快速体外产生单链 DNA。
Rapid in vitro production of single-stranded DNA.
机构信息
John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115, USA.
出版信息
Nucleic Acids Res. 2019 Dec 16;47(22):11956-11962. doi: 10.1093/nar/gkz998.
There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 μl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.
在合成生物学、生物成像和生物纳米技术领域，人们对长度超过 200 个核苷酸 (nt) 的单链 DNA (ssDNA) 的需求日益增加。现有的生产高纯度长 ssDNA 的方法在可扩展性、协议步骤的复杂性和/或产量方面存在局限性。我们提出了一种快速、高产、用户友好的体外生产高纯度 ssDNA 的方法，其长度可达至少七个千碱基。带有甲醇响应聚合物的正向引物的聚合酶链反应 (PCR) 生成一个标记的扩增子，使得在变性条件下可以选择性沉淀修饰链。我们证明，在 PCR 后约 40-50 分钟内，ssDNA 的回收率>70%，相对于输入的 PCR 扩增子，或每 100 μl PCR 反应高达 70 pmol。我们证明，回收的 ssDNA 可用于人类细胞中的 CRISPR/Cas9 同源定向修复、DNA 折纸和荧光原位杂交。
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