Isothermal strand-displacement amplification applications for high-throughput genomics
Isothermal strand-displacement amplification applications for high-throughput genomics
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DOI：
10.1006/geno.2002.7020
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发表时间：
2002-12-01
期刊：
影响因子：
4.4
通讯作者：
Richardson, PM
中科院分区：
文献类型：
--
作者：
Detter, JC;Jett, JM;Richardson, PM
源DNA的扩增几乎是分子生物学应用的普遍要求。目前研究人员可用的主要方法仅限于在大肠杆菌宿主体内进行扩增和聚合酶链式反应。滚环DNA复制是一种著名的合成噬菌体基因组的方法，近年来被用于特定靶序列的滚环扩增(RCA)以及用于克隆的环状载体。在这里，我们证明了使用带有Phi29 DNA聚合酶的随机六聚体引物的RCA可以用于包含各种插入大小的不同载体的链置换扩增，以产生一致的用于末端测序反应的模板。我们证明这一程序在高通量的质粒生产测序过程中特别有效。此外，我们证明了从细胞或少量纯化的基因组DNA中可以有效地扩增出完整的细菌基因组，而没有明显的偏向，用于下游应用，包括全基因组鸟枪法测序。
Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with Phi29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.