High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System

Abstract

DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1–10 Kbp fragment lengths with a yield of 75.30–91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.

Rotation-Induced Macromolecular Spooling of DNA

“…Since the single-file stem conformation results from shear drag on the shofar end, rotation-induced macromolecular spooling of very long strands of fragile DNA may face limitations due to shear-induced fragmentation [31][32][33], overstretching [34][35][36] or tether failing. Within the stem, the tension is largest and the rate of tether breakage events may be increased or overstretching may occur at high rotation rates.…”

Polydimethylsiloxane microstructure-induced acoustic streaming for enhanced ultrasonic DNA fragmentation on a microfluidic chip

“…The DNA shearing results are unrelated to the concentration of the initial sample. 57 Before each experiment, the DNA solution was diluted to a concentration of 2.0 μg mL −1 with Tris-EDTA buffer solution (Sigma-Aldrich/ MFCD00236359, Germany).…”

“…[24][25][26][27] Even though hydrodynamic shearing has a good performance in generating long DNA molecular fragments (several thousand bp), it cannot be improved to produce shorter and uniform ones for next-generation sequencing (NGS requires 300-500 bp) due to limited energy. [28][29][30][31] Wu et al realized DNA fragmentation in microfluidic chips with funnel geometry shapes by tensile stress, indicating that the extension rate at the microscale was the main factor of DNA fragmentation. 55 Shui et al realized salmon sperm DNA fragmentation to ∼5 kbp lengths by hydrodynamic shearing in microfluidic chips.…”

Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters

“…Enzymatic digestion, such as using site-directed restriction enzyme Sau3AI to partial digest purified DNA (Clos and Zander-Dinse, 2019), sonication, such as using ultrasound to generate > 120 kb fragments (Bhushan et al, 2011), and hydrodynamic shearing, such as repeatedly passing DNA through a syringe needle (Liu C. et al, 2016), have been widely used for constructions of large-fragment libraries. Compared with enzymatic shearing, sonication and hydrodynamic shearing, which are mechanical fragmentation methods, are more random and enable better control of the size distribution (Li et al, 2017). After fragmentation, DNA samples can be analyzed by fragment analyzer or horizontal agarose gel electrophoresis to test the extent of the yield fragments.…”