How it Works: Accurate Shearing of Chromosomal DNA
All contemporary, as well as NextGen and Third Generation sequencing methodologies are dependent on the generation of DNA fragments from initial MegaBase long chromosomal DNA molecules. General requirements are that such fragments have to be random and of similar size.
Problem: All contemporary, as well as NextGen and Third Generation sequencing methodologies are dependent on the generation of DNA fragments from initial MegaBase long chromosomal DNA molecules. General requirements are that such fragments have to be random and of similar size. Many methods of DNA fragmentation have been developed. Most of these methods include limitations and difficulties including high costs, fragmentation, broad fragment size distribution, or irreversible damage in DNA fragments. In addition, the results can become concentration dependent and/or not highly reproducible.
Figure 1. The average size of DNA fragments obtained with the HydroShear Plus system depends on the linear speed as the sample passes through the orifice, which is easily controlled by the speed code parameter.
Solution: Digilab, Inc. has developed the HydroShear, an automated, inexpensive, highly reproducible, controllable, and easy-to-use device for the generation of random and tightlydistributed DNA fragments (maximal length being less than twice the size of the smallest ones for 90 percent of the resulting fragment mixture) based solely on hydrodynamic breakage of the double-stranded DNA. This process neither generates heat nor chemically alters DNA molecules, resulting in production of DNA fragments with minimal damage (previously published data suggested that about 60 percent of the resulting DNA fragments can be ligated and cloned even without additional end repair). It has been shown that singlestranded polynucleotides (both DNA and RNA) can also be sheared by the system and were found to be sheared more readily than the double-stranded ones. Successful use of the system has been already reported in more than 400 scientific publications.
The average size of the double-stranded DNA fragments (Figure 1) depends on several parameters that the enduser can easily control (by the type of shearing assembly that the customer chooses; the precise average fragment size can be fine tuned by an adjustable speed code parameter that determines the flow rate at which the DNA molecules are passed through the shearing assembly). The system is very reproducible and, if used with the same parameters, creates DNA fragments with the same size distribution every time regardless of DNA origin, initial size, concentration, or sample volume (within 50-500 μL range). A specifically designed washing procedure, recommended to be performed between two consecutive shearing events, guarantees that all traces of DNA potentially remaining within the system are destroyed and completely flushed out so the next sample is not cross-contaminated (each wash reduces the DNA concentration within the system approximately tenfold).
Figure 2. HydroShear Plus DNA/RNA Shearing System
Digilab’s latest model, the HydroShear Plus (Figure 2), completely automates both shearing and washing procedures, and is supplied with a complimentary Wash Solutions Kit designed to optimize HydroShear Plus performance, which is certified to be nuclease- and particle-free. The HydroShear Plus is equipped with a fortified syringe that pumps the DNA sample through the orifice of a shearing assembly unit. The standard shearing assembly unit can be used to generate DNA fragments with an average size from 1.4 to 8 kB. If larger DNA fragments are to be generated, a large shearing assembly unit is recommended. Using a small shearing assembly unit is recommended for the generation of fragments with average size from 0.85 to 3 kB. Shearing of DNA to the current smallest average fragment size (700 bp) can be achieved by using a special shearing assembly unit.
For more information, visit www.digilabglobal.com.