| 摘要 |
This invention relates to nucleoside, nucleotide, and oligonucleotide analogs that incorporate non-standard nucleobase analogs, those that present a pattern of hydrogen bonds to a paired nucleobase analog in a complementary strand that is different from the pattern presented by adenine, guanine, cytosine, and thymine. Most specifically, this invention discloses and claims processes for amplifying nucleic acid analogs containing non-standard nucleobases using polymerase chain reactions, and combinations of non-standard nucleobases, analogs of standard nucleotides, and enzymes that perform this amplification. Most specifically, this invention is for the use of 2-thiothymidine triphosphate (2-thioTTP) instead of thymidine triphosphate in a six letter polymerase chain reaction that includes 2′-deoxyadenosine triphosphate, 2′-deoxyguanosine triphosphate, 2′-deoxycytidine triphosphate, 2′-deoxy-iso-guanosine triphosphate, and 2′-deoxy-iso-cytidine triphosphate, as well as their forms that contain side chain modifications. Because of the size and hydrogen bonding properties of the sulfur unit in 2-thioT, 2-thioT does not mispair effectively with the minor tautomer of isoG. This permits the PCR amplification of a six letter artificially expanded genetic information system, we examined the relative rates of misincorporation of 2-thioTTP and TTP opposite isoG using affinity electrophoresis with a fidelity-per-round of ca. 98%. The analogous PCR employing TTP has a fidelity-per-round of only ca. 93%. Therefore, this invention represents the first example of a six letter artificial genetic system that is amplifiable by a thermostable polymerase, and capable of Darwinian evolution.
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