Detection of nucleic acids
The present invention provides modified oligonucleotides and methods for their use in the detection of nucleic acids. The oligonucleotides and methods find particular application in amplifying and/or detecting areas of genetic variation in target nucleic acid sequences.
1. A double-stranded nucleic acid duplex comprising:
an oligonucleotide comprising:
a first component terminating at the 5′ end of the oligonucleotide,
a second component terminating at the 3′ end of the oligonucleotide and capable of hybridising to a second portion of a polynucleotide by complementary base pairing, and
a third component located between the first and second components; and
(ii) the polynucleotide, wherein:
the first component is hybridised to a first portion of the polynucleotide by complementary base pairing;
the second component is hybridised to a second portion of the polynucleotide by complementary base pairing, and
the third component comprises a sequence of at least two nucleotides that do not share base pair complementarity with an opposing sequence of nucleotides in the polynucleotide.
2. The double-stranded nucleic acid duplex of claim 1 , wherein the third component is located partially or completely in the 3′ half of the oligonucleotide.
3. The double-stranded nucleic acid duplex of claim 1 , wherein the third component comprises a sequence of at least four nucleotides, at least five, at least six, at least seven, or at least eight nucleotides that do not share base pair complementarity with an opposing sequence of nucleotides in the polynucleotide.
4. The double-stranded nucleic acid duplex according to claim 1 , wherein the polynucleotide comprises unhybridized nucleotides located between its first and second portions.
5. The double-stranded nucleic acid duplex according to claim 4 , wherein the number of said unhybridized nucleotides is equal to the number of nucleotides in said third component that do not share base pair complementarity with the opposing sequence.
6. The double-stranded nucleic acid duplex according to claim 4 , wherein the number of said unhybridized nucleotides exceeds the number of nucleotides in said third component that do not share base pair complementarity with the opposing sequence.
7. The double-stranded nucleic acid duplex according to claim 4 , wherein the number of said unhybridized nucleotides is less than the number of nucleotides in said third component that do not share base pair complementarity with the opposing sequence.
8. The double-stranded nucleic acid duplex according to claim 1 , wherein the number of nucleotides in said third component is between 1 and 200 nucleotides, 1 and 150 nucleotides, 1 and 100 nucleotides, 1 and 75 nucleotides, 1 and 50 nucleotides 1 and 25 nucleotides, 5 and 200 nucleotides, 5 and 150 nucleotides, 5 and 100 nucleotides, 5 and 100 nucleotides, 5 and 75 nucleotides, 5 and 50 nucleotides 5 and 25 nucleotides, 10 and 200 nucleotides, 10 and 150 nucleotides, 10 and 100 nucleotides, 10 and 100 nucleotides, 10 and 75 nucleotides, 10 and 50 nucleotides, or 10 and 25 nucleotides, less than the number of unhybridized nucleotides in the polynucleotide located between portions of the polynucleotide hybridised to said first and second components.
9. The double-stranded nucleic acid duplex according to claim 1 , wherein the oligonucleotide and/or the polynucleotide is genomic DNA, complementary DNA (cDNA), or RNA.
10. The double-stranded nucleic acid duplex according to claim 1 , wherein the melting temperature of the first component is greater than the melting temperature of the second component when hybridised to the polynucleotide strand by complementary base pairing.
11. The double-stranded nucleic acid duplex according to claim 1 , wherein the polynucleotide comprises an intermediate portion between said first and second portions, and the intermediate portion comprises nucleotides that do not share base pair complementarity with an opposing sequence of nucleotides in said third component.
12. The double-stranded nucleic acid duplex according to claim 11 , wherein the first component and the second component each hybridise by complementary base pairing to distinct non-contiguous components of the polynucleotide, thereby juxtaposing the non-contiguous components and creating a loop portion comprising said unhybridized nucleotides of the intermediate portion of the polynucleotide.
13. The double-stranded nucleic acid duplex according to claim 11 , wherein all nucleotides of the first oligonucleotide are hybridised to the polynucleotide by complementary base pairing.
14. The double-stranded nucleic acid duplex according to claim 12 , wherein the loop portion of the polynucleotide comprises between 1 and 200 nucleotides, 1 and 150 nucleotides, 1 and 100 nucleotides, 1 and 75 nucleotides, 1 and 50 nucleotides 1 and 25 nucleotides, 5 and 200 nucleotides, 5 and 150 nucleotides, 5 and 100 nucleotides, 5 and 100 nucleotides, 5 and 75 nucleotides, 5 and 50 nucleotides 5 and 25 nucleotides, 10 and 200 nucleotides, 10 and 150 nucleotides, 10 and 100 nucleotides, 10 and 100 nucleotides, 10 and 75 nucleotides, 10 and 50 nucleotides, or 10 and 25 nucleotides.
15. The double-stranded nucleic acid duplex according to claim 1 , wherein the duplex is isolated.
16. The double-stranded nucleic acid duplex according to claim 1 , wherein the oligonucleotide or, the polynucleotide, or both of the oligonucleotide and the polynucleotide, is/are synthetic or recombinantly made.
17. The double-stranded nucleic acid duplex according to claim 1 , wherein the oligonucleotide or the polynucleotide, or both the oligonucleotide and the polynucleotide, are labelled with a detectable moiety.