Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
First Claim
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1. A method of detecting at least one target nucleic acid in a sample, said method comprising:
- (A) generating at least one purified target nucleic acid by;
(A1) generating at least one double-stranded nucleic acid hybrid by hybridizing the at least one target nucleic acid in the sample to a hybrid probe set comprising at least a first nucleic acid probe specifically hybridized to the at least one target nucleic acid;
(A2) separating the at least one double-stranded nucleic acid hybrid from the sample, wherein the at least one double-stranded nucleic acid hybrid is the at least one purified target nucleic acid;
(B) amplifying at least a portion of the at least one purified target nucleic acid, thereby generating at least one amplified nucleic acid; and
(C) detecting the at least one target nucleic acid by;
(C1) contacting the at least one amplified nucleic acid with a detection probe set comprising at least three nucleic acid detection probes and forming at least one hybridization complex, wherein;
(C1(a)) each detection probe of the detection probe set bears a detectable fluorescent label;
(C1(b)) at least two detection probes of the detection probe set carry the same detectable fluorescent label;
(C1(c)) each probe carrying the same detectable fluorescent label of the detection probe set has a different melting temperature (Tm), and each probe having identical or very similar melting temperatures of the detection probe set carry a different fluorescent label; and
(C1(d) each detection probe of the detection probe set hybridizes to a different target nucleic acid sequence;
(C2) detecting and identifying the detectable fluorescent label of each detection probe of the detection probe set hybridized to the at least one amplified nucleic acid; and
(C3) detecting the temperature at which each detection probe of the detection probe set dissociates from the at least one amplified nucleic acid;
wherein the at least one target nucleic acid is detected based on the detectable fluorescent label of each detection probe of the detection probe set and the temperature which each detection probe of the detection probe set dissociates from its corresponding hybridization complex of the at least one hybridization complex.
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Abstract
Methods and materials for determining the presence of at least one nucleic acid in a sample are provided, said methods comprising (1) a purification step using sequence specific hybrid capture; (2) an amplification step; and (3) a detection step comprising contacting the target nucleic acid with a plurality of detectably labeled nucleic acid detection probes, wherein each (a) bears a different detectable label from the other detection probes, and/or (b) has a different melting temperature from probes bearing the same detectable label. Also disclosed are compositions and kits for use in such a method.
138 Citations
24 Claims
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1. A method of detecting at least one target nucleic acid in a sample, said method comprising:
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(A) generating at least one purified target nucleic acid by; (A1) generating at least one double-stranded nucleic acid hybrid by hybridizing the at least one target nucleic acid in the sample to a hybrid probe set comprising at least a first nucleic acid probe specifically hybridized to the at least one target nucleic acid; (A2) separating the at least one double-stranded nucleic acid hybrid from the sample, wherein the at least one double-stranded nucleic acid hybrid is the at least one purified target nucleic acid; (B) amplifying at least a portion of the at least one purified target nucleic acid, thereby generating at least one amplified nucleic acid; and (C) detecting the at least one target nucleic acid by; (C1) contacting the at least one amplified nucleic acid with a detection probe set comprising at least three nucleic acid detection probes and forming at least one hybridization complex, wherein; (C1(a)) each detection probe of the detection probe set bears a detectable fluorescent label; (C1(b)) at least two detection probes of the detection probe set carry the same detectable fluorescent label; (C1(c)) each probe carrying the same detectable fluorescent label of the detection probe set has a different melting temperature (Tm), and each probe having identical or very similar melting temperatures of the detection probe set carry a different fluorescent label; and (C1(d) each detection probe of the detection probe set hybridizes to a different target nucleic acid sequence; (C2) detecting and identifying the detectable fluorescent label of each detection probe of the detection probe set hybridized to the at least one amplified nucleic acid; and (C3) detecting the temperature at which each detection probe of the detection probe set dissociates from the at least one amplified nucleic acid; wherein the at least one target nucleic acid is detected based on the detectable fluorescent label of each detection probe of the detection probe set and the temperature which each detection probe of the detection probe set dissociates from its corresponding hybridization complex of the at least one hybridization complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A detection probe set comprising at least five nucleic acid probes specific for different target nucleic acids, each of the at least five nucleic acid probes comprising a detectable label, wherein:
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(A) at least two of the nucleic acid probes of the at least five nucleic acid probes comprise the same detectable label; (B) each probe carrying the same detectable label of the nucleic acid probes of the at least five nucleic acid probes has a different melting temperature; and (C) the detectable label is a fluorophore selected from the group consisting of; (C1);
a fluorophore capable of being excited by an excitation source at 365±
20 nm and detected using a 460±
15 nm detection filter;(C2);
a fluorophore capable of being excited by an excitation source at 470±
10 nm and detected using a 510±
5 nm detection filter;(C3);
a fluorophore capable of being excited by an excitation source at 530±
5 nm and detected using a 555±
5 nm detection filter;(C4);
a fluorophore capable of being excited by an excitation source at 585±
5 nm and detected using a 610±
5 nm detection filter;(C5);
a fluorophore capable of being excited by an excitation source at 625±
10 nm and detected using a of 660±
10 nm detection filter; and(C6);
a fluorophore capable of being excited by an excitation source at 680±
5 nm and detected using a 712 nm long pass detection filter. - View Dependent Claims (21, 22, 23, 24)
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Specification