Claims for Patent: 8,481,258

Summary for Patent: 8,481,258

Title:	Methods and compounds for chemical ligation
Abstract:	Compositions and methods for chemical ligation are provided. Methods for nucleic acid sequencing, nucleic acid assembly and nucleic acid synthesis are also provided.
Inventor(s):	Church; George M. (Brookline, MA), Sismour; A. Michael (Cambridge, MA)
Assignee:	President and Fellows of Harvard College (Cambridge, MA)
Application Number:	12/628,518
Patent Claims:	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.sup.2T molecules. 4. The method of claim 1, wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S.sup.2T 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.sup.2T molecules. 24. The method of claim 16, wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S.sup.2T 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.sup.2T molecules. 36. The method of claim 2, wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S.sup.2T 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.sup.2T molecules. 43. The method of claim 7, wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S.sup.2T 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.sup.2T molecules. 49. The method of claim 8, wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S.sup.2T 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.sup.2T molecules. 53. The method of claim 9, wherein one or more thymines of the first probe oligonucleotide are replaced by one or more S.sup.2T 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.

Details for Patent 8,481,258

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2028-06-02
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2028-06-02
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2028-06-02