Azevedo Flávio, Pereira Humberto, Johansson Björn
Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
Methods Mol Biol. 2017;1620:129-139. doi: 10.1007/978-1-4939-7060-5_8.
Escherichia coli and Saccharomyces cerevisiae are currently the two most important organisms in synthetic biology. E.coli is almost always used for fundamental DNA manipulation while yeast is the simplest host system for studying eukaryotic gene expression and performing large scale DNA assembly. Yeast expression studies may also require altering of the chromosomal DNA by homologous recombination. All these studies require the verification of the expected DNA sequence and the fastest method of screening is colony PCR, which is direct PCR of DNA in cells without prior DNA purification. Colony PCR is hampered by the difficulty of releasing DNA into the PCR mix and the presence of PCR inhibitors. We hereby present one protocol for E. coli and two protocols for S. cerevisiae differing in efficiency and complexity as well as an overview of past and possible future developments of efficient S. cerevisiae colony PCR protocols.
大肠杆菌和酿酒酵母是目前合成生物学中两个最重要的生物体。大肠杆菌几乎总是用于基础DNA操作，而酵母是研究真核基因表达和进行大规模DNA组装的最简单宿主系统。酵母表达研究可能还需要通过同源重组改变染色体DNA。所有这些研究都需要验证预期的DNA序列，而最快的筛选方法是菌落PCR，即无需事先纯化DNA就直接对细胞中的DNA进行PCR。菌落PCR受到DNA释放到PCR混合物中的困难以及PCR抑制剂的存在的阻碍。我们在此提出一种针对大肠杆菌的方案和两种针对酿酒酵母的方案，它们在效率和复杂性上有所不同，同时还概述了高效酿酒酵母菌落PCR方案的过去和可能的未来发展。
