Double stranded linear nucleic acid probe and uses thereof

Abstract

A double-stranded nucleic acid hybridization probe and methods of using the same are described. The probe described is particularly suited for real-time RT-PCR reactions and has high tolerance to mismatches.

1 . A nucleic acid probe comprising a first oligonucleic acid and a second oligonucleic acid, wherein 
 (a) the first oligonucleic acid 
 (i) is substantially complementary to a nucleic acid of interest, and  
 (ii) comprises a fluorophore,  
   (b) the second oligonucleic acid comprises a quencher    (c) the first oligonucleic acid and second oligonucleic acid are substantially complementary to each other such that the first oligonucleic acid and second oligonucleic acid can bind together to form a double-stranded nucleic acid,    (d) when the first oligonucleic acid is bound to the second oligonucleic acid the fluorescent emission of the fluorophore attached to the first oligonucleic acid is detectably less than the emission of the same fluorophore when the first oligonucleic acid and second oligonucleic acid are not bound together in a double-stranded nucleic acid,    (e) said nucleic acid probe characterized in that: 
 (i) the second oligonucleic acid comprises “n” nucleobases substantially complementary to the first oligonucleic acid, and the first oligonucleic acid comprises “m” nucleobases substantially complementary to a nucleic acid of interest, wherein “m” and “n” are independently selected integers, and  
 (ii) when m is less than 25, n is equal or less than m/2; when m is 26-29, n is from 8 to 13; when mis 30 to 34, n is from 8 to 15; when mis 35 to 39, n is from 8 to 20; when m is 40 to 44, n is 9 to 25; when m is 45 to 49, n is 10 to 30; when m is 50 to 54, n is 10 to 35; when m is 55 to 59, n is 10 to 40; when m is 60 to 64, n is 11 to 45; when m is 65 to 69, n is from 11 to 50; when m is 70 to 75, n is from 15 to 55.  
   
     
     
         2 . The nucleic acid of  claim 1 , wherein the first and the second oligonucleic acids further comprise additional nucleobases that are not complementary to the nucleic acid of interest and the first oligonucleotide, respectively, at the 5′ and 3′ ends.  
     
     
         3 . The nucleic acid probe of  claim 1 , wherein the ratio of the second oligonucleic acid to the first oligonucleic acid is more than 1.1.  
     
     
         4 . The nucleic acid probe of  claim 1 , wherein the ratio of the second oligonucleic acid to the first oligonucleic acid is more than 0.1 and less than 0.9.  
     
     
         5 . The nucleic acid probe of  claim 1 , wherein the first oligonucleic acid further comprises a quencher.  
     
     
         6 . The nucleic acid probe of  claim 5 , wherein the ratio of the second oligonucleic acid to the first oligonucleic acid is more than 0.1 and less than 0.9.  
     
     
         7 . The nucleic acid probe of  claim 5 , wherein the ratio of the second oligonucleic acid to the first oligonucleic acid is more than 1.1  
     
     
         8 . The nucleic acid probe of  claim 1 , wherein the first oligonucleic acid comprises at least two fluorophores to allow the fluorescence generated by the first oligonucleic acid comprising two fluorophores when bound to a nucleic acid of interest to be substantially greater than the fluorescence generated when only one fluorophore is present.  
     
     
         9 . A nucleic acid probe comprising a first oligonucleic acid and a second oligonucleic acid, wherein 
 (a) the first oligonucleic acid 
 (i) is substantially complementary to a nucleic acid of interest, and  
 (ii) comprises a quencher,  
   (b) the second oligonucleic acid comprises a fluorophore,    (c) the first oligonucleic acid and second oligonucleic acid are substantially complementary to each other such that the first oligonucleic acid and second oligonucleic acid can bind together to form a double-stranded nucleic acid,    (d) when the first oligonucleic acid is bound to the second oligonucleic acid the fluorescent emission of the fluorophore attached to the second oligonucleic acid is detectably less than the emission of the same fluorophore when the first oligonucleic acid and second oligonucleic acid are not bound together in a double-stranded nucleic acid,    (e) said nucleic acid probe characterized in that: 
 (i) the second oligonucleic acid comprises “n” nucleobases substantially complementary to the first oligonucleic acid and the first oligonucleic acid comprises “m” nucleobases substantially complementary to the nucleic acid of interest, wherein “m” and “n” are independently selected integers, and  
 (ii) when m is less than 25, n is equal or less than m/2; when m is 26-29, n is from 8 to 13; when m is 30 to 34, n is from 8 to 15; when m is 35 to 39, n is from 8 to 20; when m is 40 to 44, n is 9 to 25; when m is 45 to 49, n is 10 to 30; when m is 50 to 54, n is 10 to 35; when m is 55 to 59, n is 10 to 40; when m is 60 to 64, n is 11 to 45; when m is 65 to 69, n is from 11 to 50; when m is 70 to 75, n is from 15 to 55.  
   
