Claims for Patent: 8,546,136

Summary for Patent: 8,546,136

Title:	Compositions and methods for the assembly of polynucleotides
Abstract:	The present invention provides compositions and methods for rapid assembly of one or more assembled polynucleotides from a plurality of component polynucleotides. The methods of the invention utilize circular nucleic acid vectors that comprise a DNA segment D flanked by an annealable linker sequence, annealable linker sequence pairs LA and LB, or annealable linker sequence/primer binding segment pairs LA and PB or PA and LB. Restriction endonuclease digestion of a plurality of vectors containing the DNA segments to be assembled generates a plurality of DNA fragments comprising the elements PA-D-LB, LA-D-LB, and LA-D-PB or D-LB, LA-D-LB, and LA-D. The sequences of annealable linker sequences LA and LB provide complementary termini to the DNA fragments, which are utilized in host cell mediated homologous recombination or together with promer binding segments PA and PB in a polymerase cycling assembly reaction for the ordered assembly of the various DNA segments into one or more assembled polynucleotides.
Inventor(s):	Serber; Zach (Emeryville, CA), Lowe; Raymond (Emeryville, CA), Ubersax; Jeffrey A. (Emeryville, CA), Chandran; Sunil S. (Emeryville, CA)
Assignee:	Amyris, Inc. (Emeryville, CA)
Application Number:	13/430,322
Patent Litigation and PTAB cases:	See patent lawsuits and PTAB cases for patent 8,546,136
Patent Claims:	1. A library of nucleic acid molecules comprising at least one of each of the following nucleic acid molecules: (a) a first nucleic acid molecule wherein the first nucleic acid molecule is circular and comprises, in a 5' to 3' orientation, a first restriction site RA.sub.0, any DNA segment selected from the group D.sub.0, an annealable linker sequence LB.sub.0, and a second restriction site RB.sub.0; (b) an intermediate nucleic acid molecule wherein the intermediate nucleic acid molecule n is circular and comprises, in a 5' to 3' orientation, a first restriction site RA.sub.n, a first annealable linker sequence LA.sub.n, any DNA segment selected from the group D.sub.n, a second annealable linker sequence LB.sub.n, and a second restriction site RB.sub.n, and wherein n represents an integer from one to the number of intermediate nucleic acid molecules; and (c) a last nucleic acid molecule wherein the last nucleic acid molecule is circular and comprises, in a 5' to 3' orientation, a first restriction site RA.sub.m, an annealable linker sequence LA.sub.m, any DNA segment selected from the group D.sub.m, a second restriction site RB.sub.m, wherein m represents an integer one greater than the number of intermediate nucleic acid molecules; whereupon cleavage of restriction sites RA.sub.0 through RB.sub.m, and denaturation of the resulting linear nucleic acid molecules, each annealable linker sequence LB.sub.(p-1) is capable of hybridizing to the complement of annealable linker sequence LA.sub.p, wherein n is an integer that varies from 1 to (m-1), wherein p represents an integer from 1 to m, and wherein each group D.sub.0, . . . D.sub.n, . . . and D.sub.m independently consists of one or more DNA segments. 2. The library of claim 1, wherein at least one first nucleic acid molecule further comprises a primer binding segment PA positioned 5' to the DNA segment selected from the group D.sub.0. 3. The library of claim 1, wherein at least one last nucleic acid molecule further comprises a primer binding segment PB positioned 3' to the DNA segment selected from the group D.sub.m. 4. The library of claim 1, wherein each of restriction sites RA.sub.0 through RB.sub.m is cleavable by the same restriction endonuclease. 5. The library of claim 1, wherein each of restriction sites RA.sub.0 through RB.sub.m is cleavable by a Type IIS restriction endonuclease. 6. The library of claim 1, wherein the restriction sites RA.sub.0 through RB.sub.m are cleavable by the same Type IIS restriction endonuclease. 