The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.
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.
The amplification and digital quantification of single DNAmolecules are important in biomedicine and diagnostics. Beyond quantifying DNAmolecules in asample,the ability to express proteins from the amplified DNAw ould open even broader applications in synthetic biology,d irected evolution, and proteomics.H erein, am icrofluidic approach is reported for the production of condensed DNAn anoparticles that can serve as efficient templates for in vitro protein synthesis.Using phi29 DNAp olymerase and am ultiple displacement amplification reaction, single DNAm olecules were converted into DNAn anoparticles containing up to about 10 4 clonal gene copies of the starting template.D NA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNAs ynthesis) and magnesium ions from the buffer.T ranscription-translation reactions performed in vitro showed that individual DNAn anoparticles can serve as efficient templates for protein synthesis in vitro. Compartmentalization and amplification of single DNA molecules inside nano-or picoliter-sized wells [1] and drop-lets [2] has opened up new opportunities for biomedical and biological sciences.T he discrete nature of compartments enables digital quantification of absolute numbers of nucleic acids in as ample, [3] accurate estimation of copy-number variation, [4] detection of pathogens [5] and rare cancer mutations , [6] as well as other applications. [7] Them ost common method of amplifying DNAi n as ample involves the polymerase chain reaction (PCR). However,f or droplet microfluidics experiments,t he large temperature gradient required for PCR is am ajor drawback that can cause droplet coalescence and loss of compartmen-talization. In addition, amplification of long (> 1kb) templates is often inefficient, leading to decreased reaction yields. In contrast, DNAa mplification under isothermal reaction conditions has been shown to generate large amounts of material from as ingle-copy DNAt emplate, [8] circumventing potential problems associated with emulsion stability.M ore-over, the ability to amplify DNAa nd then express proteins from the clonally amplified template would greatly increase the scope of potential applications.F or example,s ynthetic biology,d irected evolution, and large-scale proteomics screens would benefit from techniques that do not rely on protein expression in living systems. Amajor challenge for in vitro expression of proteins is the relatively large amounts of DNAt emplate needed-on the order of 500 ng DNA(% 10 9 gene copies) per 50 mL reaction [9]
Synthesis of DNA and magnesium pyrophosphate particles and their use for protein expression in vitro
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