     
     
         10 . The nucleic acid of  claim 9 , wherein the first and the second oligonucleotides further comprise additional nucleobases that are not complementary to the nucleic acid of interest and the first oligonucleotide, respectively, at the 5′ and 3′ ends.  
     
     
         11 . The nucleic acid probe according to  claim 9 , wherein the ratio of the first oligonucleic acid to the second oligonucleic acid is more than 1.1.  
     
     
         12 . The nucleic acid probe according to  claim 9 , wherein the ratio of the first oligonucleic acid to the second oligonucleic acid is more than 0.1 and less than 0.9.  
     
     
         13 . The nucleic acid probe of  claim 9 , wherein the second oligonucleic acid further comprises a quencher.  
     
     
         14 . The nucleic acid probe of  claim 13 , wherein the ratio of the first oligonucleotide to the second oligonucleotide is more than 0.1 and less than 0.9.  
     
     
         15 . The nucleic acid probe of  claim 13 , wherein the ratio of the first oligonucleotide to the second oligonucleotide is more than 1.1.  
     
     
         16 . A method of detecting a nucleic acid of interest in a test sample, said method comprising 
 (a) mixing test sample with DNA amplification reagents, optionally including reverse transcription reagents and the fluorescent probe of  claim 1  to create a mixture in a reaction vessel,    (b) optionally incubating the reaction for a suitable time and under suitable reverse transcription conditions to reverse transcribe the RNA into a cDNA,    (c) incubating the reaction for a suitable time and under suitable DNA amplification conditions to amplify a portion of the nucleic acid of interest, and    (d) measuring fluorescence from the fluorescent probe as an indication of whether the test sample contains the nucleic acid of interest, wherein 
 an additional quantity of the oligonucleic acid comprising a quencher is added to the vessel prior to closing the vessel to obtain a molar ratio between the second oligonucleotide and the first oligonucleotide that is greater than 1.1 and less than 20.  
   
     
     
         17 . A method of detecting a nucleic acid of interest in a test sample, said method comprising 
 (a) mixing test sample with DNA amplification reagents, optionally including reverse transcription reagents and the fluorescent probe of  claim 9  to create a mixture in a reaction vessel,    (b) optionally incubating the reaction for a suitable time and under suitable reverse transcription conditions to reverse transcribe the RNA into a cDNA,    (c) incubating the reaction for a suitable time and under suitable DNA amplification conditions to amplify a portion of the nucleic acid of interest, and    (d) measuring fluorescence from the fluorescent probe as an indication of whether the test sample contains the nucleic acid of interest, wherein 
 an additional quantity of the oligonucleic acid comprising a quencher is added to the vessel prior to closing the vessel to obtain a molar ratio between the first oligonucleotide and the second oligonucleotide that is greater than 1.1 and less than 20.  
   
     
     
         18 . A method for quantifying RNA in a test sample, the method comprising 
 (A) mixing test sample with amplification reagents, reverse transcription reagents and a nucleic acid probe of  claim 1  to create a mixture in a reaction vessel, wherein the nucleic acid probe comprises a first oligonucleic acid and a second oligonucleic acid, wherein 
 (a) the first oligonucleic acid is substantially complementary to a nucleic acid of interest, and comprises a fluorophore,  
 (b) the second oligonucleic acid comprises a quencher,  
 (c) the first oligonucleic acid and second oligonucleic acid are substantially complementary to each other such that the first oligonucleic acid and second oligonucleic acid can bind together to form a double-stranded nucleic acid,  
 (d) when the first oligonucleic acid is bound to the second oligonucleic acid the fluorescent emission of the fluorophore attached to the first oligonucleic acid is detectably less than the emission of the same fluorophore when the first oligonucleic acid and second oligonucleic acid are not bound together in a double-stranded nucleic acid,  
 (e) said nucleic acid probe characterized in that: 
 (i) the second oligonucleic acid comprises “n” nucleobases substantially complementary to the first oligonucleic acid, and the first oligonucleic acid comprises “m” nucleobases substantially complementary to a nucleic acid of interest, wherein “m” and “n” are independently selected integers, and,  
 (ii) when m is less than 25, n is equal or less than m/2; when m is 26-29, n is from 8 to 13; when m is 30 to 34, n is from 8 to 15; when m is 35 to 39, n is from 8 to 20; when m is 40 to 44, n is 9 to 25; when m is 45 to 49, n is 10 to 30; when m is 50 to 54, n is 10 to 35; when m is 55 to 59, n is 10 to 40; when m is 60 to 64, n is 11 to 45; when m is 65 to 69, n is from 11 to 50; when m is 70 to 75, n is from 15 to 55,  
 
