Methods and compounds for chemical ligation
Compositions and methods for chemical ligation are provided. Methods for nucleic acid sequencing, nucleic acid assembly and nucleic acid synthesis are also provided.
1. A method of ligating a probe to a reference oligonucleotide comprising the steps of:
providing a reference oligonucleotide having an anchor primer bound thereto;
providing a first probe oligonucleotide having a preactivated chemical ligation compound attached thereto;
allowing the first probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized first probe oligonucleotide and the anchor primer, wherein the preactivated chemical ligation compound is a phosphoramidate.
2. A method of ligating a probe to a reference oligonucleotide comprising the steps of:
providing a reference oligonucleotide having an anchor primer bound thereto;
providing a first probe oligonucleotide having a preactivated chemical ligation compound attached thereto;
allowing the first probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized first probe oligonucleotide and the anchor primer, wherein the preactivated chemical ligation compound is a phosphoramidate which includes an R group selected from the group consisting of pyridine, aniline, imidazole, ethanolamine, azaoxybenzotriazolide, N-oxysuccinimide and succinimide.
3. The method of claim 1 , wherein the first probe oligonucleotide further comprises one or more S 2 T molecules.
4. The method of claim 1 , wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S 2 T molecules.
5. The method of claim 1 , wherein the first probe oligonucleotide further comprises one or more diaminopurines.
6. The method of claim 1 , wherein one or more adenines of the first probe oligonucleotide are replaced by one or more diaminopurines.
7. A method of ligating a probe to a reference oligonucleotide comprising the steps of:
providing a reference oligonucleotide having an anchor primer bound thereto;
providing a first probe oligonucleotide having two preactivated chemical ligation compounds attached thereto;
allowing the first probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized first probe oligonucleotide and the anchor primer, wherein the preactivated chemical ligation compound is a phosphoramidate.
8. A method of ligating a probe to a reference oligonucleotide comprising the steps of:
providing a reference oligonucleotide having an anchor primer bound thereto;
providing a first probe oligonucleotide having a preactivated chemical ligation compound attached thereto;
allowing the first probe oligonucleotide to hybridize to the reference oligonucleotide;
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized first probe oligonucleotide and the anchor primer;
providing a second probe oligonucleotide;
allowing the second probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized second probe oligonucleotide and the hybridized first probe oligonucleotide, wherein the preactivated chemical ligation compound is a phosphoramidate.
9. A method of ligating a probe to a reference oligonucleotide comprising the steps of:
providing a reference oligonucleotide having an anchor primer bound thereto;
providing a first probe oligonucleotide having a preactivated chemical ligation compound attached thereto;
allowing the first probe oligonucleotide to hybridize to the reference oligonucleotide;
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized first probe oligonucleotide and the anchor primer;
providing a second probe oligonucleotide;
allowing the second probe oligonucleotide to hybridize to the reference oligonucleotide, wherein the second probe oligonucleotide has a preactivated chemical ligation compound attached thereto; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized second probe oligonucleotide and the hybridized first probe oligonucleotide, wherein the preactivated chemical ligation compound is a phosphoramidate.
10. A method of assembling a polynucleotide sequence comprising the steps of:
providing a plurality of overlapping oligonucleotides, at least a portion of which have a preactivated chemical ligation compound attached thereto;
allowing hybridization of at least a portion of the overlapping oligonucleotides to each other; and
allowing the preactivated chemical ligation compounds to mediate ligation between adjacent 5′ and 3′ ends of hybridized oligonucleotides to assemble a polynucleotide sequence, wherein the preactivated chemical ligation compound is a phosphoramidate.
11. The method of claim 10 , wherein the polynucleotide sequence is DNA.
12. The method of claim 11 , wherein the DNA is selected from the group consisting of a vector, a gene, a gene fragment, an exon, an intron and an intergenic DNA sequence.
13. A method of ligating a first oligonucleotide to a second oligonucleotide comprising the steps of:
providing a first oligonucleotide having a preactivated chemical ligation compound attached thereto;
providing a second oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the first oligonucleotide and the second oligonucleotide, wherein the preactivated chemical ligation compound is a phosphoramidate.
14. The method of claim 13 , further comprising the step of allowing the first oligonucleotide to bind a reference oligonucleotide.
15. The method of claim 14 , wherein the reference oligonucleotide is attached to a substrate.
16. A method of ligating a probe to a reference oligonucleotide comprising the steps of:
providing a reference oligonucleotide having an anchor primer bound thereto;
providing a first probe oligonucleotide having two preactivated chemical ligation compounds attached thereto;
allowing the first probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized first probe oligonucleotide and the anchor primer, wherein the preactivated chemical ligation compound is a member selected from the group consisting of a cyanogen bromide-based compound, a carbodiimide-based compound, an amidate-based compound and an imine.
17. The method of claim 16 , wherein the cyanogen bromide-based compound is a phosphocyanate.
18. The method of claim 17 , wherein the phosphocyanate is 2-(N-Morpholino)Ethane Sulfonic Acid-cyanogen.
19. The method of claim 16 , wherein the carbodiimide-based compound is an O-phospho-isocarbamide compound.
20. The method of claim 19 , wherein the O-phospho-isocarbamide compound is phospho-1-Ethyl-3-[3-Dimethylaminopropyl]Carbodiimide.
