PCR-based accurate synthesis of long DNA sequences.
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.
Xiong
Ai-Sheng
AS
Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, The People's Republic of China.
Yao
Quan-Hong
QH
Peng
Ri-He
RH
Duan
Hui
H
Li
Xian
X
Fan
Hui-Qin
HQ
Cheng
Zong-Ming
ZM
Li
Yi
Y
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*
D004247:DNA