NUCLEIC ACID MONOMERS WITH 2'-CHEMICAL MOIETIES
The invention provides nucleic acid monomers with a 2′-modification that are useful for the incorporation of dyes or blocking groups. The monomers can be incorporated on the 3′-end of a dual labeled probe to inhibit PCR polymerase extension during PCR. The polymerase is inhibited from extending the probe at the 3′-hydroxyl group when the monomer is present; there is no need to add a chemical moiety to the 3′-hydroxyl or remove the 3′-hydroxyl. The monomers can also be incorporated internally or at the 5′-end of the oligonucleotide. A detectable label, such as a fluorescent or quenching dye, can be incorporated on the 2′-position of such monomers.
1 . A chemical composition having a structure of Formula 9:
wherein B is a nucleobase;
Y is a fluorescent dye, a quenching dye or an acetal group;
and W is selected from a group comprising a phosphodiester bond, a hydroxyl group, a protected hydroxyl group, a nucleotide, an oligonucleotide chain, an —SH— group, a protected —SH— group or a phosphorothioate bond.
2 . An oligonucleotide between about 10 and about 75 monomers in length wherein at least one of said monomers is the composition of claim 1 .
3 . The oligonucleotide of claim 2 wherein the claim 1 composition is at the 3′-end of the oligonucleotide.
4 . The oligonucleotide of claim 2 wherein the oligonucleotide is complementary to a target nucleic acid sequence for which the oligonucleotide is a probe.
5 . The oligonucleotide of claim 4 wherein the oligonucleotide is modified with a reporter group.
6 . The oligonucleotide of claim 5 wherein the reporter group is a fluorophore.
7 . The oligonucleotide of claim 5 wherein the oligonucleotide also contains a quencher.
8 . A method of detecting a target nucleic acid sequence wherein the oligonucleotide of claim 4 is used as a probe.
9 . The method of claim 8 wherein the probe is used to detect a product of an amplification reaction.
10 . The method of claim 9 wherein said amplification reaction is PCR or a polynomial amplification reaction.
11 . The method of claim 10 wherein the amplification reaction utilizes an enzymatic cleavage of the oligonucleotide.
12 . The method of claim 11 wherein the enzymatic cleavage of the oligonucleotide is effected by a 5′-nuclease activity of a DNA polymerase.
13 . A method of detecting a polynomial amplification product wherein an oligonucleotide of claims 2 is used as a template for polymerase extension of said polynomial amplification product.
14 . The method of claim 13 wherein said oligonucleotide is cleaved enzymatically.
15 . An oligonucleotide between about 10 to about 75 monomers in length, wherein at least one monomer has a chemical composition having a structure of Formula 10:
wherein B is a nucleobase;
Y is a fluorescent dye, a quenching dye, a silyl group, a ketone group or an acetal group;
and W is selected from a group comprising a phosphodiester bond, a hydroxyl group, a protected hydroxyl group, a nucleotide, an oligonucleotide chain, an —SH— group, a protected —SH— group or a phosphorothioate bond.
16 . The oligonucleotide of claim 15 wherein a monomer having a chemical composition having a structure of Formula 10 is incorporated at the 3′-end of the oligonucleotide.
17 . The oligonucleotide of claim 15 wherein the oligonucleotide is complementary to a target nucleic acid sequence for which the oligonucleotide is a probe.
18 . The oligonucleotide of claim 17 wherein the oligonucleotide is modified with a reporter group.
19 . The oligonucleotide of claim 18 wherein the reporter group is a fluorophore.
20 . The oligonucleotide of claim 18 wherein the oligonucleotide also contains a quencher.
21 . The oligonucleotide of claim 16 wherein Y of the 3′-end monomer inhibits extension of the oligonucleotide by a polymerase.
22 . The oligonucleotide of claims 15 wherein Y is a triisopropyl silyl or a tert-butyldiphenylsilyl group.
23 . A method of detecting a target nucleic acid sequence wherein the oligonucleotide of claim 16 is used as a probe.
24 . The method of claim 23 wherein the probe is used to detect a product of an amplification reaction.
25 . The method of claim 24 wherein said amplification reaction is PCR or a polynomial amplification reaction.
26 . The method of claim 25 wherein the amplification reaction utilizes an enzymatic cleavage of the oligonucleotide.
27 . The method of claim 26 wherein the enzymatic cleavage of the oligonucleotide is effected by a 5′-nuclease activity of a DNA polymerase.
28 . A method of detecting a polynomial amplification product wherein an oligonucleotide of claims 15 is used as a template for polymerase extension of said polynomial amplification product.
29 . The method of claim 28 wherein said oligonucleotide is cleaved enzymatically.
