Processes replacing standard nucleotides by non-standard nucleotides and non-standard nucleotides by standard nucleotides in DNA

摘要

This invention concerns non-standard nucleotides that can form non-standard Watson-Crick nucleobase pairs having geometries similar to the geometries of standard nucleotide pairs, but that are joined by a non-standard hydrogen bonding schemes. Disclosed are processes that yield oligonucleotides that are semi-complementary to a standard oligonucleotide, where the region of semi-complementarity pairs one or more standard nucleotides with a non-standard nucleotide, or vice versa. Duplexes formed from two semi-complementary oligonucleotides are also inventions disclosed. The processes extending a primer annealed to an oligonucleotide template with a polymerase in the presence of a non-standard nucleoside triphosphate and a mixture of standard nucleoside triphosphates, where the non-standard triphosphate is incorporated opposite the standard triphosphate because it has available a protonated or deprotonated form, or a minor tautomeric form, that is complementary in the Watson-Crick sense to the standard nucleotide, even though in its normal form (neither protonated or deprotonated, or as its major tautomer) it is complementary in the Watson-Crick sense to a non-standard nucleobase. Also disclosed are processes that exploit an intermediary nucleoside, one whose nucleobase is partially complementary to both a standard nucleotide and a non-standard nucleotide at two of their three hydrogen bonding units. Also disclosed are intermediary nucleotides whose hydrogen bonding patterns are changed by chemical reagents. Also disclosed are the vice versa processes and process pairs where standard nucleotides are incorporated opposite non-standard nucleotides, yielding clonable products that can be sequenced to determine where non-standard nucleotides were present in the parent template oligonucleotide.

主权项

1. A process for the addition of a nucleotide having, after incorporation, the structure to the 3′-end of an oligonucleotide primer having a preselected sequence, wherein X is selected from the group consisting of —H and —OH, Z is the point of attachment to the 3′-end of said oligonucleotide primer, and B is selected from the group consisting of said process comprising contacting said primer in buffered aqueous solution with a template under conditions where said primer forms a duplex with said template, and incubating said duplex with a mixture of polymerase and polymerase substrates comprising the triphosphate wherein X and B are defined as above, and wherein said added nucleotide is incorporated by said polymerase opposite a non-complementary standard nucleoside in the template selected from the group consisting of adenosine, guanosine, cytidine, uridine, and thymidine.