7. The library of claim 1, wherein the restrictions sites RA.sub.0 through RB.sub.m are cleavable by SapI or LguI restriction endonuclease. 8. The library of claim 1, wherein upon denaturation of the linear nucleic acid molecules, each annealable linker sequence LB.sub.(p-1) is capable of selectively hybridizing to the complement of annealable linker sequence LA.sub.p compared to the other annealable linker sequences, or their complements, in the composition. 9. The library of claim 1, wherein each annealable linker LB.sub.(p-1) is identical in sequence to annealable linker sequence LA.sub.p, or the complements thereof. 10. The library of claim 1, wherein each of two or more annealable linker sequences is independently at least 24 nucleotides in length and has a melting temperature of at least 60.degree. C. 11. The library of claim 1, wherein each of two or more annealable linker sequences independently has a G-C content of at least 70% and a melting temperature of at least 70.degree. C. 12. The library of claim 1, wherein each of two or more annealable linker sequences independently has an A-T content of at least 30% and a melting temperature of at least 65.degree. C., and comprises a sequence motif 5'ANNNNNNNNANNNAANTANNTTNANA-3', [SEQ ID NO:221] wherein A stands for adenine, N for any nucleotide, and T for thymine. 13. The library of claim 1, wherein two or more of the annealable linker sequences are independently selected from the group consisting of SEQ ID NOS: 1 to 8, and complements thereof. 14. The library of claim 1, wherein each of the annealable linker sequences is independently selected from the group consisting of SEQ ID NOS: 1 to 8, and complements thereof. 15. The library of claim 1, wherein each of the primer binding segments is independently selected from the group consisting of SEQ ID NOS: 9 and 10, and complements thereof. 16. The library of claim 1, wherein any DNA segment comprises a sequence selected from the group consisting of a protein-coding sequence, a reporter gene, a fluorescent marker, a promoter, an enhancer, a terminator, an intron, an exon, a poly-A tail, a multiple cloning site, a nuclear localization signal, a nuclear export signal, a mRNA stabilization signal, a selectable marker, an integration loci, an epitope tag, and a degradation signal. 17. The library of claim 16, wherein the DNA segment comprises a promoter selected from the group consisting of a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasone-inducible MMTV promoter, a SV40 promoter, a MRP pol III promoter, a constitutive MPSV promoter, an RSV promoter, a tetracycline-inducible CMV promoter, a constitutive CMV promoter, PGAL3, PGAL7, PCTR3, PMET3, PPGK1, PTDH1, PTDH3, PFBA1, PTEF1, PENO1, PENO2, PCYC1, PTDH2, PCUP1, PGAL80, PGAL2, PBNA6, PTMA29, PSBP1, PPUP3, PACS2, PTPO1, PRPT1, PAAT2, PAHP1, PSSE1, PTEF2, PNPL3, PPET9, PTUB2, POLE1, PCPR1, PIPPP1, and PSOD1. 18. The library of claim 17, wherein the DNA segment comprises a terminator selected from the group consisting of TADH1, TENO1, TENO2, TCYC1, TNDT80, TTDH3, TTDH1, and TPGK1. 19. The library of claim 1, wherein the library comprises more than one first nucleic acid molecule, and each first nucleic acid molecule comprises the same annealable linker sequence LB.sub.0. 20. The library of claim 1, wherein the library comprises more than one first nucleic acid molecule, and each first nucleic acid molecule comprises the same DNA segment selected from the group D.sub.0. 21. The library of claim 1, wherein the library comprises more than one last nucleic acid molecule, and each last nucleic acid molecule comprises the same annealable linker sequence LA.sub.m. 22. The library of claim 1, wherein the library comprises more than one last nucleic acid molecule, and each last nucleic acid molecule comprises the same DNA segment selected from the group D.sub.m. 23. The library of claim 1, wherein the library comprises more than one intermediate nucleic acid molecule, and each intermediate nucleic acid molecule comprises the same annealable linker sequence LA.sub.n. 24. The library of claim 1, wherein the library comprises