Published online 7 July 2004
Nucleic Acids Research, Vol. 32 No. 12 © Oxford University Press 2004; all rights reserved
A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences
Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, The People's Republic of China, 1 Department of Plant Sciences, University of Tennessee, Knoxville TN 37996, USA and 2 Department of Plant Science, University of Connecticut, Storrs CT 06269, USA
* To whom correspondence should be addressed. Tel: +86 21 62208660 3207; Fax: +86 21 62205704; Email: yaoquanhong{at}yahoo.com
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received March 11, 2004; Revised May 12, 2004; Accepted June 9, 2004
Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are 
500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5–7 days) and suitable for synthesizing long segments of DNA (5–6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.
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