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Nucleic Acids Research Advance Access published online on March 5, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp118
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis

Hongye Ye, Mo Chao Huang, Mo-Huang Li* and Jackie Y. Ying

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669

*To whom correspondence should be addressed. Tel: +65 6824 7119; Fax: +65 6478 9085; Email: mhli{at}ibn.a-star.edu.sg

Received January 8, 2009. Accepted February 11, 2009.

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10°C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.


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M. Bode, S. Khor, H. Ye, M.-H. Li, and J. Y. Ying
TmPrime: fast, flexible oligonucleotide design software for gene synthesis
Nucleic Acids Res., July 1, 2009; 37(suppl_2): W214 - W221.
[Abstract] [Full Text] [PDF]


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