title: PCR-based accurate synthesis of long DNA sequences.
abstract: Here we describe a simple and rapid method for assembly and PCR-based accurate synthesis (PAS) of long DNA sequences. The PAS protocol involves the following five steps: (i) design of the DNA sequence to be synthesized and of 60-bp overlapping oligonucleotides to cover the entire DNA sequence; (ii) purification of the oligonucleotides by PAGE; (iii) first PCR, to synthesize DNA fragments of 400-500 bp in length using 10 inner (template) and two outer (primer) oligonucleotides; (iv) second PCR, to assemble the products of the first PCR into the full-length DNA sequence; and (v) cloning and verification of the synthetic DNA by sequencing and, if needed, error correction using an overlap-extension PCR technique. This method, which takes approximately 1 wk, is suitable for synthesizing diverse types of long DNA molecule. We have successfully synthesized DNA fragments from 0.5 to 12.0 kb, with high G+C content, repetitive sequences or complex secondary structures. The PAS protocol therefore provides a simple, rapid, reliable and relatively inexpensive method for synthesizing long, accurate DNA sequences.
authors.affiliation: Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, The People's Republic of China.
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Hui
authors.initials: H
authors.lastname: Duan
authors.forename: Xian
authors.initials: X
authors.lastname: Li
authors.forename: Hui-Qin
authors.initials: HQ
authors.lastname: Fan
authors.forename: Zong-Ming
authors.initials: ZM
authors.lastname: Cheng
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics*; D005813:Genes, Synthetic / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004247:DNA