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 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 terminal deoxynucleotidyl transferase so that the initiator or elongated fragments of the initiator are elongated by incorporation of a 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 having the predetermined sequence is formed; and
c) treating the polynucleotide with an endonuclease V activity to cleave the polynucleotide from the initiator, such that the polynucleotide cleaved from the initiator has a 5′-monophosphate.
2. The method of claim 1 , wherein said endonuclease V activity is provided by a prokaryotic endonuclease V.
3. The method of claim 2 , wherein said prokaryotic endonuclease V is an E. coli endonuclease V.
4. The method of claim 2 , further comprising a step of removing said prokaryotic endonuclease V from said cleaved polynucleotide.
5. The method of claim 1 , wherein said initiator is attached to a support by a 5′ end.
6. The method of claim 5 , wherein said support is a solid support.
7. The method of claim 1 , wherein said initiator has a 3′-terminal sequence of 5′-dI-dT-3′.
8. The method of claim 1 , wherein said blocking group attached to said 3′-oxygen is azidomethyl.
9. The method of claim 1 , wherein said blocking group attached to said 3′-oxygen is amino.
10. The method of claim 1 , wherein said blocking group attached to said 3′-oxygen is allyl.