Real-time linear detection probes: sensitive 5'-minor groove binder-containing probes for PCR analysis
First Claim
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1. A method for continuous monitoring of polynucleotide amplification, comprising:
- (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 MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore, and said ODN portion has a sequence complementary to a portion of said target sequence being amplified, 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.
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Abstract
Oligonucleotide probes/conjugates are provided along with method for their use in assays to monitor amplification wherein the signal produced does not rely on 5′ nuclease digestion.
29 Citations
21 Claims
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1. 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 MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore, and said ODN portion has a sequence complementary to a portion of said target sequence being amplified, 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 (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. 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 MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore. - View Dependent Claims (12, 13, 14, 15)
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16. 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 MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore, 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 (17, 18, 19, 20)
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21-39. -39. (canceled)
Specification