Compositions and methods for the assembly of polynucleotides
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.
1. A composition comprising:
(a) one or more first nucleic acid molecules, wherein each first nucleic acid molecule is circular and comprises, in a 5′ to 3′ orientation, a first restriction site RA 0 , any DNA segment selected from the group D 0 , an annealable linker sequence LB 0 , and a second restriction site RB 0 ;
(b) one or more intermediate nucleic acid molecules, wherein each intermediate nucleic acid molecule n is circular and comprises, in a 5′ to 3′ orientation, a first restriction site RA n , a first annealable linker sequence LA n , any DNA segment selected from the group D n , a second annealable linker sequence LB n , and a second restriction site RB n , and wherein n represents an integer from one to the number of intermediate nucleic acid molecules; and
(c) one or more last nucleic acid molecules, wherein each last nucleic acid molecule is circular and comprises, in a 5′ to 3′ orientation, a first restriction site RA m , an annealable linker sequence LA m , any DNA segment selected from the group D m , a second restriction site RB m , wherein m represents an integer one greater than the number of intermediate nucleic acid molecules;
whereupon cleavage of restriction sites RA 0 through RB m and denaturation of the resulting linear nucleic acid molecules, each annealable linker sequence LB (p-1) is capable of hybridizing to the complement of annealable linker sequence LA 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 0 , . . . D n , . . . D m independently consists of one or more DNA segments.
2. The composition of claim 1 , wherein each of said one or more first nucleic acid molecules further comprises any primer binding segment selected from the group PA positioned 5′ to the DNA segment selected from the group D 0 , wherein each of said one or more last nucleic acid molecules further comprises any primer binding segment selected from the group PB positioned 3′ to the DNA segment selected from the group D m .
3. The composition of claim 1 , wherein upon cleavage of restriction sites RA 0 through RB m and denaturation of the resulting linear nucleic acid molecules, each annealable linker sequence LB (p-1) is capable of selectively hybridizing to the complement of annealable linker sequence LA p compared to the other annealable linker sequences, or their complements, in the composition.
4. The composition of claim 1 , wherein the restrictions sites RA 0 through RB m , are cleavable by SapI or LguI restriction endonuclease.
5. The composition of claim 1 , wherein two or more annealable linker sequences are at least 24 nucleotides in length and have a melting temperature of at least 60° C.
6. A composition comprising a plurality of linear nucleic acid molecules formed by digesting the composition of claim 1 with one or more restriction endonucleases capable of cleaving the restriction sites RA 0 through RB m .
7. A method of generating an assembled polynucleotide from a plurality of component polynucleotides comprising the steps of:
(a) digesting an assembly composition with one or more restriction endonucleases to generate a components composition, the assembly composition comprising:
(i) one or more first nucleic acid molecules, wherein each first nucleic acid molecule is circular and comprises, in a 5′ to 3′ orientation, a first restriction site RA 0 , any primer binding segment selected from the group PA, any DNA segment selected from the group D 0 , an annealable linker sequence LB 0 , and a second restriction site RB 0 ;
(ii) one or more intermediate nucleic acid molecules, wherein each intermediate nucleic acid molecule n is circular and comprises, in a 5′ to 3′ orientation, a first restriction site RA n , a first annealable linker sequence LA n , any DNA segment selected from the group D n , a second annealable linker sequence LB n , and a second restriction site RB n , and wherein n represents an integer from one to the number of intermediate nucleic acid molecules; and
(iii) one or more last nucleic acid molecules, wherein each last nucleic acid molecule is circular and comprises, in a 5′ to 3′ orientation, a first restriction site RA m , an annealable linker sequence LA m , any DNA segment selected from the group Dm, any primer binding segment selected from the group PB, a second restriction site RB m wherein m represents an integer one greater than the number of intermediate nucleic acid molecules;
whereupon cleavage of restriction sites RA 0 through RB m and denaturation of the resulting linear nucleic acid molecules, each annealable linker sequence LB (p-1) is capable of hybridizing to the complement of annealable linker sequence LA 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 0 , . . . D n , . . . and D m consists of one or more DNA segments; and
(b) contacting the components composition with DNA polymerase, deoxyribonucleoside triphosphates and one or more first primers and one or more second primers, under conditions suitable for denaturation of the nucleic acid molecules, annealing of annealable linker sequence LB (p-1) to annealable linker sequence LA p , and extension therefrom; wherein each said first primer is capable of hybridizing to one of said primer binding segments selected from the group PA and each said second primer is capable of hybridizing to one of said primer binding segments selected from the group PB; and subjecting the components composition to polymerase chain reaction,
wherein a polynucleotide is assembled which comprises, in a 5′ to 3′ orientation, one DNA segment selected from each of the groups D 0 , . . . D n , . . . and D m .
