| 摘要 |
3-alkynyl inosine analogs and their uses as universal bases are provided. The inosine analogues can be incorporated into nucleic acid primers and probes. They do not significantly destabilize nucleic acid duplexes. As a result, the novel nucleic acid primers and probes incorporating the inosine analogues can be used in a variety of methods. The analogs function unexpectedly well as universal bases. Not only do they stabilize duplexes substantially more than hypoxanthine opposite A, C, T, and G but they are also recognized in primers by polymerases, allowing efficient amplification. |
| 主权项 |
1. A method for the polynucleotide amplification of target nucleic acid sequences comprising:
a) providing a mixture including a sample containing one or more target nucleic acids and at least one oligonucleotide that is substantially complementary to a target nucleic acid sequence, wherein the target nucleic acids comprise the target nucleic acid sequence and at least one target nucleic acid sequence variation, wherein the target nucleic acid sequence variation differs from the target nucleic acid sequence by at least one polymorphic base, wherein the at least one polymorphic base is A, C, or T,
wherein said oligonucleotide comprises at least one 3-alkynyl-1H-pyrazolo[3,4-d]pirimidine-4(5H)-one analogue positioned opposite the at least one polymorphic base that forms a mismatched duplex with the target nucleic acid sequence and the target nucleic acid sequence variation having substantially the same stability as a complementary duplex, regardless which polymorphic base is positioned opposite to the 3-alkynyl-1H-pyrazolo[3,4-d]pirimidine-4(5H)-one base; b) incubating the mixture under hybridization conditions, wherein the hybridization conditions comprise conditions favorable for polymerization and wherein the at least one oligonucleotide hybridizes to the target nucleic acid sequence and the target nucleic acid sequence variation to form mismatched duplexes having substantially the same stability as a complementary duplex, to produce amplified target nucleic acids from the target nucleic acid sequence and the target nucleic acid sequence variation; and c) detecting the amplified target nucleic acids, wherein the amplified target nucleic acids are not distinguished. |
