Efficient product cleavage in template-free enzymatic synthesis of polynucleotides
The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides that include or enable a step of efficiently cleaving the polynucleotide products from its initiator using an endonuclease V activity and initiator with a 3′-penultimate deoxyinosine.
1. A method of synthesizing a polynucleotide having a predetermined sequence, the method comprising the steps of:
a) providing an initiator having a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having a free 3′-hydroxyl;
b) repeating cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of the 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls, until the polynucleotide is formed, wherein said template-independent DNA polymerase is a terminal deoxynucleotidyl transferase; and
c) treating the polynucleotide with an endonuclease V activity to cleave the polynucleotide from the initiator, wherein said endonuclease V activity is provided by a prokaryotic endonuclease V.
2. The method of claim 1 , wherein said prokaryotic endonuclease V is an E. coli endonuclease V.
3. The method of claim 1 , further comprising a step of removing said prokaryotic endonuclease V from said cleaved polynucleotide.
4. The method of claim 1 , wherein said initiator is attached to a support by a 5′ end.
5. The method of claim 4 , wherein said support is a solid support.
6. The method of claim 1 , wherein said initiator has a 3′-terminal sequence of 5′-dI-dT-3′.
7. The method of claim 1 , wherein said polynucleotide cleaved from said initiator has a 5′-monophosphate.
8. A kit for enzymatically synthesizing a polynucleotide comprising an initiator attached to a support by a 5′ end and having a 3′-penultimate deoxyinosine; a 3′-terminal nucleotide having a free 3′-hydroxyl; an endonuclease V capable of cleaving an initiator-polynucleotide conjugate 3′ of a terminal nucleotide of the initiator; the template-free polymerase terminal deoxynucleotidyl transferase; and a 3′-O-blocked nucleoside triphosphate for one or more of deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
9. The kit of claim 8 , further comprising said initiator with a 3′-terminal sequence of 5′-dI-dT-3′.
10. The kit of claim 8 , wherein said support is a solid support.
11. The kit of claim 8 , further comprising a de-blocking agent.
12. The kit of claim 8 wherein said 3′-O-blocked nucleoside triphosphate is 3′-O-NH 2 -nucleoside triphosphate.
13. The method of claim 2 , further comprising a step of removing said prokaryotic endonuclease V from said cleaved polynucleotide.
14. The method of claim 2 , wherein said initiator is attached to a support by a 5′ end.
15. The method of claim 14 , wherein said support is a solid support.