Stanford
Genome Technology Center
Technology
Development Group
| Instruments
Hydrodynamic DNA Shearer |
Point-sink
Shearer: a Hydrodynamic DNA Shearing Device
Random DNA fragments are required to construct efficient libraries for genomic sequencing & gene cloning strategies. We have built and tested an automated device that generates random DNA fragments by hydrodynamic shearing. The method improves on other fragmentation techniques currently being used. It is inexpensive, easy to use, reproducible, and versatile. The new device uses a syringe pump to create hydrodynamic shear forces by pushing a DNA sample through a small abrupt contraction. We have used commercially available components for simplicity and have reduced the optimal volume for shearing down to 100-250µL, and processing time to less than 15 minutes. Shearing of the samples is completely automated by computer control. Loading and unloading are currently manual steps and washing is semi-automated, but plans are under way to automate these steps as well. The sheared DNA fragments fall within a tight size distribution that is extremely repeatable. Only three parameters are critical: the exact flow geometry, the flow speed, and a minimum number of passes through the contraction. Shearing is reproducible over a wide range of temperatures, DNA concentrations, and initial DNA size. The cloning efficiency of the sheared DNA is very good even without end repair. There is not a significant sequence bias at the ends of sheared fragments that have been cloned. Size selection is unnecessary because 90% of the fragments fall within a two-fold size range. A manuscript detailing the
development and application of the Point-sink
Thorstenson, YR, SP Hunicke-Smith, PJ Oefner, RW Davis. 1998. "An Automated Hydrodynamic Process for Controlled, Unbiased DNA Shearing" Genome Research, Vol 8, No 8, Pg 848-855. The article itself may be
accessed
at the publishers webpage (though you
may need a subscription to the journal).
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