   (B) placing the test sample under conditions permissive of reverse transcription, which optionally can also be permissive of amplification, such that a cDNA is produced,    (C) thermocycling the test mixture such that cDNA is amplified, and    (D) measuring the fluorescence of the test mixture during the amplification reaction as an indication of the quantity of RNA in the test sample.    
     
     
         19 . A method for quantifying RNA in a test sample, the method comprising 
 (A) mixing test sample with amplification reagents, reverse transcription reagents and a nucleic acid probe of  claim 9  to create a mixture in a reaction vessel, wherein the nucleic acid probe comprises a first oligonucleic acid and a second oligonucleic acid, wherein 
 (a) the first oligonucleic acid is substantially complementary to a nucleic acid of interest, and comprises a quencher,  
 (b) the second oligonucleic acid comprises a fluorophore,  
 (c) the first oligonucleic acid and second oligonucleic acid are substantially complementary to each other such that the first oligonucleic acid and second oligonucleic acid can bind together to form a double-stranded nucleic acid,  
 (d) when the first oligonucleic acid is bound to the second oligonucleic acid the fluorescent emission of the fluorophore attached to the second oligonucleic acid is detectably less than the emission of the same fluorophore when the first oligonucleic acid and second oligonucleic acid are not bound together in a double-stranded nucleic acid,  
 (e) said nucleic acid probe characterized in that: 
 (i) the second oligonucleic acid comprises “n” nucleobases substantially complementary to the first oligonucleic acid, and the first oligonucleic acid comprises “m” nucleobases substantially complementary to a nucleic acid of interest, wherein “m” and “n” are independently selected integers, and,  
 (ii) when m is less than 25, n is equal or less than m/2; when m is 26-29, n is from 8 to 13; when m is 30 to 34, n is from 8 to 15; when m is 35 to 39, n is from 8 to 20; when m is 40 to 44, n is 9 to 25; when m is 45 to 49, n is 10 to 30; when m is 50 to 54, n is 10 to 35; when m is 55 to 59, n is 10 to 40; when m is 60 to 64, n is 11 to 45; when m is 65 to 69, n is from 11 to 50; when m is 70 to 75, n is from 15 to 55,  
 
   (B) placing the test sample under conditions permissive of reverse transcription, which optionally can also be permissive of amplification, such that a cDNA is produced,    (C) thermocycling the test mixture such that cDNA is amplified, and    (D) measuring the fluorescence of the test mixture during the amplification reaction as an indication of the quantity of RNA in the test sample.    
     
     
         20 . The method of  claim 18  or  19 , wherein the first oligonucleic acid and the second oligonucleic acid of the nucleic acid probe do not bind together at the reverse transcription temperature.  
     
     
         21 . The method of  claim 18  or  19 , wherein neither the first oligonucleic acid nor the second oligonucleic acid bind substantially to the RNA of interest at the reverse transcription temperature.  
     
     
         22 . A method of detecting a nucleic acid of interest in a test sample, said method comprising: 
 (i) mixing test sample with DNA amplification reagents,    (ii) incubating the reaction for a suitable time and under suitable DNA amplification conditions to amplify a portion of the nucleic acid of interest,    (iii) adding a first oligonucleic acid that is single-stranded, and comprises a fluorophore and a quencher,    (iv) before, after or at the same time as step (iii) adding a second oligonucleic acid comprising a quencher, wherein the first oligonucleic acid and second oligonucleic acid are complementary such that they are capable of forming a duplex in solution, wherein the ratio of the second oligonucleic acid to the first oligonucleic acid in the test sample is more than 0.1 and less than 0.9,    (v) measuring the fluorescence from the fluorescent probe as an indication of whether the test sample contains the nucleic acid of interest.