorescence; and
wherein the conjugate forms a stable hybrid only with said target sequence that is perfectly complementary to the ODN portion of said conjugate, and the conjugate does not form a stable hybrid with any of the other polynucleotides; and
(b) measuring the fluorescence produced on hybrid formation, whereby hybrid formation indicates the presence of said target sequence. - View Dependent Claims (25, 26)
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27. A method for distinguishing between wild-type, mutant and heterozygous target polynucleotides, said method comprising:
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(a) contacting a sample containing a target polynucleotide with two probes wherein a first probe is specific for said wild-type target polynucleotide and a second probe is specific for said mutant target polynucleotide, each of said probes having a formula;
wherein MB is a minor groove binder, Q is a quencher, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group, Fl is a fluorophore and W is a trivalent linking group that provides sufficient spacing between the MB, F and ODN components such that;
i) MB binds in the minor groove when said oligonucleotide conjugate hybridizes to said target sequence;
ii) in an unhybridized form, the fluorescence of Fl in said oligonucleotide conjugate is less than 10% of unquenched fluorescence; and
iii) when said oligonucleotide conjugate hybridizes to said target sequence, the fluorescence of Fl is at least 50% of its unquenched fluorescence; and
wherein the conjugate forms a stable hybrid only with said target sequence that is perfectly complementary to the ODN portion of said conjugate, and the conjugate does not form a stable hybrid with any of the other polynucleotides; and
(b) measuring the fluorescence produced on hybrid formation, whereby hybrid formation indicates the presence or absence of each of said wild-type, mutant and heterozygous target polynucleotides. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34)
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Specification