Abstract
Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL) volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme β-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications.
Highlights
Droplet microfluidics offers a powerful tool to isolate, amplify and quantify nucleic acid molecules in a massively parallel manner [1,2]
Single-DNA molecule isolation, amplification and digital quantification have gained broad interest for the evaluation of rare diseases [30,31,32], quantifying the absolute number of nucleic acids in a sample [33,34] or disease diagnostics [35,36]. In these and others examples, the sample containing diluted suspension of nucleic acids is encapsulated in microfluidic droplets and amplified using the polymerase chain reaction (PCR) reaction followed by fluorescence readout
The template size in most of the reports is in the range of ~200–300 nucleotides, which prevents their use for other important applications, such as directed evolution or drug screening
Summary
Droplet microfluidics offers a powerful tool to isolate, amplify and quantify nucleic acid molecules in a massively parallel manner [1,2]. For biological assays in particular, the droplet format provides significant savings for the cost of the reagents, and increased analytical sensitivity and ultra-high throughput capabilities [3,4]. These features have led to a growing number of applications of droplet microfluidics technology for DNA amplification and detection [5,6,7], library preparation [8,9], in vivo directed evolution [10,11,12], single-cell assays [13,14,15] and others [16,17,18]. It was shown that during the multiple displacement amplification (MDA) reaction, the magnesium ions from the buffer and DNA replication reaction byproduct inorganic
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