TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing.
TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing.
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DOI：
10.2144/jun0208
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发表时间：
2002-06
期刊：
影响因子：
2.7
通讯作者：
J. Nelson;Yuyang Cai;T. Giesler;J. Farchaus;Shanmuuga Sundaram;M. Ortiz-Rivera;Louis P. Hosta;Peter L. Hewitt;J. Mamone;Chockalingam Palaniappan;C. Fuller
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文献类型：
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作者：
J. Nelson;Yuyang Cai;T. Giesler;J. Farchaus;Shanmuuga Sundaram;M. Ortiz-Rivera;Louis P. Hosta;Peter L. Hewitt;J. Mamone;Chockalingam Palaniappan;C. Fuller
我们开发了一种新颖的等温DNA扩增策略，该策略使用phi29 DNA聚合酶和滚环扩增来生成用于DNA测序反应的高质量模板。Templiphone DNA扩增试剂盒利用了这样一个事实，即克隆的DNA通常是在环状载体中获得的，这些环状载体很容易使用phi29 DNA聚合酶通过滚环机制在体外复制。这种单亚基校对DNA聚合酶具有优异的加工性和链置换特性，可产生多个串联的环状DNA双链拷贝，产生多达10(7)倍的扩增。在短短4小时内即可获得大量产品(1-3微克)。输入的DNA可以是0.01 ng纯化的质粒DNA，一个细菌菌落，或者1微L饱和的过夜培养。此外，phi29 DNA聚合酶内相关的校对功能的存在确保了高保真扩增。一旦完成，产品DNA就可以直接用于测序反应。此外，还讨论了phi29 DNA聚合酶的性质及其在人类基因组DNA扩增中的应用。
We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.