Multiple fluorophore detector system

Abstract

A detector oligonucleotide comprises multiple pairs of a donor fluorophore and a quencher molecule, which donor fluorophores and quencher molecules are separated by a site that is capable of being cleaved when in double-stranded form. The detector oligonucleotide may be made double-stranded in a manner that depends on the presence of a target nucleic acid, allowing the cleavage sites to be cleaved. Separation of the donor fluorophores and the quencher molecules decreases fluorescence quenching and generates a detectable change in a fluorescence parameter of the fluorophores of the detector oligonucleotide. By using multiple donor/quencher pairs, the present detector oligonucleotide advantageously generates a high signal to noise ratio and high efficiency in detection of a target nucleic acid.

1 . A detector oligonucleotide, comprising at least two pairs of a donor fluorophore and a quencher molecule in close proximity, wherein said donor fluorophore and said quencher molecule in each said pair are separated by a cleavage site that is cleavable in a double-stranded form, and wherein cleavage at said cleavage site is capable of creating a detectable signal that indicates the presence of a target nucleic acid.  
   
   
       2 . A single-stranded first detector oligonucleotide, comprising at least two pairs of a donor fluorophore and a quencher molecule in close proximity, wherein said donor fluorophore and said quencher molecule in each said pair are separated by a cleavage site that is cleavable in a double-stranded form, and wherein the cleavage site is double-stranded when the first detector oligonucleotide forms a duplex with a second oligonucleotide that is capable of being formed in the presence of a target nucleic acid.  
   
   
       3 . The detector oligonucleotide of  claim 2 , wherein at least one said donor fluorophore is selected from the group consisting of fluorescein, sulforhodamine 101, pyrenebutanoate, acridine, ethenoadenosine, eosin, rhodamine, and erythrosine.  
   
   
       4 . The oligonucleotide of  claim 2 , wherein at least one said quencher molecule is selected from the group consisting of DABCYL, DAMBI, DABSYL and methyl red.  
   
   
       5 . The oligonucleotide of  claim 2 , wherein said donor fluorophores and said quencher molecules are separated by about 5 to 20 nucleic acid bases that comprise a cleavage site.  
   
   
       6 . The oligonucleotide of  claim 5 , wherein said donor fluorophores and said quencher molecules are separated by about 6 to 8 nucleic acid bases that comprise a cleavage site.  
   
   
       7 . The oligonucleotide of  claim 2 , wherein said cleavage site is cleavable by a chemical cleavage reagent.  
   
   
       8 . The oligonucleotide of  claim 2 , wherein said cleavage site is cleavable by an endonuclease.  
   
   
       9 . The oligonucleotide of  claim 8 , wherein said endonuclease is selected from the group consisting of Hinc II, Nci I, and BsoB1.  
   
   
       10 . The oligonucleotide of  claim 2 , comprising ten donor/quencher pairs.  
   
   
       11 . The oligonucleotide of  claim 10 , comprising 50 donor/quencher pairs.  
   
   
       12 . The oligonucleotide of  claim 2 , wherein the first detector oligonucleotide comprises a first portion at a 5′ terminus that is capable of forming a duplex with a first portion at a 3′ terminus of a second oligonucleotide, wherein the second oligonucleotide comprises a second portion that is complementary to a target nucleic acid and a third portion at a 5′ terminus of the second oligonucleotide that comprises one strand of an endonuclease recognition site.  
   
   
       13 . The oligonucleotide of  claim 2 , wherein said first detector oligonucleotide comprises a first portion capable of forming a duplex with a third oligonucleotide, wherein said third oligonucleotide comprises two portions: (1) a first portion having a sequence capable of forming a duplex with a target nucleic acid and (2) a second portion capable of forming a duplex with said first portion of said first detector oligonucleotide.  
   
   
       14 . The detector oligonucleotide of  claim 13 , where in said first portion is at the 5′ terminus of said first detector oligonucleotide.  
   
   
       15 . The oligonucleotide of  claim 2 , wherein said first detector oligonucleotide comprises a first portion capable of forming a duplex with a third oligonucleotide, wherein said third oligonucleotide comprises two portions: (1) a first portion having a sequence complementary to a target nucleic acid and (2) a second portion capable of forming a duplex with said first portion of said first detector oligonucleotide.  
   
   
       16 . A partially double-stranded detector oligonucleotide, comprising at least two pairs of a donor fluorophore and a quencher molecule in close proximity, wherein said donor fluorophore and said quencher molecule in each said pair are separated by a cleavage site, wherein said partially double-stranded detector oligonucleotide comprises a single-stranded portion that is capable of forming a duplex with a target nucleic acid.  
   
   
       17 . The oligonucleotide of  claim 16 , wherein at least one of said cleavage sites is cleavable by a chemical cleavage reagent.  
   
