Compositions and methods for multiplex analysis of polynucleotides
First Claim
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1. A method of analyzing a polynucleotide sample for one or more target sequences, comprising the steps of:
- contacting a polynucleotide sample suspected of comprising one or more target sequences with;
(i) a first signal probe which is capable of hybridizing to at least a portion of a first target sequence and producing a first detectable signal when hybridized thereto;
(ii) a first quencher probe which is capable of hybridizing in quenching proximity to the first signal probe and quenching the signal of the first signal probe when hybridized in quenching proximity thereto, said first quencher probe having a Tm below that of the first signal probe;
(iii) at least a second signal probe which is capable of hybridizing to at least a portion of a second target sequence and producing a second detectable signal when hybridized thereto; and
(iv) an optional second quencher probe which is capable of hybridizing in quenching proximity to the second signal probe and quenching the signal of the second signal probe when hybridized in quenching proximity thereto, said optional second quencher probe having a Tm below that of the second signal probe;
monitoring the detectable signals of the signal probes as a function of temperature; and
determining therefrom the presence or absence of one or more target sequences in said polynucleotide sample.
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Abstract
Provided herein are compositions and methods for the multiplex analysis and/or detection of polynucleotides having one or more distinguishable target sequences. The methods employ signal-quencher probe pairs having specific relative differential thermal melting temperatures that permit the detection of one or more target sequences on one or more polynucleotides.
40 Citations
51 Claims
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1. A method of analyzing a polynucleotide sample for one or more target sequences, comprising the steps of:
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contacting a polynucleotide sample suspected of comprising one or more target sequences with;
(i) a first signal probe which is capable of hybridizing to at least a portion of a first target sequence and producing a first detectable signal when hybridized thereto;
(ii) a first quencher probe which is capable of hybridizing in quenching proximity to the first signal probe and quenching the signal of the first signal probe when hybridized in quenching proximity thereto, said first quencher probe having a Tm below that of the first signal probe;
(iii) at least a second signal probe which is capable of hybridizing to at least a portion of a second target sequence and producing a second detectable signal when hybridized thereto; and
(iv) an optional second quencher probe which is capable of hybridizing in quenching proximity to the second signal probe and quenching the signal of the second signal probe when hybridized in quenching proximity thereto, said optional second quencher probe having a Tm below that of the second signal probe;
monitoring the detectable signals of the signal probes as a function of temperature; and
determining therefrom the presence or absence of one or more target sequences in said polynucleotide sample. - 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)
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43. A method of analyzing a polynucleotide sample for one or more target sequences, comprising the steps of:
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contacting a polynucleotide sample with;
(1) a first set of m signal-quencher probe pairs, each of which comprises (i) a signal probe capable of hybridizing to a portion of a target sequence and producing a first detectable signal when hybridized thereto and (ii) a corresponding quencher probe capable of hybridizing in quenching proximity to the signal probe and quenching its detectable signal when hybridized in quenching proximity thereto, wherein the first signal probe has the highest Tm and the Tm of each quencher probe is lower than the Tm of its corresponding signal probe and the Tm of each signal probe is lower than the Tm of the quencher probe of the preceding signal-quencher probe pair, and further wherein the quencher probe of the signal-quencher probe pair of the first set having the lowest Tm is optional; and
(2) a second set of n signal-quencher probe pairs, each of which comprises (i) a signal probe capable of hybridizing to a portion of a target sequence and producing a second detectable signal distinguishable from the first detectable signal when hybridized thereto and (ii) a corresponding quencher probe capable of hybridizing in quenching proximity to the signal probe and quenching its detectable signal when hybridized in quenching proximity thereto, wherein the Tm of each quencher probe is lower than the Tm of its corresponding signal probe and the Tm of each signal probe is lower than the Tm of the quencher probe of the preceding signal-quencher probe pair, and further wherein the quencher probe of the signal-quencher probe pair of the second set having the lowest Tm is optional;
monitoring the first and second detectable signals as a function of temperature; and
determining the presence or absence of one or more target sequences in said polynucleotide sample.
