Kim Samuel C, Premasekharan Gayatri, Clark Iain C, Gemeda Hawi B, Paris Pamela L, Abate Adam R
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco (UCSF), California Institute for Quantitative Biosciences (QB3) San Francisco, California 94158, USA.
Department of Urology, Division of Hematology & Oncology, University of California, San Francisco (UCSF), San Francisco, California 94158, USA.
Microsyst Nanoeng. 2017;3. doi: 10.1038/micronano.2017.18. Epub 2017 Jun 19.
Uniform amplification of low input DNA is important for applications across biology, including single-cell genomics, forensic science, and microbial and viral sequencing. However, the requisite biochemical amplification methods are prone to bias, skewing sequence proportions and obscuring signals relating to copy number. Digital droplet multiple displacement amplification enables uniform amplification, but requires expert knowledge of microfluidics to generate monodisperse emulsions. In addition, existing microfluidic methods are tedious and labor intensive for preparing many samples. Here, we introduce rapid emulsification multiple displacement amplification, a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter. While conventional microfluidic devices require >10 minutes to emulsify a sample, our system takes tens of seconds and yields data of equivalent quality. We demonstrate the approach by using it to accurately measure copy number variation in single cancer cells.
低输入量DNA的均匀扩增对于生物学的诸多应用都很重要，包括单细胞基因组学、法医学以及微生物和病毒测序。然而，所需的生化扩增方法容易产生偏差，导致序列比例扭曲，并掩盖与拷贝数相关的信号。数字液滴多重置换扩增能够实现均匀扩增，但需要微流体方面的专业知识才能生成单分散乳液。此外，现有的微流体方法在制备多个样本时既繁琐又费力。在此，我们介绍快速乳化多重置换扩增，这是一种使用手持注射器和分级液滴分离器生成单分散液滴的方法。传统的微流体设备乳化一个样本需要超过10分钟，而我们的系统只需几十秒，并且能产生质量相当的数据。我们通过使用该方法精确测量单个癌细胞中的拷贝数变异来证明这一方法。