8. The method of claim 7 , wherein two or more annealable linker sequences are at least 24 nucleotides in length and have a melting temperature of at least 60° C.
9. The method of claim 7 , wherein each annealable linker sequence LB (p-1) is identical in sequence to annealable linker sequence LA p .
10. The method of claim 7 , wherein the restriction sites RA 0 through RB m are cleavable by the same restriction endonuclease.
11. The method of claim 7 , wherein the restrictions sites RA 0 through RB m are cleavable by SapI or LguI restriction endonuclease, and the restriction endonuclease of step (a) is SapI or LguI.
12. A method of generating a host cell comprising a polynucleotide, the method comprising the steps of:
(a) transforming a host cell with a polynucleotide assembled according to claim 7 ; and
(b) selecting a host cell comprising the assembled polynucleotide.
13. The method of claim 12 , wherein the assembled polynucleotide comprises a selectable marker and step (b) comprises propagating the transformed host cell on selectable media.
14. The method of claim 12 , further comprising transforming the host cell with a linearized plasmid comprising:
(i) a first region of homology with primer binding segment PA; and
(ii) a second region of homology with primer binding segment PB,
wherein said first and second regions of homology are of sufficient length to initiate host cell mediated homologous recombination between said polynucleotide and said plasmid to form a circularized plasmid in the host cell.
15. A method of generating a host cell comprising a polynucleotide, the method comprising the steps of:
(a) transforming a host cell with the composition of claim 6 , wherein each annealable linker sequence LB (p-1) comprises a region of homology with annealable linker sequence LA p of sufficient length to initiate host cell mediated homologous recombination between LB (p-1) and LA p wherein p represents an integer from 1 to m, wherein said homologous recombination results in the assembly of a polynucleotide; and
(b) selecting a host cell comprising an assembled polynucleotide, wherein the assembled polynucleotide comprises in a 5′ to 3′ orientation, one DNA segment selected from each of the groups D 0 , . . . D n , . . . and D m .
16. The method of claim 15 , wherein
(a) each of the one or more first linear nucleic acid molecules further comprises a first region of homology with a first integration site of the host cell genome; and
(b) each of the one or more last linear nucleic acid molecules further comprises a second region of homology with a second integration site of the host cell genome,
wherein said first and second regions of homology are of sufficient length to initiate host cell mediated homologous recombination with said first and second integration sites, respectively, wherein said homologous recombination results in integration of an assembled polynucleotide into the host cell genome.
17. The method of claim 15 , wherein at least one homologous recombination of an annealable linker sequence LB (p-1) and LA p forms a nucleic acid sequence that encodes a selectable marker gene.
18. The method of claim 15 , further comprising transforming the host cell with a linearized plasmid comprising:
(i) a first region of homology with the one or more first linear nucleic acid molecules; and
(ii) a second region of homology with the one or more last linear nucleic acid molecules, wherein said first and second regions of homology are of sufficient length to initiate host cell mediated homologous recombination between an assembled polynucleotide and said plasmid to form a circularized plasmid in the host cell.