OLIGONUCLEOTIDES CONTAINING PYRAZOLO[3,4-D] PYRIMIDINES FOR HYBRIDIZATION AND MISMATCH DISCRIMINATION
Oligonucleotides in which one or more purine residues are substituted by pyrazolo[3,4-d]pyrimidines exhibit improved hybridization properties. Oligonucleotides containing pyrazolo[3,4-d]pyrimidine base analogues have higher melting temperatures than unsubstituted oligonucleotides of identical sequence. Thus, in assays involving hybridization of an oligonucleotide probe to a target polynucleotide sequence, higher signals are obtained. In addition, mismatch discrimination is enhanced when pyrazolo[3,4-d]pyrimidine-containing oligonucleotides are used as hybridization probes, making them useful as probes and primers for hybridization, amplification and sequencing procedures, particularly those in which single- or multiple-nucleotide mismatch discrimination is required.
1 - 47 . (canceled)
48 . An oligonucleotide probe capable of hybridizing with a target sequence comprising a modified target binding sequence wherein one or more purine residues of said target binding sequence are substituted by a pyrazolo[3,4-d]pyrimidine residue, and further comprising an attached minor groove binder, said modified target binding sequence corresponding to and capable of binding with a target sequence containing no pyrazolo[3,4-d]pyrimidine residues.
49 . An oligonucleotide probe of claim 48 , wherein the minor groove binder is selected from the group consisting of a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC5).
50 . An oligonucleotide probe of claim 48 , further comprising a detectable label.
51 . An oligonucleotide probe of claim 50 wherein the detectable label is selected from radioactive isotopes, chromophores, fluorophores, chemiluminescent agents, electrochemiluminescent agents, magnetic labels, immunologic labels, ligands and enzymatic labels.
52 . An oligonucleotide probe of claim 50 , wherein the label is located is located at the oligonucleotide 5′ end.
53 . An oligonucleotide probe of claim 50 , wherein the label is located is located at the oligonucleotide 3′ end.
54 . An oligonucleotide probe of claim 50 , wherein the detectable label is a fluorescent label.
55 . An oligonucleotide probe of claim 54 further comprising a quenching agent which quenches the fluorescence emission of the fluorescent label.
56 . An oligonucleotide probe of claim 50 , comprising multiple fluorescent labels.
57 . An oligonucleotide probe of claim 56 , wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.
58 . An oligonucleotide probe according to claim 48 , wherein the purine residues are selected from guanine residues and adenine residues.
59 . An oligonucleotide probe according to claim 48 , wherein the purine residues are guanine residues, and all of the guanine residues are substituted by a pyrazolo[3,4-d]pyrimidine residue.
60 . An oligonucleotide probe according to claim 48 , comprising four guanine residues in sequence that are each substituted by a pyrazolo[3,4-d]pyrimidine residue.
61 . An oligonucleotide probe according to claim 48 , comprising six guanine residues in sequence that are each substituted by a pyrazolo[3,4-d]pyrimidine residue.
62 . An oligonucleotide probe according to claim 48 comprising at least 12 but less than 21 bases.