Rouillard Jean-Marie, Lee Woonghee, Truan Gilles, Gao Xiaolian, Zhou Xiaochuan, Gulari Erdogan
Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA.
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W176-80. doi: 10.1093/nar/gkh401.
There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis. Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR. Ideally, such oligonucleotides should represent both strands of the final DNA molecule. They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly. This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set. Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences. We have developed Gene2Oligo (http://berry.engin.umich.edu/gene2oligo/), a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands. The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis. We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.
人们对实施一种生物信息学工具有着浓厚兴趣，该工具能够设计寡核苷酸以支持体外基因合成的发展。目前制备长合成DNA分子的方案依赖于通过连接酶链式反应（LCR）或组装PCR对一组短寡核苷酸进行体外组装。理想情况下，此类寡核苷酸应代表最终DNA分子的两条链。它们应在同一条链上相邻，并与第二条链的互补寡核苷酸重叠，以确保组装过程中的良好杂交。这意味着每组寡核苷酸的热力学性质必须一致。此外，任何给定的寡核苷酸必须对其靶标具有完全特异性，以避免产生错误组装的序列。我们开发了Gene2Oligo（http://berry.engin.umich.edu/gene2oligo/），这是一种基于网络的工具，可将长输入DNA序列划分为一组代表两条DNA链的相邻寡核苷酸。寡核苷酸的长度会动态优化，以确保体外基因合成所需的特异性和统一解链温度。我们已成功设计并使用一组寡核苷酸，通过LCR和组装PCR合成了酿酒酵母细胞色素b5。
