Effect of jet nebulization on DNA: identifying the dominant degradation mechanism and mitigation methods

DNA Acts as a Nucleation Site for Transient Cavitation in the Ultrasonic Nebulizer

“…This shear rate is well above rates that have been reported to degrade plasmid DNA. 39,52,53 Although these shear rates are substantial in magnitude, they operate over a large area compared to the diameter of the plasmids under investigation (e.g., wavelengths of approximately 600 mm; particle diameters of approximately 300 nm), and therefore are not likely to contribute substantially to the observed degradation of plasmid DNA. 54 In contrast, the length of the linear strands of genomic DNA may be sufficient to be affected by these disturbances.…”

“…LNQ01 was transformed in E. coli DH5a and isolated using Qiagen Plasmid Giga kits (Qiagen, Inc., Valencia, CA) as previously described. 39 The cosmid DNA had an A 260 /A 280 ratio of 1.86 AE 0.08. All forms and sizes of DNA were dissolved to a final concentration of 0.1 mg/mL in buffer (10 mM TrisHCl, 200 mM DTPA, pH 8.5).…”

“…These experimental conditions resulted in droplet sizes of 6.2 AE 3.1 mm as measured with a Phase Doppler Particle Analyzer (Aerometrics, Inc., Sunnyvale, CA) as described previously. 39 …”

The arrival of genetic engineering

“…In order to assess the level of damage experienced by pDNA due to the aerosolisation process, the integrity of the supercoiled structure in the aerosols after aerosolisation has been studied and a summary of findings of a number of studies is presented in Table 6. From the table, jet nebulisation resulted in damage to a 5 kb and 9.8 kb plasmid, the loss of the sc content being attributed to hydrodynamic shear and shock waves [137]. Damage to a small plasmid of size 4.8 kb in the aerosols of a jet nebuliser was reported by Kleemann et al [138] due to the shearing effects of aerosolisation.…”

LCAT DNA Shearing

“…Nebulization, which aerosolizes the DNA solution, poses the problem of postfragmentation collection and also the difficulty of automation. 6 Liu et al 9 developed a micromixer based on cavitation microstreaming that requires only a piezoelectric transducer, which can be reused repeatedly. Cavitation microstreaming occurs when an air bubble undergoes oscillations in a fluid, creating velocity and pressure fluctuations in the surrounding fluid (Fig.…”