阅读说明：本技术 酶法合成寡核苷酸的方法和体系 (Method and system for enzymatic synthesis of oligonucleotides ) 是由 肯德尔·霍夫 米歇尔·哈尔佩因 詹卡洛·加尔巴尼亚蒂 周巍 于 2018-10-01 设计创作，主要内容包括：本发明涉及在聚合酶存在下由单链固定化引物酶法合成寡核苷酸的方法、过程和系统。利用本发明的方法,在脱氧核糖核苷酸三磷酸或核糖核苷酸三磷酸存在下,可从单链固定化引物酶法合成单链寡核苷酸。双脱氧核糖核苷酸三磷酸酯、带有可逆终止子的脱氧核糖核苷酸三磷酸酯或带有可逆终止子的核糖核苷酸三磷酸酯可以酶法添加到引物或其延伸产物的末端。根据本发明的方法,单链引物可与模板结合,所形成的双链结构可使聚合酶在3’端延伸引物。(The present invention relates to methods, processes and systems for the enzymatic synthesis of oligonucleotides from single-stranded immobilized primers in the presence of a polymerase. Using the method of the present invention, a single-stranded oligonucleotide can be enzymatically synthesized from a single-stranded immobilized primer in the presence of deoxyribonucleotide triphosphate or ribonucleotide triphosphate. A dideoxyribonucleotide triphosphate, a deoxyribonucleotide triphosphate with a reversible terminator or a ribonucleotide triphosphate with a reversible terminator may be enzymatically added to the end of a primer or its extension product. According to the method of the present invention, a single-stranded primer can be bound to a template, and the double-stranded structure formed allows a polymerase to extend the primer at the 3' end.)

1. A method of synthesizing a single stranded oligonucleotide comprising:

(a) providing a single-stranded primer comprising a free 3' end, a polymerase, and a nucleotide reagent; and

(b) extending the single-stranded primer from the free 3' end with the nucleotide reagent by the polymerase;

wherein the polymerase requires no more than 7 base pairings to extend the single stranded primer.

2. The method according to claim 1, wherein the nucleotide reagent is a reversible terminator nucleotide with a 3' terminal terminator.

3. The method according to claim 1, wherein the single-stranded primer further comprises a 5' -end attached to a substrate.

4. The method according to claim 1, wherein the polymerase is a modified/improved/modified polymerase.

5. The method of claim 2, further comprising:

(c) removing the 3' terminal terminator from the reversible terminator nucleotide.

6. The method of claim 5, further comprising:

(d) repeating steps (a) - (c).

7. The method of claim 5, further comprising after (b) and before (c):

(b1) treating the single-stranded primer with a dideoxynucleotide reagent in the presence of the polymerase or terminal deoxynucleotidyl transferase (TdT).

8. The method of claim 1, further comprising after (a) and before (b):

(a1) hybridizing a hybridization site on said single-stranded primer to a template, wherein said hybridization site is at said 3' end and comprises no more than 7 bases.

9. The method according to claim 8, wherein the template is one of the single-stranded primer, an adjacent single-stranded nucleic acid attached to the substrate, or a plurality of single-stranded nucleic acid templates in solution.

10. The method according to claim 9, wherein the template is the adjacent single-stranded nucleic acid, and wherein the adjacent single-stranded nucleic acid has a sequence identity of no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, no more than 50%, no more than 55%, no more than 60%, no more than 65%, no more than 70%, no more than 75%, no more than 80%, no more than 85%, no more than 90%, or no more than 95% to the single-stranded primer.

11. The method according to claim 9, wherein said template is said adjacent single-stranded nucleic acid, and wherein said adjacent single-stranded nucleic acid has 100% sequence identity to said single-stranded primer.

12. The method according to claim 9, wherein the template is a member of the plurality of single-stranded nucleic acid templates in solution, and wherein the plurality of single-stranded nucleic acid templates in solution comprises a plurality of dimers, a plurality of trimers, a plurality of tetramers, a plurality of pentamers, a plurality of hexamers, a plurality of heptamers, a plurality of octamers, a plurality of nonamers, a plurality of decamers, a plurality of undecamers, or a plurality of dodecamers.

13. The method of claim 9, wherein the plurality of single-stranded nucleic acid templates comprise random sequences.

14. The method according to claim 8, wherein the hybridization site comprises 1, 2, 3, 4, 5, 6 or 7 bases.

15. The method of claim 8, wherein the efficiency of said extending in (b) is increased in the presence of said template in (a 1).

16. The method according to claim 8, wherein the polymerase requires 1, 2, 3, 4, 5, 6 or 7 base pairs to extend the single stranded primer.

17. The method according to claim 1, wherein the extension is performed between about 20 ℃ and about 99 ℃.

18. The method according to claim 17, wherein the extension is performed between about 50 ℃ to about 75 ℃.

19. The method of claim 17, wherein the extending is performed between about 55 ℃ to about 65 ℃.

20. The method according to claim 17, wherein the extension is performed at about 60 ℃.

21. The method according to claim 4, wherein the modified polymerase is a modified deoxyribonucleic acid (DNA) polymerase or a modified reverse transcriptase.

22. The method according to claim 21, wherein the modified deoxyribonucleic acid (DNA) polymerase is a thermophilic DNA polymerase which reduces 3 '→ 5' proof reading exonuclease activity relative to the unmodified DNA polymerase.

23. The method of claim 22, wherein the modified DNA polymerase has no more than 6% 3 'to 5' proof-reading exonuclease activity as compared to the unmodified DNA polymerase.

24. The method of claim 22, wherein the modified DNA polymerase has no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5% 3 'to 5' proof-reading exonuclease activity as compared to the unmodified DNA polymerase.

25. The method according to claim 21, wherein the modified reverse transcriptase is a moloney murine leukemia virus (M-MLV) reverse transcriptase or a human immunodeficiency virus type 1 reverse transcriptase.

26. The method according to claim 21, wherein the modified reverse transcriptase is a modified moloney murine leukemia virus (M-MLV) reverse transcriptase or a modified human immunodeficiency virus type 1 reverse transcriptase.

27. The method according to claim 1, wherein the single stranded primer comprises uracil.

28. The method of claim 26, further comprising: cleaving the single-stranded primer on the uracil.

29. The method according to claim 26, wherein said uracil is located at the last base of the 3' end of said single-stranded primer.

30. The method according to claim 6, wherein said repeating in (d) extends said single-stranded primer from said free 3' end, thereby synthesizing a single-stranded oligonucleotide product comprising said single-stranded primer.

31. The method of any of claims 1 to 30, further comprising: cleaving the single-stranded oligonucleotide product.

32. The method according to claim 31, wherein said cleaving is performed at a position within said single-stranded primer.

33. The method according to claim 31, wherein the cleavage removal sequence comprises at least a portion of the single-stranded primer.

34. The method of claim 32, wherein the removed sequence comprises the single-stranded primer.