30 . A chemical composition having the structure of Formula 11:
wherein B is a nucleobase,
Y 3 is XR wherein X is a heteroatom or an alkyl group and R is a substituted alkyl or acetal chain with a ketone;
W is selected from a group comprising a phosphodiester bond, a hydroxyl group, a protected hydroxyl group, a nucleotide, an oligonucleotide chain, an —SH— group, a protected —SH— group or a phosphorothioate bond;
And Z is selected from a group comprising a hydroxyl group, one or more nucleotides, a solid support or a linking group.
31 . The chemical composition of claim 30 wherein the linking group is a phosphoramidite, a succinate monoester, H-phosphonate or a phosphate diester.
32 . The composition of claim 30 wherein R is (CH 2 ) n —X 2 —(CH 2 ) n —(X 3 ) m , wherein X 2 is a heteroatom, a ketone group or CH 2 , n is 1-5, X 3 is a COCH 3 group or a ketone group, and m is 0 when X 2 is a ketone or 1 when X 2 is not a ketone group.
33 . The composition of claim 30 wherein Y 3 is
34 . The composition in claims 30 wherein the ketone group is conjugated to an aminooxy label.
35 . The composition of claim 34 wherein the aminooxy label is a fluorophore.
36 . The composition of claim 34 wherein the aminooxy label is a quencher.
37 . The composition of claim 34 wherein the composition is Formula 12:
38 . An oligonucleotide between about 10 and about 75 monomers in length wherein at least one of said monomers is the composition of claim 30 .
39 . The oligonucleotide of claim 38 wherein the claim 30 composition is at the 3′-end of the oligonucleotide.
40 . The oligonucleotide of claim 38 wherein the oligonucleotide is complementary to a target nucleic acid sequence for which the oligonucleotide is a probe.
41 . The oligonucleotide of claim 40 wherein the oligonucleotide is modified with a reporter group.
42 . The oligonucleotide of claim 41 wherein the reporter group is a fluorophore.
43 . The oligonucleotide of claim 42 wherein the oligonucleotide also contains a quencher.
44 . A method of detecting a target nucleic acid sequence wherein the oligonucleotide of claim 41 is used as a probe.
45 . The method of claim 44 wherein the probe is used to detect a product of an amplification reaction.
46 . The method of claim 45 wherein said amplification reaction is PCR or a polynomial amplification reaction.
47 . The method of claim 46 wherein the amplification reaction utilizes an enzymatic cleavage of the oligonucleotide.
48 . The method of claim 47 wherein the enzymatic cleavage of the oligonucleotide is effected by a 5′-nuclease activity of a DNA polymerase.
49 . A method of detecting a polynomial amplification product wherein an oligonucleotide of claims 38 is used as a template for polymerase extension of said polynomial amplification product.
50 . The method of claim 49 wherein said oligonucleotide is cleaved enzymatically.
51 . An oligonucleotide between about 10 to about 75 monomers for use as a probe in a hybridization assay, said oligonucleotide being complementary to a target nucleic acid sequence, wherein said oligonucleotide contains a reporter group, and wherein a monomer at the 3′-end of the oligonucleotide is Formula 13:
wherein Y is a blocking group that inhibits extension of the oligonucleotide by a polymerase.
52 . The oligonucleotide of claim 51 wherein the reporter group is a fluorophore.
53 . The oligonucleotide of claim 51 wherein the oligonucleotide also contains a quencher.
54 . A method of detecting a target nucleic acid sequence wherein the oligonucleotide of claim 52 is used as a probe.
55 . The method of claim 54 wherein the probe is used to detect a product of an amplification reaction.
56 . The method of claim 55 wherein said amplification reaction is PCR or a polynomial amplification reaction.
57 . The method of claim 56 wherein the amplification reaction utilizes an enzymatic cleavage of the oligonucleotide.
58 . The method of claim 57 wherein the enzymatic cleavage of the oligonucleotide is effected by a 5′-nuclease activity of a DNA polymerase.
59 . A method of detecting a polynomial amplification product wherein an oligonucleotide of claims 51 is used as a template for polymerase extension of said polynomial amplification product.
60 . The method of claim 59 wherein said oligonucleotide is cleaved enzymatically.
61 . A kit for an amplification of a target sequence, the kit comprising:
a) the oligonucleotide probe of claim 51;
b) a set of one or more target sequence primers;
c) a polymerase enzyme.
62 . A nucleic acid comprising: (a) a cleavage domain comprising a single-stranded region, said single-stranded region comprising at least one internucleotide linkage 3′ to an adenosine residue, at least one internucleotide linkage 3′ to a cytosine residue, at least one internucleotide linkage 3′ to a guanosine residue, and at least one internucleotide linkage 3′ to a uridine residue, and wherein said cleavage domain does not comprise a deoxyribonuclease-cleavable internucleotide linkage; (b) a fluorescence reporter group on one side of the internucleotide linkages; and (c) the monomer of claim 37 on the other side of the internucleotide linkages.