Breaksite batch mapping, a rapid method for assay and identification of DNA breaksites in mammalian cells
Breaksite batch mapping, a rapid method for assay and identification of DNA breaksites in mammalian cells
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DOI：
10.1093/nar/29.6.e33
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发表时间：
2001-03-15
影响因子：
14.9
通讯作者：
Maizels, Nancy
中科院分区：
文献类型：
--
作者：
Kong, Qingzhong;Maizels, Nancy
DNA断裂发生在哺乳动物细胞的许多过程中，包括重组、修复、诱变和凋亡。本文报道了一种简单、快速的DNA断裂分析和断裂位点鉴定方法。断裂位点首先被标记并通过连接介导的PCR（LM-PCR）扩增，使用巢式PCR引物以增加扩增的特异性和灵敏度。然后通过批量测序LM-PCR产物定位断裂位点。这允许容易地鉴定每个反应的多个断裂位点，而不需要通过凝胶电泳或克隆对PCR产物进行繁琐的分级分离。断裂位点批次作图需要很少的起始材料，可用于鉴定单链或双链断裂。
DNA breaks occur during many processes in mammalian cells, including recombination, repair, mutagenesis and apoptosis. Here we report a simple and rapid method for assaying DNA breaks and identifying DNA breaksites. Breaksites are first tagged and amplified by ligation-mediated PCR (LM-PCR), using nested PCR primers to increase the specificity and sensitivity of amplification. Breaksites are then mapped by batch sequencing LM-PCR products. This allows easy identification of multiple breaksites per reaction without tedious fractionation of PCR products by gel electrophoresis or cloning. Breaksite batch mapping requires little starting material and can be used to identify either single- or double-strand breaks.