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44. A method of genotyping an organism, comprising the steps of:
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contacting a polynucleotide sample from the organism, or an amplification product thereof, with a first plurality of signal-quencher probe pairs, each of which is capable of hybridizing, in quenching proximity, to a different genotype-specific sequence and producing a resolvable, temperature-dependent on-off hybridization profile;
obtaining temperature-dependent on-off hybridization profiles for the signal-quencher probe pairs; and
determining therefrom the genotype of the organism.
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45. A method of genotyping a virus, comprising the steps of:
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contacting a polynucleotide sample from a virus, or an amplification product thereof, with a first plurality of signal-quencher probe pairs, each of which is capable of hybridizing, in quenching proximity, to a different virus genotype-specific sequence and producing a resolvable, temperature-dependent on-off hybridization profile;
obtaining temperature-dependent on-off hybridization profiles for the signal-quencher probe pairs; and
determining therefrom the genotype of the virus.
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46. A method of analyzing a sample for the presence of a polynucleotide sequence of interest, comprising the steps of:
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contacting a polynucleotide from the sample, or an amplification product thereof, with a first plurality of signal-quencher probe pairs, wherein each said signal-quencher probe pair is capable of hybridizing, in quenching proximity, to a different known target sequence and producing a resolvable, temperature-dependent, on-off hybridization profile;
obtaining temperature-dependent, on-off hybridization profiles for the signal-quencher probe pairs; and
determining the presence or absence of one or more different target sequences.
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47. A multiplex method of genotyping a polynucleotide of an organism, comprising the steps of:
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amplifying the polynucleotide in the presence of amplification primers suitable for producing a plurality of genotype-specific amplicons and a plurality of signal-quencher probe pairs, wherein each said signal-quencher probe pair is capable of hybridizing, in quenching proximity, to a different genotype-specific amplicon and producing a resolvable, temperature-dependent, on-off hybridization profile;
obtaining temperature-dependent, on-off hybridization profiles for the signal-quencher probe pairs; and
determining therefrom the genotype of the organism.
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48. A multiplex method of diagnosing a patient for a malady of interest, comprising the steps of:
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incubating a polynucleotide sample derived from the patient in the presence of a plurality of signal-quencher probe pairs, wherein each said signal-quencher probe pair is capable of hybridizing, in quenching proximity, to a different target sequence indicative of a particular malady of interest and producing a resolvable, temperature-dependent, on-off hybridization profile when hybridized thereto;
obtaining temperature-dependent, on-off hybridization profiles for the signal-quencher probe pairs; and
determining therefrom whether the patient has the malady of interest.
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49. A multiplex method of diagnosing a patient for a malady of interest, comprising the steps of:
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amplifying a polynculeotide sample derived from the patient in the presence of amplification primers suitable for producing a plurality of different amplicons, each of which correlates to a different malady of interest, and a plurality of signal-quencher probe pairs, wherein each said signal-quencher probe pair is capable of hybridizing, in quenching proximity, to a different amplicon and producing a resolvable, temperature-dependent, on-off hybridization profile;
obtaining temperature-dependent, on-off hybridization profiles for the signal-quencher probe pairs; and
determining therefrom whether the patient has the malady of interest.
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50. A kit for multiplex polynucleotide analysis, comprising a plurality of signal-quencher probe pairs, each of which pairs is capable of hybridizing, in quenching proximity, to a different target sequence and producing a resolvable, temperature-dependent on-off hybridization profile in the presence of its respective target sequence.
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51. A kit for determining the presence or absence of mutations or polymorphisms at multiple loci of one or more polynucleotides, comprising a first signal probe which is capable of hybridizing to a polynucleotide at a region of a first target sequence and producing a first detectable fluorescent signal when hybridized thereto, a first quencher probe capable of hybridizing, in quenching proximity, to a different region of the same target sequence as the first signal probe and quenching the signal of the first signal probe when hybridized in quenching proximity thereto, said first quencher probe having a Tm below that of the first signal probe, a second signal probe which is capable of hybridizing to the same or different polynucleotide at a region of a second target sequence and producing a second detectable fluorescent signal when hybridized thereto, said second signal probe having a Tm below that of the first quencher probe, and an optional second quencher probe which is capable of hybridizing, in quenching proximity, to the a different region of the same target sequence as the second signal probe and quenching the signal of the second signal probe when hybridized in quenching proximity thereto, said optional second quencher probe having a Tm below that of the second signal probe.
Specification