High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System

Lanhui Li,Albert Van Den Berg,Jan C T Eijkel,Guofu Zhou,Shuting Xie,Chenglong Sun,Mingliang Jin,Xiaoxue Wang,Lingling Shui

doi:10.1038/srep40745

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.

Highlights

Results demonstrate that the length of DNAs after ligation are about twice of their corresponding DNAs before ligation, suggesting that the relatively smaller fragmented DNA molecules are ligated into larger ones. These results show that the ends of the fragmented DNAs are not damaged by hydrodynamic shearing and they are usable for step process
When N2 is introduced into the DNA solution via a small tube, bubbles are generated from the tube tip, and rise up to the solution surface where they burst to release N2 to air and DNA solution back to bulk solution
Lentz et al have investigated the mechanism of DNA fragmentation by jet nebulization, and found that hydrodynamic shear is responsible for DNA degradation[13]

Summary

Introduction

Shear stresses are generated during the bubbling process, such as resulting from gas-liquid friction, drainage during bubble film change, and hydrodynamic forces during bubble bursting. When gas is introduced into the DNA solution via a tube, bubbles form at the orifice, rising up, merging and bursting at the air-liquid surface[27,28]. By controlling gas pressure, bubbling time and temperature, we have obtained size-controllable DNA fragments ranging from 10 to 1 Kbp with a narrow size distribution.

Results

Conclusion