Miklos Aleksandr E, Hughes Randall A, Ellington Andrew D
The University of Texas at Austin, Applied Research Laboratories, Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology, Austin, Texas, USA.
Curr Protoc Mol Biol. 2012 Jul;Chapter 3:Unit3.23. doi: 10.1002/0471142727.mb0323s99.
The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing "in-house" gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides.
定制合成基因长度的DNA产品的出现消除了研究工作中的诸多瓶颈，使基因合成成为一种越来越常见的商业服务。然而，将合成寡核苷酸组装成大型定制DNA构建体并非特别困难，进行“内部”基因合成具有时间和成本优势。本单元将探讨基因合成中设计和物理组装两方面的问题，包括如何设计用于合成的DNA序列以及重叠寡核苷酸方案的设计，以确保能轻松组装成最终产物。组装通过一系列可靠的PCR反应完成，并包含一个故障排除组装方案，该方案不仅能组装困难序列，还能将故障源确定到一对寡核苷酸。
