Ligation-mediated PCR for quantitative in vivo footprinting
Ligation-mediated PCR for quantitative in vivo footprinting
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DOI：
10.1038/80323
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发表时间：
2000-10-01
影响因子：
46.9
通讯作者：
Flanagan, SD
中科院分区：
文献类型：
--
作者：
Dai, SM;Chen, HH;Flanagan, SD
连接介导的聚合酶链反应（LM-PCR）是一种基因组分析技术，用于测定（1）主要DNA核苷酸序列（2）胞嘧啶甲基化模式（3）DNA损伤形成和修复，以及（4）体内蛋白质-DNA足迹1，2，3，4。然而，LM-PCR可能受到所需的多个步骤和每个反应可以分析的相对短的序列段（通常< 200 bp）的限制。我们在这里报告了一个简化的，为期一天的LM-PCR协议，其中所有移液步骤可以由机器人工作站进行，而且，通过使用非放射性检测和LI-COR DNA测序仪，提供了更长的读取（> 350 bp）和增强的信号质量。使用5′-末端用红外荧光染料标记的寡核苷酸引物实现了与放射性标记相当的灵敏度。我们表明，该技术可用于敏感和可重复的人磷酸甘油酸激酶1（PGK 1）启动子的体内光足迹，以及提供良好的Maxam-Gilbert序列信息。这里描述的方法应该允许高通量，高分辨率的转录因子结合和染色质结构的分析，也可能是有用的测序的差距是难克隆。
Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein–DNA footprints 1, 2, 3, 4. However, LM-PCR can be limited by the multiple steps required and the relatively short stretch of sequence (usually< 200 bp) that can be analyzed per reaction. We report here a simplified, one-day LM-PCR protocol in which all pipetting steps can be performed by a robotic workstation and which, moreover, provides longer reads (> 350 bp) and enhanced signal quality by use of nonradioactive detection and a LI-COR DNA sequencing instrument. Sensitivity comparable to radiolabeling is achieved using oligonucleotide primers that are 5′-end labeled with infrared fluorochromes. We showed that the technique could be used for sensitive and reproducible in vivo photofootprinting of the human phosphoglycerate kinase 1 (PGK1) promoter, as well as providing good Maxam–Gilbert sequence information. The methods described here should allow high-throughput, high-resolution analysis of transcription factor binding and chromatin structure, and also may be useful for sequencing gaps that are refractory to cloning.