Scott Adrian C, Ludlow Catherine L, Cromie Gareth A, Dudley Aimée M
Pacific Northwest Diabetes Research Institute.
Pacific Northwest Diabetes Research Institute;
J Vis Exp. 2014 May 1(87):51401. doi: 10.3791/51401.
Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.
四分子分析是酵母遗传学中的一种重要工具，但该过程繁琐的手工操作特性阻碍了其大规模应用。四分子条形码测序（BEST）[1]取代了分离、破坏和间隔四分子的手工操作。BEST通过一种孢子形成特异性GFP融合蛋白来分离四分子，该蛋白允许通过荧光激活细胞分选将四分子直接分选到琼脂平板上，在平板上，子囊被酶解，孢子被破坏并通过玻璃珠铺板随机排列。然后，利用基因分型过程中读取的分子条形码，为单倍体菌落确定姐妹孢子关系，即关于哪些孢子来自同一四分子的信息。通过消除手工解剖的瓶颈，几分钟内就可以分离出数百甚至数千个四分子。在这里，我们详细描述了在酿酒酵母中进行BEST所需的实验步骤，从杂合二倍体菌株开始，直至分离出单倍体减数分裂后代衍生的菌落。
