An improved method for photofootprinting yeast genes in vivo using Taq polymerase.
An improved method for photofootprinting yeast genes in vivo using Taq polymerase.
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一种使用 Taq 聚合酶进行体内光足迹酵母基因的改进方法。
DOI：
10.1093/nar/17.1.171
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发表时间：
1989
影响因子：
14.9
通讯作者：
Majors,J
中科院分区：
文献类型：
--
作者：
Axelrod,JD;Majors,J
我们已经开发了一种改进的方法，光足迹在体内利用热稳定的DNA聚合酶（Taq）的引物延伸试验。紫外线用于将光产物引入完整酵母细胞的基因组DNA中。然后，通过使用Taq聚合的多轮退火和延伸，在核苷酸水平上检测和绘制光产物，该聚合被模板DNA中的光产物阻断。该方法比先前描述的化学光足迹法（Nature 325）更快速、敏感、可重复性好。173 - 177年)。并且检测光产物的特异性与前面描述的程序相似，但不相同。利用引物延伸光足迹法证明了GAL4蛋白与GAL1-10上游激活序列中的结合位点的结合。引物延伸试验也可用于绘制由其他足迹方法产生的DNA链断裂，并直接从酵母基因组确定DNA序列。
We have developed an improved method for photofootprintingin vivowhich utilizes the thermostable DNA polymerase fromT aquancus(Taq) in a primer extension assay. UV light is used to introduce photoproducts into the genomic DNA of intact yeast cells. The photoproducts are then detected and mapped at the nucleotide level by multiple rounds of annealing and extension using Taq polymerise, which is blocked by photoproducts in the template DNA. The method is more rapid, sensitive, and reproducible than the previously described chemical photofootprinting procedure developed In this laboratory(Nature 325. 173–177.). and detects photoproducts with a specificity which is similar, but not identical to that of the previously described procedure. Binding of GAL4 protein to its binding sites within the GAL1–10 upstream activating sequence is demonstrated using the primer extension photofootprinting method. The primer extension assay can also be used to map DNA strand breakage generated by other footprinting methods, and to determine DNA sequence directly from the yeast genome.