   
       18 . The oligonucleotide of  claim 16 , wherein at least one of said cleavage sites is cleavable by an endonuclease.  
   
   
       19 . A method for detecting a target nucleic acid, comprising: 
 a. contacting (i) a first detector oligonucleotide comprising a single-stranded portion that comprises at least two pairs of a donor fluorophore and a quencher molecule in close proximity, wherein said donor fluorophore and said quencher molecule in each said pair are separated by a cleavage site, with (ii) a single-stranded second oligonucleotide, the presence of which is indicative of the presence of the target nucleic acid, to form a duplex between said first and second oligonucleotides;    b. extending the duplex to make said single-stranded portion of the first detector oligonucleotide double-stranded;    c. cleaving at least one of said cleavage sites; and    d. detecting said donor fluorophores,    wherein a detectable change in a fluorescence parameter of said donor fluorophores is indicative of the presence of said target nucleic acid.    
   
   
       20 . The method of  claim 18 , wherein the first oligonucleotide comprises a first portion at a 5′ terminus of the first oligonucleotide that forms a duplex with a first portion at a 3′ terminus of the second oligonucleotide.  
   
   
       21 . The method of  claim 18 , further comprising amplifying the second oligonucleotide.  
   
   
       22 . The method of  claim 21 , wherein the second oligonucleotide is amplified before step (a).  
   
   
       23 . The method of  claim 21 , wherein the second oligonucleotide comprises one strand of an endonuclease recognition site in a third portion of the second oligonucleotide that is 5′ of said second portion that is complementary to a target nucleic acid.  
   
   
       24 . The method of  claim 21 , wherein the amplifying is by a method selected from the group consisting of strand displacement amplification (SDA), polymerase chain reaction (PCR), ligase chain reaction, self-sustained sequence replication (3SR), Q beta replicase-based amplification, solid phase amplification, nucleic acid sequence-based amplification (NASBA), rolling circle amplification, and transcription mediated amplification (TMA).  
   
   
       25 . The method of  claim 23 , wherein the amplifying is by the method of strand displacement amplification.  
   
   
       26 . The method of  claim 19 , wherein said detecting comprises measuring a fluorescent emission of said donor fluorophores.  
   
   
       27 . The method of  claim 19 , wherein said at least one of said pairs is selected from the group of donor and quencher molecules consisting of fluorescein/Rhodamine X, Rhodamine X/Cy5, or fluorescein/DABCYL.  
   
   
       28 . A method for detecting a target nucleic acid, comprising: 
 a. (i) hybridizing a primer P 1  to said target nucleic acid and (ii) extending P 1  by use of a polymerase to form a Strand 1, wherein the primer P 1  comprises an endonuclease recognition site at a 5′ portion of said primer P 1  that does not hybridize to the target nucleic acid;    b. (i) hybridizing a bumper B 1  to said target nucleic acid upstream from said primer P 1  and (ii) extending the bumper B 1  and removing Strand 1 from said target nucleic acid;    c. hybridizing an adaptor to Strand 1 and a primer P 2  to Strand 1, wherein the primer P 2  hybridizes upstream of the adapter;    d. (i) extending the adapter to form a Strand 2, and (ii) extending the primer P 2  to remove Strand 2 from Strand 1;    e. (i) hybridizing the primer P 1  to Strand 2 and (ii) extending the primer P 1  to form a primer P 1 -extended strand;    f. (i) nicking the primer P 1 -extended strand at the endonuclease recognition site incorporated into the primer P 1 -extended strand and (ii) extending from the nick site to form a Strand 3 and to bump the primer P 1 -extended strand that is downstream of the nick site;    g. hybridizing Strand 3 to a portion of an oligonucleotide, wherein the oligonucleotide comprises multiple pairs of donor fluorophores and quenchers, wherein the donor fluorophore and the quencher in each said pair are separated by a site that is cleavable when said cleavage site is double-stranded;    h. (i) extending Strand 3 to make the cleavage sites double-stranded and (ii) cleaving at least one of the cleavage sites; and    i. detecting said donor fluorophores,    wherein a detectable change in a fluorescence parameter of said fluorophores is indicative of the presence of said target nucleic acid.    
   
   
       29 . A kit, comprising a single-stranded first detector oligonucleotide, comprising at least two pairs of a donor fluorophore and a quencher molecule in close proximity, wherein said donor fluorophore and said quencher molecule in each said pair are separated by a cleavage site that is cleavable in a double-stranded form, and wherein the cleavage site is double-stranded when the first detector oligonucleotide forms a duplex with a second oligonucleotide capable of being formed in the presence of a target nucleic acid.  
   
   
       29 . The kit of  claim 29 , further comprising an adapter oligonucleotide that comprises a first portion, which is capable of forming a duplex with the complement of a target oligonucleotide, and a second portion, the complement of which is capable of forming a duplex with the first portion of said detector oligonucleotide.