Hydrodynamic DNA Shearer
Shearer: a Hydrodynamic DNA Shearing Device
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
shearer has been published.
The citation is
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
at the publishers webpage (though you
may need a subscription to the journal).