Droplet generation techniques allow researchers to perform large-scale studies using a minimum sample volume. This method can make DNA sequencing more efficient and cost-effective. Furthermore, it can help identify small variations within single cells relevant to cancer diagnosis. This article talks about droplet generation applied to DNA analysis. It also shows the basic setup you need to start your experiments with droplet microfluidics.
DNA analysis opportunities and challenges
Scientists analyse DNA to identify mutations in cancer, characterize human antibodies, and distinguish microorganisms, among other applications. DNA analysis includes several techniques. It may involve using arrays of probes of known sequence or sequencing a novel genome using Next Generation platforms, .
Next Generation platforms have the advantage of using chip-based arrays, which enables analysis of hundreds of millions of molecules in parallel. This capacity is essential to lower costs and increase efficiency, .
But with arrays, the number of sequences that can be analyzed is limited. Also, sequence analysis requires systems for handling fluids and mixing the reagents introduced onto the chip. Consequently, it adds more steps to the process, limiting total throughput. Furthermore, you must use the entire array to achieve a low cost per base. This often requires running samples together and parallel processing. While barcoding could minimize this problem, it adds time-consuming steps to the workflow, .
Droplet-based microfluidics uses non-miscible phases (usually aqueous and oil-based) to create discrete volumes. It enables researchers to generate and manipulate droplets using microfluidic pumps producing tiny droplets at high speed, . The main advantages of this method are:
Low sample volume
High automation degree
Microfluidic devices can potentially increase DNA analysis throughput since they handle fluid volumes quickly and precisely. For example, droplet-based microfluidic techniques generate and sort droplets at a rate of thousands per second, .
Each drop is a compartment in which a reaction occurs. Possible applications are molecule counting with digital PCR, protein crystallization screening, and gene expression characterization. Another advantage of this method is that the number of reactions scales with the droplet rate and decreases with droplet volume, allowing adjustments according to the analysis, .
Overall, this technique allows researchers to perform hundreds of millions of reactions using few reagent volumes, making it exceptionally efficient and cost-effective. However, as opposed to spots on a chip-based array, drops move through microchannels. This hinders using their location as a marker for the reactions it holds. Because of that, the technique requires alternative labeling methods, .
Single-cell genome sequencing at high throughput
Traditional sequencing is often unable to point out minor variations in the genome. So, scientists use single-cell whole-genome sequencing to identify copy number variations and single-nucleotide variations within single cells, .
Researchers can use the microfluidics approach to obtain high throughput and scalable single-cell sequencing. For example, one study barcoded amplified genomic DNA from individual cancer cells confined to droplets. This method allowed identifying cells with pathogenic mutations during complete remission and revealed complex clonal evolution within acute myeloid leukemia (AML) tumours that were not observable with traditional sequencing, .
Therefore, the technique may improve cancer genome heterogeneity analysis and therapy selection, .
What do you need to get started?
Droplet generation offers a cost-effective and high-throughput analysis. If you´d like to start your experiments with droplet microfluidics, this is what you´ll need:
2-3 Microfluidic pumps (or 2-3 channels on one pump such as the 4U pump) – control the flow of the continuous (oil) phase and dispersed (water) phase. Depending on the application, we recommend the 4U pressure pump or 2-3x ExiGo microfluidic syringe pumps. The 4U pressure pump has a stable and accurate flow rate and enables independent control of 4 different channels, controlling both pressure and flow.
2-3 x Flow sensor - provides feedback on the flow control of both oil and water phases.
Microfluidic Chip - creates droplets to ensure optimal droplet size.
Stable Channel Surface Chemistry - ensures droplet stability.
Surfactant - stabilizes the interface between the oil and water phase, giving stability to the droplets.
Oil - improves droplet stability.
Tubing - connects your pumps to the microfluidic chip.
1. Abate, Adam R et al. “DNA sequence analysis with droplet-based microfluidics.” Lab on a chip vol. 13,24 (2013): 4864-9. doi:10.1039/c3lc50905b
2. Pellegrino, Maurizio et al. “High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics.” Genome research vol. 28,9 (2018): 1345-1352. doi:10.1101/gr.232272.117
3. Sohrabi, S., & Moraveji, M. K. (2020). Droplet microfluidics: Fundamentals and its advanced applications. RSC Advances, 10(46), 27560-27574.