Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries.
Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries.
复制标题
从芯片合成的寡核苷酸文库中可扩展地扩增链子集。
DOI：
10.1038/ncomms9634
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发表时间：
2015-11-16
影响因子：
16.6
通讯作者：
Shih WM
中科院分区：
文献类型：
--
作者：
Schmidt TL;Beliveau BJ;Uca YO;Theilmann M;Da Cruz F;Wu CT;Shih WM
合成寡核苷酸是DNA纳米技术、遗传学和合成生物学研究的主要成本因素，这些研究都需要数千种高质量的寡核苷酸。廉价的芯片合成的寡核苷酸文库可以包含数十万个不同的序列，然而每条链的数量仅为亚飞摩尔。在这里，我们提出了一种选择性的寡核苷酸扩增方法，基于三轮滚环扩增，每毫升反应产生纳摩尔量的单链寡核苷酸。在多步一锅法中，可以选择性地扩增和纯化数百或数千个具有不同长度的单链DNA的子集。这些寡核苷酸用于折叠几种DNA纳米结构，并作为主要的荧光原位杂交探针。扩增成本低于其他报道的方法（通常约20美元/纳摩尔总寡核苷酸生产），并且主要是使用商业酶。 合成寡核苷酸是DNA纳米技术研究的主要成本因素。在这里，作者提出了一种选择性寡核苷酸扩增方法，基于三轮滚环扩增，每毫升反应产生纳摩尔量的单链寡核苷酸。
Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes. Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology. Here, the authors present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction.