Hughes Randall A, Miklos Aleksandr E, Ellington Andrew D
Applied Research Laboratories, The University of Texas at Austin, Austin, Texas, USA.
Methods Enzymol. 2011;498:277-309. doi: 10.1016/B978-0-12-385120-8.00012-7.
DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.
DNA合成技术正迅速成为现代分子生物学的基石，并在合成生物学领域发挥着关键作用。合成完整基因、新型遗传途径乃至整个基因组的能力已不再是30年前的梦想。只需使用一台热循环仪、商业合成的寡核苷酸和DNA聚合酶，一个标准的分子生物学实验室就能利用现有技术在一周内合成几千碱基对的合成DNA。在此，我们回顾了合成DNA所使用的技术，从寡核苷酸的化学合成到将它们组装成长的定制序列。探讨了有助于实施这些方法的软件和网站，并讨论了DNA合成技术在基因表达和合成生物学中的应用。最后，提供了一个我们自己实验室的自动化基因合成实例。
