Horecka J, Jigami Y
Department of Molecular Biology, NIBH, AIST, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Yeast. 2000 Jul;16(10):967-70. doi: 10.1002/1097-0061(200007)16:10<967::AID-YEA597>3.0.CO;2-G.
Tagged transposons are powerful tools for large-scale studies of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. The current techniques used to identify transposon insertion sites in the yeast genome require a DNA amplification step that can be time-consuming and problematic. We show that the DNA amplification step can be bypassed. Insertion sites can be identified rapidly and reliably by direct genomic sequencing using a transposon-specific primer, BigDye-labelled terminators, and an automated sequencer. Direct genomic sequencing can also save time on the genetic analysis phase of transposon-based projects.
带标签的转座子是用于大规模研究酿酒酵母基因表达、蛋白质定位和基因破坏的强大工具。目前用于鉴定酵母基因组中转座子插入位点的技术需要一个DNA扩增步骤,这可能既耗时又有问题。我们表明,可以绕过DNA扩增步骤。使用转座子特异性引物、BigDye标记的终止剂和自动测序仪,通过直接基因组测序可以快速可靠地鉴定插入位点。直接基因组测序还可以在基于转座子的项目的遗传分析阶段节省时间。