Data from: PCR duplication: a one-step cloning-free method to generate duplicated chromosomal loci and interference-free expression reporters in yeast

Here, we report on a novel PCR targeting-based strategy called ‘PCR duplication’ that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.

本研究报道了一种基于聚合酶链式反应（Polymerase Chain Reaction，PCR）靶向的新型策略，命名为‘PCR复制（PCR duplication）’，该策略可通过简便的PCR操作实现酵母基因组中特定基因组区域的靶向复制。为验证该策略的应用效果，研究团队首先对酵母FAR1基因的启动子区域进行复制，并同时在其下游插入绿色荧光蛋白（Green Fluorescent Protein，GFP）。该操作既构建了可检测启动子活性的报告系统，又完整保留了FAR1基因的固有结构与功能。在另一项实验中，研究团队利用PCR复制策略以离散方式将单个基因的拷贝数从1倍提升至2倍。以编码酵母γ-微管蛋白的TUB4基因为例，实验证实该操作可相应提高其信使RNA（mRNA）与蛋白质的表达水平。PCR复制是一种简便的单步操作流程，可通过多种方式适配应用，无需体外（ex vivo）克隆即可实现对各类基因组调控元件的快速、无干扰研究。