Homogeneous multiplex hybridization analysis by color and Tm
First Claim
1. A method for analyzing a nucleic acid sample comprising three or more loci each having at least two different allelic sequences, said method comprising:
- (a) combining at least a first, a second and a third pair of oligonucleotide probes with said nucleic acid, each of the members of said pairs being capable of hybridizing in proximity to each other to a segment of said nucleic acid comprising at least one of said three or more loci, wherein (i) the first member of each pair comprises a FRET donor and the second member comprises a FRET acceptor, wherein the FRET acceptor of the second member in said first pair has an emission spectrum which is different from the emission spectrum of the FRET acceptor of said second and third oligonucleotide probe pairs, (ii) when said second and third probe pairs have the same FRET acceptor, each of said second and third probe pairs has a different Tm from each other for each different allele within the nucleic acid segment to which each member hybridizes (iii) upon hybridization, the proximity of the members of a probe pair is sufficient to allow fluorescence resonance energy transfer between said FRET donor and said FRET acceptor, and (iv) at least one of said members of each pair has a sequence which results in the differential hybridization of that member with at least two different alleles which may be present at said loci;
(b) measuring the emission of each of said FRET acceptors at a first temperature; and
(c) repeating said emission measurements at a second temperature;
wherein the emission of said FRET acceptors at different temperatures provides an indication of the alleles present at said three or more loci.
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Abstract
The invention provides methods and devices for analyzing sequence variations in nucleic acid samples comprising multiple loci, each having two, three or more possible allelic sequences. The method involves combining at least a first and second pair of oligonucleotide probes with the nucleic acid sample. The first pair of probes is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the first locus and the second pair is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the second locus. The first member of each probe pair comprises a FRET donor and the second member comprises a FRET acceptor, the FRET acceptor of the first probe pair member having a different emission spectrum from the FRET acceptor of the second probe pair. Upon hybridization, the proximity of the first and second member of each probe pair is sufficient to allow fluorescence resonance energy transfer between the FRET donor and the FRET acceptor.
186 Citations
11 Claims
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1. A method for analyzing a nucleic acid sample comprising three or more loci each having at least two different allelic sequences, said method comprising:
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(a) combining at least a first, a second and a third pair of oligonucleotide probes with said nucleic acid, each of the members of said pairs being capable of hybridizing in proximity to each other to a segment of said nucleic acid comprising at least one of said three or more loci, wherein (i) the first member of each pair comprises a FRET donor and the second member comprises a FRET acceptor, wherein the FRET acceptor of the second member in said first pair has an emission spectrum which is different from the emission spectrum of the FRET acceptor of said second and third oligonucleotide probe pairs, (ii) when said second and third probe pairs have the same FRET acceptor, each of said second and third probe pairs has a different Tm from each other for each different allele within the nucleic acid segment to which each member hybridizes (iii) upon hybridization, the proximity of the members of a probe pair is sufficient to allow fluorescence resonance energy transfer between said FRET donor and said FRET acceptor, and (iv) at least one of said members of each pair has a sequence which results in the differential hybridization of that member with at least two different alleles which may be present at said loci;
(b) measuring the emission of each of said FRET acceptors at a first temperature; and
(c) repeating said emission measurements at a second temperature;
wherein the emission of said FRET acceptors at different temperatures provides an indication of the alleles present at said three or more loci.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification