Double stranded linear nucleic acid probe
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 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 comprising a first oligonucleic acid and a second oligonucleic acid, wherein;
i) the first oligonucleic acid is 30 to 34 bases (m) in length and having a complementarity of 80% or more to a nucleic acid of interest, and further comprises a fluorophore;
ii) the second oligonucleic acid is n bases in length wherein n is from 8 to 15, and further comprises a quencher;
iii) the first oligonucleic acid and second oligonucleic acid are characterized in that:
(1) the second oligonucleic acid is has a complementarity of 80% or more to the first oligonucleic acid, and
(2) the first oligonucleic acid and second oligonucleic acid can bind together in the absence of the nucleic acid of interest under appropriate conditions to form a double-stranded nucleic acid such that the fluorescent emission of the fluorophore attached to the first oligonucleic acid when the first oligonucleic acid is bound to the second oligonucleic acid is detectable less than the emission of the same fluorophore when the first oligonucleic acid and second oligonucleic acid are not bound together;
a) placing the test sample under conditions permissive of reverse transcription, which optionally can also be permissive of amplification, such that a cDNA is produced;
b) thermocycling the test mixture such that cDNA is amplified; and
c) measuring the fluorescence of the test mixture during the amplification reaction as an indication of the quantity of RNA in the test sample.
2. The method of claim 1 , wherein the ratio of the second oligonucleic acid to the first oligonucleic acid is more than 1.1.
3. The method 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.
4. The method of claim 1 , wherein in said nucleic acid probe the first oligonucleic acid further comprises a quencher.
5. The method of claim 4 , wherein the ratio of the second oligonucleic acid to the first oligonucleic acid is more than 0.1 and less than 0.9.
6. The method of claim 4 , wherein the ratio of the second oligonucleic acid to the first oligonucleic acid is more than 1.1.
7. The method of claim 1 , wherein in said nucleic acid probe 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.
8. The method of claim 1 , wherein said first oligonucleic acid is SEQ ID NO: 5.
9. method of claim 1 , wherein said second oligonucleic acid is SEQ ID NO: 11.
10. 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 comprising a first oligonucleic acid and a second oligonucleic acid, wherein;
i) the first oligonucleic acid is 30 to 34 bases (m) in length and has a complementarity of 80% or more to a nucleic acid of interest, and comprises a quencher,
ii) the second oligonucleic, acid is n bases in length wherein n is from 8 to 15, and further comprises a fluorophore;
iii) the first oligonucleic acid and second oligonucleic acid is characterized in that:
(1) the second oligonucleic acid has a complementaritv of 80% or more to the first oligonucleic acid, and
(2) the first oligonucleic acid and second oligonucleic acid can bind together in the absence of the nucleic acid of interest under appropriate conditions to form a double-stranded nucleic acid such that the fluorescent emission of the fluorophore attached to the second oligonucleic acid when the first oligonucleic acid is bound to the second oligonucleic acid is detectable less than the emission of the same fluorophore when the first oligonucleic acid and second oligonucleic acid are not bound together;
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.
11. The method of claim 10 , wherein the ratio of the first oligonucleic acid to the second oligonucleic acid is more than 1.1.
12. The method of claim 10 , 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 method of claim 10 , wherein the second oligonucleic acid further comprises a quencher.
14. The method of claim 13 , wherein the ratio of the first oligonucleic acid to the second oligonucleic acid is more than 0.1 and less than 0.9.
15. The method of claim 13 , wherein the ratio of the first oligonucleic acid to the second oligonucleic acid is more than 1.1.
16. The method of claim 10 , wherein in said nucleic acid probe the second oligonucleic acid comprises at least two fluorophores to allow the fluorescence generated by the second 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.
17. The method of claim 10 , wherein said first oligonucleic acid is SEQ ID NO: 5.
18. The method of claim 10 , wherein said second oligonucleic acid is SEQ ID NO: 11.