Compositions and methods for the assembly of polynucleotides

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 RA0, any primer binding segment selected from the group PA, any DNA segment selected from the group D0, an annealable linker sequence LB0, and a second restriction site RB0;
• (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 RAn, a first annealable linker sequence LAn, any DNA segment selected from the group Dn, a second annealable linker sequence LBn, and a second restriction site RBn, 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 RAm, an annealable linker sequence LAm, any DNA segment selected from the group Dm, any primer binding segment selected from the group PB, a second restriction site RBm wherein m represents an integer one greater than the number of intermediate nucleic acid molecules;
• whereupon cleavage of restriction sites RA0 through RBm 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 LAp, 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 D0, . . . , Dn, . . . and Dm 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 LAp, 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 D0, . . . Dn, . . . and Dm.