more than one intermediate nucleic acid molecule, and each intermediate nucleic acid molecule comprises the same annealable linker sequence LB.sub.n. 25. The library of claim 1, wherein the library comprises more than one intermediate nucleic acid molecule, and each intermediate nucleic acid molecule comprises the same annealable linker sequence LA.sub.n and annealable linker sequence LB.sub.n. 26. The library of claim 1, wherein the library comprises more than one intermediate nucleic acid molecule, and each intermediate nucleic acid molecule comprises the same DNA segment selected from the group D.sub.n. 27. The library of claim 2, wherein the library comprises more than one first nucleic acid molecule, and each first nucleic acid molecule comprises the same primer binding segment PA. 28. The library of claim 3, wherein the library comprises more than one last nucleic acid molecule, and each last nucleic acid molecule comprises the same primer binding segment PB. 29. The library of claim 1, comprising: (a) two first nucleic acid molecules, wherein one first nucleic acid molecule comprises, in a 5' to 3' orientation, a first restriction site RA.sub.0, a primer binding segment PA, a DNA segment D.sub.01, an annealable linker sequence LB.sub.0, and a second restriction site RB.sub.0, wherein another first nucleic acid molecule comprises, in a 5' to 3' orientation, a first restriction site RA.sub.0, a primer binding segment PA, a DNA segment D.sub.02, an annealable linker sequence LB.sub.0, and a second restriction site RB.sub.0, wherein DNA segment D.sub.01 encodes a first genomic targeting sequence, wherein DNA segment D.sub.02 encodes a second genomic targeting sequence located downstream of the first genomic targeting sequence in a target genome, and wherein DNA segment D.sub.02 is positioned in opposite orientation as DNA segment D.sub.01 relative to primer binding segment PA and annealable linker sequence LB.sub.0; (b) at least one intermediate nucleic acid molecule comprising, in a 5' to 3' orientation, a first restriction site RA.sub.n, a first annealable linker sequence LA.sub.n, a DNA segment D.sub.n, a second annealable linker sequence LB.sub.n, and a second restriction site RB.sub.n, wherein n represents an integer from one to the number of intermediate nucleic acid molecules; and (c) two last nucleic acid molecules, wherein one last nucleic acid molecule comprises, in a 5' to 3' orientation, a first restriction site RA.sub.m, an annealable linker sequence LA.sub.m, a DNA segment D.sub.m1, a primer binding segment PB, and a second restriction site RB.sub.m, wherein another last nucleic acid molecule comprises, in a 5' to 3' orientation, a first restriction site RA.sub.m, an annealable linker sequence LA.sub.m, a DNA segment D.sub.m2, a primer binding segment PB, and a second restriction site RB.sub.m, wherein m represents an integer one greater than the number of intermediate nucleic acid molecules, wherein DNA segment D.sub.m1 encodes a first segment of a selectable marker, wherein DNA segment D.sub.m2 encodes a second segment of the selectable marker, wherein DNA segment D.sub.m2 is positioned in opposite orientation as DNA segment D.sub.m1 relative to annealable linker sequence LA.sub.m and primer binding segment PB, wherein neither DNA segment D.sub.m1 nor DNA segment D.sub.m2 produces a functional selectable marker but whereupon homologous recombination of DNA segments D.sub.m1 and D.sub.m2 a functional selectable marker is generated; wherein each annealable linker sequence LB.sub.(p-1) is identical to annealable linker sequence LA.sub.p, wherein p represents the integers from 1 to m. 30. The library of claim 1, wherein each first nucleic acid molecule, each intermediate nucleic acid molecule, and each last nucleic acid molecule is contained in a separate container.

Details for Patent 8,546,136

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		2028-11-19
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2028-11-19
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2028-11-19