Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument.
Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument.
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发表时间：
2006-04
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通讯作者：
M. Zianni;K. Tessanne;M. Merighi;R. Laguna;F. Tabita
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作者：
M. Zianni;K. Tessanne;M. Merighi;R. Laguna;F. Tabita
我们已经将DNA足迹分析技术应用于一台应用生物系统3730DNA分析仪。荧光标记引物的使用消除了对放射性标记核苷酸和平板凝胶电泳的需要，并利用了常见的自动荧光毛细管电泳仪。用荧光标记的引物和双脱氧核苷酸DNA测序，我们已经证明，每个消化片段的末端碱基可以用基于毛细管的仪器准确地鉴定。用6FAM标记的引物进行聚合酶链式反应(PCR)扩增典型的靶启动子区域。然后将该PCR产物与转录激活蛋白或牛血清白蛋白孵育，然后用DNase I部分消化。克隆的启动子用热序列酶染料人工循环测序试剂盒(USB)和FAM标记的引物进行测序。通过使用Genemapper软件，Thermo Sequenase和DNasei酶切产物被准确比对，为DNA足迹中的每个峰分配正确的核苷酸提供了一种现成的方法。该方法被用来表征两种不同的转录激活蛋白与其各自的启动子区域的结合。
We have adapted the techniques of DNA footprint analysis to an Applied Biosystems 3730 DNA Analyzer. The use of fluorescently labeled primers eliminates the need for radioactively labeled nucleotides, as well as slab gel electrophoresis, and takes advantage of commonly available automated fluorescent capillary electrophoresis instruments. With fluorescently labeled primers and dideoxynucleotide DNA sequencing, we have shown that the terminal base of each digested fragment may be accurately identified with a capillary-based instrument. Polymerase chain reaction (PCR) was performed with a 6FAM-labeled primer to amplify a typical target promoter region. This PCR product was then incubated with a transcriptional activator protein, or bovine serum albumin as a control, and then partially digested with DNase I. A clone of the promoter was sequenced with the Thermo Sequenase Dye Primer Manual Cycle Sequencing kit (USB) and the FAM-labeled primer. Through the use of Genemapper software, the Thermo sequenase and DNasei digestion products were accurately aligned, providing a ready means to assign correct nucleotides to each peak from the DNA footprint. This method was used to characterize the binding of two different transcriptional activator proteins to their respective promoter regions.