Synthon formation

Patent metadata

Field	Value
Publication number	US-9447445-B2
Application number	US-201514837820-A
Country	US
Kind code	B2
Filing date	Aug 27, 2015
Priority date	Aug 27, 2014
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

This disclosure provides, among other things, a composition comprising: a 5′ exonuclease; a strand-displacing polymerase; and optionally a single strand DNA binding protein and/or a ligase. A method for polynucleotide assembly to form a synthon, as well as a kit for performing the same, are also described.

First claim

What is claimed is:

1. A preparation, comprising:
a fusion protein comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:3, wherein the fusion protein comprises:
(i) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:1; and
(ii) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2.

2. The preparation according to claim 1 , further comprising a 5′-3′ exonuclease.

3. The preparation according to claim 2 , wherein the 5′-3′ exonuclease is T5 exonuclease.

4. The preparation according to claim 1 , further comprising a single strand DNA binding protein.

5. The preparation according to claim 4 , wherein the single strand DNA binding protein is ET SSB (Extreme Thermostable Single-Stranded DNA Binding Protein), E. coli recA, T7 gene 2.5 product, phage lambda RedB or Rac prophage RecT.

6. The preparation according to claim 1 , further comprising a ligase.

7. The preparation of claim 1 , wherein the preparation does not comprise a crowding agent and/or a non strand-displacing polymerase.

8. The preparation of claim 1 , further comprising a potassium salt.

9. The preparation according to claim 1 , further comprising a plurality of polynucleotides in a set wherein at least a polynucleotide in the set has a sequence that overlaps with another polynucleotide in the set; and wherein the polynucleotides are selected from: (i) double stranded polynucleotides; (ii) single stranded oligonucleotides; (iii) at least one double strand polynucleotide and at least one single strand oligonucleotide; and (iv) a subpopulation of polynucleotides that are otherwise identical to one another except for a sequence that varies between the members of the sub-population.

10. The preparation according to claim 9 , wherein the set of polynucleotides has at least 3 members or at least 4 members or at least 5 members.

11. A method for producing a synthon, comprising:
incubating a preparation according to claim 2 further optionally comprising a ligase and a single strand DNA binding protein, with a plurality of polynucleotides that form a set, wherein members of the set have sequences that overlap under suitable reaction conditions; and
joining at least two the polynucleotides to produce a synthon.

12. The method according to claim 11 , wherein the set of polynucleotides contains at least 3 members or at least 4 members or at least 5 members.

13. The method according to claim 11 , wherein the preparation further comprises a ligase.

14. The method according to claim 11 , wherein the preparation further comprises a single strand DNA binding protein.

15. The method according to claim 11 , wherein the polynucleotides are double stranded and the double strand polynucleotides are overlapping PCR products, overlapping restriction fragments or assembled from single stranded oligonucleotides.

16. The method according to claim 11 , wherein the polynucleotides are single stranded oligonucleotides.

17. The method according to claim 11 , wherein the set of polynucleotides comprises at least one double strand polynucleotide and at least one single strand oligonucleotide.

18. A kit for polynucleotide assembly, comprising:
(a) a preparation according to claim 1 ; and
(b) a 5′-3′ exonuclease.

19. A kit according to claim 18 , further comprising a single strand binding protein.

20. The kit according to claim 18 , further comprising a ligase.

21. The kit according to claim 18 , further comprising a buffering agent.

22. The kit according to claim 18 , wherein the 5′-3′ exonuclease is T5 exonuclease.

23. The kit according to claim 18 , does not include a crowding agent.

24. The kit according to claim 18 , wherein (a)-(b) are in the same vessel.

25. The preparation according to claim 1 , wherein the fusion protein comprises an amino acid sequence that has at least 95% identity to SEQ ID NO:3, wherein the fusion protein comprises:
(i) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:1; and
(ii) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:2.

26. The preparation according to claim 1 , wherein the fusion protein comprising the amino acid sequence of SEQ ID NO:3.

Assignees

• New England Biolabs Inc

Inventors

• Hsieh Pei-Chung
• Sun Luo
• Evans Jr Thomas C
• Davis Theodore B
• Gardner Andrew

Classifications

• C12N9/1252

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12N15/64

  General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title