Applications for biversal nucleoside analogs
This invention relates to the use of analogs of purines that have two tautomeric forms, one formally complementary to thymidine, the other formally complementary to cytidine, and analogs of pyrimidines that have two tautomeric forms, one formally complementary to guanosine, the other formally complementary to adenosine. These uses include the polymerase chain reaction amplification of targets having polymorphisms in primer binding sites and ligation.
1. A process for ligating a donor fragment and an acceptor fragment together, said fragments being DNA, RNA or DNA-RNA oligonucleotides, said process comprising
(a) contacting said fragments with a DNA or RNA template that comprises two adjacent segments to form a complex, a first segment that is 5′-adjacent to a second segment, wherein said first segment is substantially complementary to the donor fragment that carries a 5′-phosphate group, and said second segment is substantially complementary to the acceptor fragment, under conditions where said donor fragment binds to form a duplex with said first segment, and said acceptor fragment binds to form a duplex with said second segment, and
(b) incubating said complex with a ligase under conditions where the two fragments become joined by a phosphodiester bond,
wherein said duplexes contain at least one nucleobase pair selected from the group consisting of
wherein Q is either CH 3 or H, and R is the point of attachment of the heterocycle nucleobase to the rest of the oligonucleotide.
2. The process of claim 1 wherein said ligase is the Thermus aquaticus DNA ligase, 9° N DNA ligase, Pfu DNA ligase, T4 DNA ligase, Splint R® ligase, T4 RNA ligase, or High Fidelity Taq DNA ligase.
3. The process of claim 1 wherein both of said duplexes contain at least one nucleobase pair selected from the group consisting of
4. The process of claim 1 wherein both of said duplexes contain multiple nucleobase pairs selected from the group consisting of
5. The process of claim 1 , where multiple fragments are ligated on a template that contains multiple adjacent segments.
6. A process for synthesizing a DNA molecule that is complementary to a preselected template, said process comprising
(a) annealing said template to a DNA OR RNA primer and probe that is substantially complementary to a segment of said template to form a duplex, and
(b) incubating said duplex with a DNA polymerase and 2′-deoxynucleoside triphosphates under conditions where said primer is extended,
wherein said primer includes at least one nucleotide comprising the heterocycle
7. The process of claim 6 , wherein said polymerase is Taq DNA polymerase, reverse transcriptase, or AmpliTaq Gold DNA polymerase.
8. The process of claim 6 wherein said primer includes at least two nucleotides comprising the heterocycle
9. The process of claim 6 wherein said probe includes at least one nucleotides comprising the heterocycle
10. A process for increasing the number of copies of two complementary DNA target strands, wherein said process comprises:
(a) adding one or both of said target strands to an aqueous mixture that comprises a DNA polymerase, wherein
(b) said mixture also contains 2′-deoxynucleoside triphosphates that complement each nucleotide in said complementary DNA strands, wherein
(c) said aqueous mixture also contains primers comprising a first primer that is substantially complementary in sequence to a segment at or near the 3′-end of the first of said DNA strands, and a second primer that is substantially complementary in sequence to a segment at or near the 3′-end of the second of said DNA strands, and
wherein at least one primer comprises at least one nucleotide comprising the heterocycle
11. The process of claim 10 wherein one or both of said target strands is RNA.
12. The process of claim 10 wherein at least one primer comprises at least two nucleotides comprising the heterocycle
13. The process of claim 10 , wherein said polymerase is Taq DNA polymerase, KlenTaq DNA polymerase, or AmpliTaq Gold DNA polymerase.
14. A process for determining what nucleotide occupies a site in a DNA target molecule, said process comprising
(a) contacting said target sequence with a probe that is substantially complementary to the target sequence to form a complex, said probe containing a single ribonucleotide complementary to a nucleotide that might occupy said site, the remainder of the nucleotides in said probe being 2′-deoxyribonucleotides, under conditions where said probe and said target hybridize to form a duplex, and
(b) incubating said complex with a ribonuclease H under conditions where the probe is cleaved should the probe ribonucleotide perfectly match the nucleotide in said site,
wherein said duplexes contains at least one nucleobase pair selected from the group consisting of
wherein Q is either CH 3 or H, and R is the point of attachment of the heterocycle nucleobase to the rest of the oligonucleotide.
15. The process of claim 14 wherein said ribonuclease H is ribonuclease H2.
16. The process of claim 14 wherein said duplexes contains at least two nucleobase pairs selected from the group consisting of