In situ localized amplification and contact replication of many individual DNA molecules
In situ localized amplification and contact replication of many individual DNA molecules
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DOI：
10.1093/nar/27.24.e34
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发表时间：
1999-12-01
影响因子：
14.9
通讯作者：
Church, George M.
中科院分区：
文献类型：
--
作者：
Mitra, Robi D.;Church, George M.
我们描述了一种通过在倒在玻璃显微镜载玻片上的聚丙烯酰胺薄膜上进行聚合酶链式反应 (PCR) 来克隆和扩增 DNA 的方法。聚丙烯酰胺基质阻碍线性 DNA 分子的扩散，从而使扩增产物保持位于其各自模板附近。反应结束时，形成了许多 PCR 菌落或“聚合酶菌落”，每个菌落均由单个模板分子生长而来。在一张载玻片上可以并行扩增多达 500 万个克隆。如果引物之一的 5' 末端包含 Acrydite 修饰，则扩增的 DNA 将共价连接至聚丙烯酰胺基质，从而可以同时对所有克隆进行进一步的酶促操作。我们描述了制作这些聚合酶切片的复制品的技术，以及该技术的高通量测序方案。还讨论了其他应用。
We describe a method to clone and amplify DNA by performing the polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the linear DNA molecules so that the amplification products remain localized near their respective templates. At the end of the reaction, a number of PCR colonies, or 'polonies', have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. If an Acrydite modification is included at the 5' end of one of the primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simultaneously. We describe techniques to make replicas of these polony slides, and high throughput sequencing protocols for this technology. Other applications are also discussed.