Oligonucleotide probes and primers comprising universal bases for diagnostic purposes
View Patent ↗Aspects of the invention relate to novel oligonucleotides comprising universal and generic bases for use as primers and probes, as well as, methods of using said oligonucleotides for the diagnosis of disease.
1. An oligonucleotide probe for detecting a first target nucleic acid sequence comprising the formula:
XRY
wherein X comprises between about 5 and 20 modified nucleic acid bases complementary to a first region of said first target nucleic acid sequence;
wherein R comprises between about 5 and 10 universal or generic bases juxtaposed to one another and said universal or generic bases align with bases of a second region of said first target nucleic acid sequence;
wherein Y comprises between about 5 and about 20 nucleic acid bases complementary to a third region of said first target nucleic acid sequence;
wherein X, R, and Y are covalently joined;
wherein greater than 20% of the total number of bases in said oligonucleotide probe are said universal or generic bases juxtaposed to one another;
wherein said oligonucleotide probe further comprises a detectable label; and
wherein said oligonucleotide probe is capable of hybridizing to and detecting said first target nucleic acid sequence but not able to detect a second target nucleic acid sequence that differs from said first target nucleic acid sequence by a single nucleotide base mismatch in the first or third region of the target nucleic acid sequence.
2. An oligonucleotide probe for distinguishing a first target nucleic acid sequence from a second target nucleic acid sequence that differs from said first target nucleic acid sequence by a nucleotide base mismatch, said oligonucleotide probe comprising
a site complementary to said first target nucleic acid sequence, said site having a 5′ and 3′ side thereto and having a non-complementary base to said mismatch of said second target nucleic acid sequence;
at least five universal or generic bases juxtaposed to one another, said universal or generic bases being located on the 5′ or 3′ side of said site and aligned with bases of the first target nucleic acid sequence;
a first region complementary to a first region of the first target nucleic acid and a second region complementary to a second region of the first target nucleic acid, said first and second regions being located on the 5′ and 3′ side of said site, respectively;
wherein said oligonucleotide probe has between about 10 and 25 bases;
wherein said oligonucleotide probe further comprises a detectable label;
and wherein said probe detects said first target nucleic acid sequence and does not detect said second target sequence.
3. The oligonucleotide probe of claim 1 , wherein at least 6 of said bases are universal or generic bases juxtaposed to one another.
4. The oligonucleotide probe of claim 1 , wherein at least 7 of said bases are universal or generic bases juxtaposed to one another.
5. The oligonucleotide probe of claim 1 wherein said universal bases are selected from the group consisting of: 2-deoxyinosine, 5-nitroindole, 3-nitropyrrole, and 2-deoxynebularine.
6. The oligonucleotide probe of claim 1 , wherein said oligonucleotide probe comprises between about 10 and about 25 bases.
7. The oligonucleotide probe of claim 1 , wherein said oligonucleotide probe comprises between about 10 and about 50 bases.
8. The oligonucleotide probe of claim 1 , further comprising a non-nucleic acid linker.
9. The oligonucleotide probe of claim 1 , wherein 30% or more of said bases are universal or generic bases.
10. A hybrid consisting of the oligonucleotide probe of claim 1 and a target nucleic acid sequence.
11. The oligonucleotide probe of claim 1 , wherein said oligonucleotide probe has an increased ability to distinguish said first target nucleic acid sequence from said second target nucleic acid sequence that differs from said first target nucleic acid sequence by a nucleotide base mismatch as compared to an oligonucleotide probe that does not have any universal or generic bases.
12. The oligonucleotide probe of claim 2 , wherein at least 6 of said bases are universal or generic bases juxtaposed to one another.
13. The oligonucleotide probe of claim 2 , wherein at least 7 of said bases are universal or generic bases juxtaposed to one another.
14. The oligonucleotide probe of claim 2 wherein said universal bases are selected from the group consisting of: 2-deoxyinosine, 5-nitroindole, 3-nitropyrrole, and 2-deoxynebularine.
15. The oligonucleotide probe of claim 2 , wherein said oligonucleotide probe comprises between about 10 and about 50 bases.
16. The oligonucleotide probe of claim 2 , further comprising a non-nucleic acid linker.
17. The oligonucleotide probe of claim 2 , wherein greater than 20% of said bases are universal or generic bases.
18. The oligonucleotide probe of claim 2 , wherein 30% or more of said bases are universal or generic bases.
19. A hybrid consisting of the oligonucleotide probe of claim 2 and a target nucleic acid sequence.
20. The oligonucleotide probe of claim 2 , wherein said oligonucleotide probe has an increased ability to distinguish said first target nucleic acid sequence from said second target nucleic acid sequence that differs from said first target nucleic acid sequence by a nucleotide base mismatch as compared to an oligonucleotide probe that does not have any universal or generic bases.
21. The oligonucleotide probe of claim 1 or 2 , wherein said universal bases are selected from the group consisting of: 2-deoxyinosine and 2-deoxynebularine.
22. The oligonucleotide probe of claim 1 or 2 , wherein said universal bases are 3-nitropyrrole.
23. The oligonucleotide probe of claim 1 or 2 , wherein said universal bases are 5-nitroindole.