Template switch-based methods for producing a product nucleic acid
Provided are methods of producing a product nucleic acid. The methods include combining a template deoxyribonucleic acid (DNA), a polymerase, a template switch oligonucleotide, and dNTPs into a reaction mixture. The components are combined into the reaction mixture under conditions sufficient to produce a product nucleic acid that includes the template DNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.
1. A kit comprising:
a 3′ overhang-generating enzyme that generates, in a template DNA, a 3′ overhang comprising more than one unpaired nucleotide;
a template switching polymerase having terminal transferase activity;
a template switch nucleic acid having a first 5′ adapter sequence and a 3′ end complementary to a nucleotide stretch produced by the template switching polymerase via non-templated nucleotide addition; and
a first strand primer comprising a 3′ domain that hybridizes to the template DNA and a second 5′ adapter sequence that does not hybridize to the template DNA, wherein the first and second 5′ adapter sequences are different.
2. The kit according to claim 1 , wherein the 3′ overhang-generating enzyme is a restriction endonuclease.
3. The kit according to claim 1 , wherein the 3′ overhang-generating enzyme is a terminal transferase.
4. The kit according to claim 1 , further comprising a primer comprising the first 5′ adapter sequence.
5. The kit according to claim 1 , wherein the template switch nucleic acid comprises a cloning vector element.
6. The kit according to claim 5 , wherein the cloning vector element is selected from the group consisting of: a selectable marker, a reporter, a promoter, an origin of replication, a multiple cloning site, and combinations thereof.
7. A kit comprising:
a template switching polymerase;
a template switch oligonucleotide comprising a 3′ hybridization domain and a 5′ adapter sequence;
a terminal transferase; and
a template DNA.
8. The kit according to claim 7 , wherein the kit further comprises a first primer.
9. The kit according to claim 8 , wherein the first primer comprises a first domain that hybridizes to a template DNA and a second domain that does not hybridize to the template DNA.
10. The kit according to claim 9 , wherein the first domain has a defined sequence.
11. The kit according to claim 9 , wherein the first domain has an arbitrary sequence.
12. The kit according to claim 11 , wherein the second domain comprises a nucleotide sequence that is identical to a nucleotide sequence present in the template switch oligonucleotide.
13. The kit according to claim 11 , wherein the second domain comprises a nucleotide sequence that is not present in the template switch oligonucleotide.
14. The kit according to claim 1 , further comprising an amplification polymerase.
15. The kit according to claim 1 , wherein the 3′ domain of the first strand primer comprises a defined sequence.
16. The kit according to claim 1 , wherein the 3′ domain of the first strand primer comprises an arbitrary sequence.
17. The kit according to claim 1 , wherein the template switch nucleic acid is a DNA oligonucleotide and the 3′ end of the DNA oligonucleotide comprises at least one ribonucleotide.
18. The kit according to claim 17 , wherein the 3′ end of the DNA oligonucleotide comprises a homonucleotide stretch.
19. The kit according to claim 18 , wherein the homonucleotide stretch is a homo-trinucleotide stretch.
20. The kit according to claim 1 , wherein the template switch nucleic acid comprises a modification that prevents the template switching polymerase from switching from the template switch nucleic acid to another template after synthesizing the compliment of the 5′ end of the template switch nucleic acid.
21. The kit according to claim 1 , further comprising instructions for performing a method to produce a product nucleic acid comprising the template DNA and the template switch nucleic acid each hybridized to adjacent regions of a single hybrid nucleic acid polymerized in part by the template switching polymerase from dNTPs.
22. The kit according to claim 1 , wherein the 3′ overhang-generating enzyme and the template switching polymerase are present in separate containers.
23. The kit according to claim 1 , wherein the template switching polymerase is a retroviral reverse transcriptase.
24. The kit according to claim 1 , further comprising a template DNA.
25. The kit according to claim 7 , wherein the template switch oligonucleotide is a DNA oligonucleotide and the 3′ end of the DNA oligonucleotide comprises at least one ribonucleotide.