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 ∼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.
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Acknowledgements
This research was supported by the Shanghai Project for International Scientific and Technological Cooperation, the Fund of the National Natural Science Foundation of China, the Shanghai Rising-Star Program, the Shanghai Natural Science Foundation, the Shanghai Key Basic Research Project, the Key Laboratory of Shanghai Project, the University of Connecticut Agricultural Experiment Station (Storrs) and the University of Tennessee Agricultural Experiment Station. We thank X. Yang and L. Osburn for critical review of the manuscript.
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Xiong, AS., Yao, QH., Peng, RH. et al. PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1, 791–797 (2006). https://doi.org/10.1038/nprot.2006.103
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DOI: https://doi.org/10.1038/nprot.2006.103
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