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一种利用Taq聚合酶在体内对酵母基因进行光足迹分析的改进方法。

An improved method for photofootprinting yeast genes in vivo using Taq polymerase.

作者信息

Axelrod J D, Majors J

机构信息

Department of Biological Chemistry, Washington University School of Medicine, St Louis, MO 63110.

出版信息

Nucleic Acids Res. 1989 Jan 11;17(1):171-83. doi: 10.1093/nar/17.1.171.

DOI:10.1093/nar/17.1.171
PMID:2643080
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC331543/
Abstract

We have developed an improved method for photofootprinting in vivo which utilizes the thermostable DNA polymerase from T. aquaticus (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 polymerase, 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.

摘要

我们开发了一种改进的体内光足迹法，该方法在引物延伸分析中利用了来自嗜热水生栖热菌（Taq）的耐热DNA聚合酶。紫外光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聚合酶进行多轮退火和延伸，在核苷酸水平上检测并定位光产物，Taq聚合酶会被模板DNA中的光产物阻断。该方法比本实验室先前描述的化学光足迹法（《自然》325. 173 - 177）更快、更灵敏且更具可重复性，并且检测光产物的特异性与先前描述的方法相似，但不完全相同。使用引物延伸光足迹法证明了GAL4蛋白与其在GAL1 - 10上游激活序列内的结合位点的结合。引物延伸分析还可用于定位由其他足迹法产生的DNA链断裂，并直接从酵母基因组中确定DNA序列。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/f97033aa90a5/nar00210-0191-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/a4325dc2dee3/nar00210-0185-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/9c4dffeff323/nar00210-0187-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/eb5be2b91e13/nar00210-0188-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/ce7c0059a2b1/nar00210-0189-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/3d37b2717b5c/nar00210-0190-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/f97033aa90a5/nar00210-0191-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/a4325dc2dee3/nar00210-0185-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/9c4dffeff323/nar00210-0187-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/eb5be2b91e13/nar00210-0188-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/ce7c0059a2b1/nar00210-0189-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/3d37b2717b5c/nar00210-0190-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/79cc/331543/f97033aa90a5/nar00210-0191-a.jpg

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