DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis
DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis
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DOI：
10.1093/nar/30.10.e43
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发表时间：
2002-05-15
影响因子：
14.9
通讯作者：
Lubkowski, J
中科院分区：
文献类型：
--
作者：
Hoover, DM;Lubkowski, J
整个基因组序列的可用性大大增加了蛋白质靶点的数量，其中许多蛋白质靶点需要在原始DNA来源以外的细胞中过表达。基因合成通常提供快速且经济有效的方法。合成基因可以被优化用于表达，并且被构建用于容易的突变操作，而不考虑亲本基因组。然而，设计和构建合成基因，特别是那些编码大蛋白质的基因，可能是一个缓慢、困难和令人困惑的过程。我们已经编写了一个计算机程序，可以自动设计用于基因合成的寡核苷酸。我们的程序需要简单的输入信息，即目标蛋白的氨基酸序列和合成寡核苷酸的解链温度（基因组装所需）。该程序输出一系列寡核苷酸序列，其密码子针对在所选生物体中的表达进行了优化。这些寡核苷酸的特征是高度均匀的解链温度和最小化的发夹形成趋势。在此程序和两步PCR方法的帮助下，我们已经成功地构建了大量的合成基因，范围从139到1042 bp。这里介绍的方法简化了从各种生物体中生产蛋白质的基因组学为基础的研究。
The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Yet design and construction of synthetic genes, especially those coding for large proteins, can be a slow, difficult and confusing process. We have written a computer program that automates the design of oligonucleotides for gene synthesis. Our program requires simple input information, i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program outputs a series of oligonucleotide sequences with codons optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogeneous melting temperatures and a minimized tendency for hairpin formation. With the help of this program and a two-step PCR method, we have successfully constructed numerous synthetic genes, ranging from 139 to 1042 bp. The approach presented here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.