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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Cell-free cloning using φ29 DNA polymerase
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Cell-free cloning using φ29 DNA polymerase

机译：使用φ29DNA聚合酶进行无细胞克隆

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摘要

We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5-7 kb in size by > 10~9-Fold, using φ29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using φ29 DNA polymerase is "background" DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using φ29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the φ29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using φ29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopoly-mer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning.
机译：我们使用φ29DNA聚合酶描述了大小为5-7 kb的单个DNA分子滚环扩增（RCA）的条件，其大小> 10〜9倍。使用φ29DNA聚合酶通过RCA扩增少量模板的主要困难是“背景” DNA合成，通常在省略模板或低模板浓度时发生。在保持模板量固定的同时减少反应体积会增加模板浓度，从而导致背景合成受到抑制。通过使用很小的体积（通常为600 nl）进行扩增反应，可以使用φ29DNA聚合酶实现单个环状分子的无细胞克隆。该程序允许无细胞克隆无法在大肠杆菌中克隆的单个合成DNA分子，例如携带致命突变的合成噬菌体基因组。它还允许从细菌分离的基因组DNA的无细胞克隆。可以直接从φ29DNA聚合酶反应中测序该DNA，而无需进一步扩增。与PCR扩增相反，使用φ29DNA聚合酶的RCA不会产生突变累积奖金，并且该酶的高加工性消除了均聚物链上的卡顿现象。无细胞克隆对天然和合成DNA都有许多潜在的应用。其中包括已证明难以克隆的环境DNA样品和编码有毒产物的合成基因。该方法还可以通过消除生物克隆的需要来加快基因组测序。

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