Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction
Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction
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DOI：
10.1006/meth.1997.0529
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发表时间：
1997-11-01
期刊：
影响因子：
--
通讯作者：
Falaschi, A
中科院分区：
文献类型：
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作者：
Giacca, M;Pelizon, C;Falaschi, A
基于确定在特定基因组区域中新生DNA链的相对丰度，开发了一种方法来绘制哺乳动物细胞染色体中DNA复制的起源。该方法需要纯化来自最近活化起源的新生DNA短链，并通过竞争性聚合酶链反应技术在该样品中定量不同相邻DNA片段的相对丰度舞蹈。预计在DNA复制开始的位置，起源区域内定义标记的丰度是最大的。这种起源图谱绘制程序(i)允许对单拷贝基因组区域进行分析，（ii）可以在没有任何化学处理的情况下对培养细胞和原代细胞进行分析，（iii）不需要细胞同步，（iv）允许在几百个碱基对内绘制起源图谱。这种高分辨率允许对原点函数所需的顺式和反式元素进行研究。将该方法应用于哺乳动物细胞的单拷贝序列，已经确定了与人类层蛋白B2基因结构域的500-bp片段相似的复制起点，以及与仓鼠二氢叶酸还原酶基因位点的800 -bp片段相似的复制起点。(C) 1997学术出版社。
A procedure was developed for mapping origins of DNA replication in mammalian cell chromosomes based on determining the relative abundance of nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from recently activated origins and the quantification, within this sample, of the relative abundances dances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin map ping procedure (i) allows analysis of single-copy genomic regions, (ii) can be performed on cultured and primary cells in the absence of any chemical treatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of the cis- and trans-acting elements required for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an similar to 500-bp segment in the human lamin B2 gene domain and within an similar to 8OO-bp segment in the hamster dihydrofotate reductase gene locus. (C) 1997 Academic Press.