21. The method of claim 16 , wherein the amidate-based compound is a phosphoramidate.
22. The method of claim 21 , wherein the phosphoramidate includes an R group selected from the group consisting of pyridine, aniline, imidazole, ethanolamine, azaoxybenzotriazolide, N-oxysuccinimide and succinimide.
23. The method of claim 16 , wherein the first probe oligonucleotide further comprises one or more S 2 T molecules.
24. The method of claim 16 , wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S 2 T molecules.
25. The method of claim 16 , wherein the first probe oligonucleotide further comprises one or more diaminopurines.
26. The method of claim 16 , wherein one or more adenines of the first probe oligonucleotide are replaced by one or more diaminopurines.
27. The method of claim 16 , further comprising the steps of:
providing a second probe oligonucleotide;
allowing the second probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized second probe oligonucleotide and the hybridized first probe oligonucleotide.
28. The method of claim 27 , wherein the second probe oligonucleotide has a preactivated chemical ligation compound attached thereto.
29. A method of assembling a polynucleotide sequence comprising the steps of:
providing a plurality of overlapping oligonucleotides having at least a portion of which have a preactivated chemical ligation compound attached thereto and at least one of which has two preactivated chemical ligation compounds attached thereto;
allowing hybridization of at least a portion of the overlapping oligonucleotides to each other; and
allowing the preactivated chemical ligation compounds to mediate ligation between adjacent 5 and 3′ ends of hybridized oligonucleotides to assemble a polynucleotide sequence, wherein the preactivated chemical ligation compound is a member selected from the group consisting of a cyanogen bromide-based compound, a carbodiimide-based compound, an amidate-based compound and an imine.
30. The method of claim 29 , wherein the polynucleotide sequence is DNA.
31. The method of claim 30 , wherein the DNA is selected from the group consisting of a vector, a gene, a gene fragment, an exon, an intron and an intergenic DNA sequence.
32. A method of ligating a first oligonucleotide to a second oligonucleotide comprising the steps of:
providing a first oligonucleotide having two preactivated chemical ligation compounds attached thereto;
providing a second oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the first oligonucleotide and the second oligonucleotide, wherein the preactivated chemical ligation compound is a member selected from the group consisting of a cyanogen bromide-based compound, a carbodiimide-based compound, an amidate-based compound and an imine.
33. The method of claim 32 , further comprising the step of allowing the first oligonucleotide to bind a reference oligonucleotide.
34. The method of claim 32 , wherein the reference oligonucleotide is attached to a substrate.
35. The method of claim 2 , wherein the first probe oligonucleotide further comprises one or more S 2 T molecules.
36. The method of claim 2 , wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S 2 T molecules.
37. The method of claim 2 , wherein the first probe oligonucleotide further comprises one or more diaminopurines.
38. The method of claim 2 , wherein one or more adenines of the first probe oligonucleotide are replaced by one or more diaminopurines.
39. The method of claim 2 , wherein the first probe oligonucleotide has two preactivated chemical ligation compounds attached thereto.
40. The method of claim 2 , further comprising the steps of:
providing a second probe oligonucleotide;
allowing the second probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized second probe oligonucleotide and the hybridized first probe oligonucleotide.
41. The method of claim 40 , wherein the second probe oligonucleotide has a preactivated chemical ligation compound attached thereto.
42. The method of claim 7 , wherein the first probe oligonucleotide further comprises one or more S 2 T molecules.
43. The method of claim 7 , wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S 2 T molecules.
44. The method of claim 7 , wherein the first probe oligonucleotide further comprises one or more diaminopurines.
45. The method of claim 7 , wherein one or more adenines of the first probe oligonucleotide are replaced by one or more diaminopurines.
46. The method of claim 7 , further comprising the steps of:
providing a second probe oligonucleotide;
allowing the second probe oligonucleotide to hybridize to the reference oligonucleotide; and
allowing the preactivated chemical ligation compound to mediate ligation between the hybridized second probe oligonucleotide and the hybridized first probe oligonucleotide.
47. The method of claim 46 , wherein the second probe oligonucleotide has a preactivated chemical ligation compound attached thereto.
48. The method of claim 8 , wherein the first probe oligonucleotide further comprises one or more S 2 T molecules.
49. The method of claim 8 , wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S 2 T molecules.
50. The method of claim 8 , wherein the first probe oligonucleotide further comprises one or more diaminopurines.
51. The method of claim 8 , wherein one or more adenines of the first probe oligonucleotide are replaced by one or more diaminopurines.
52. The method of claim 9 , wherein the first probe oligonucleotide further comprises one or more S 2 T molecules.
53. The method of claim 9 , wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S 2 T molecules.
54. The method of claim 9 , wherein the first probe oligonucleotide further comprises one or more diaminopurines.
55. The method of claim 9 , wherein one or more adenines of the first probe oligonucleotide are replaced by one or more diaminopurines.