| 主权项 |
1. A method for nucleic acid assembly, the method comprising:
a) providing a predetermined nucleic acid sequence; b) providing a plurality of precursor double-stranded nucleic acid fragments, each precursor double-stranded nucleic acid fragment having two strands, wherein each of the two strands comprises a sticky end sequence of 5′-A (Nx) T-3′ (SEQ ID NO.: 1) or 5′-G (Nx) C-3′ (SEQ ID NO.: 16), wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two precursor double-stranded nucleic acid fragments comprise the same sticky end sequence; c) providing primers comprising a nicking endonuclease recognition site and a sequence comprising (i) 5′-A (Nx) U-3′ (SEQ ID NO.: 80) corresponding to each of the different sticky end sequences of 5′-A (Nx) T-3′(SEQ ID NO.: 1) or (ii) 5′-G (Nx) U-3′ (SEQ ID NO.: 81) corresponding to each of the different sticky end sequences of 5′-G (N′) C-3′ (SEQ ID NO.: 16); d) performing a polynucleotide extension reaction to form double-stranded nucleic acid fragments; e) subjecting the polynucleotide extension reaction product to nicking and cleavage reactions to form double-stranded nucleic acid fragments with 3′ overhangs; and f) annealing the double-stranded nucleic acid fragments to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site. |
