An automated hydrodynamic process for controlled, unbiased DNA shearing.
An automated, inexpensive, easy-to-use, and reproducible technique for controlled, random DNA fragmentation has been developed. The technique is based on point-sink hydrodynamics that result when a DNA sample is forced through a small hole by a syringe pump. Commercially available components are used to reduce the cost and complexity of the instrument. The design is optimized to reduce the volume of sample required and to speed processing time. Shearing of the samples can be completely automated by computer control. Ninety percent of sheared DNA fragments fall within a twofold size distribution that is highly reproducible. Three parameters are critical: the flow geometry, the flow rate, and a minimum number of iterations. 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, the distribution of assembled sequences is random, and there is no sequence bias at the ends of sheared fragments that have been cloned. The instrument, called the Point-sink Shearer (PtS), has already been exported successfully to many other laboratories.
Stanford DNA Sequencing and Technology Center, Palo Alto, California 94304, USA. yvonne@sequence.stanford.edu
Y R
YR
Thorstenson
S P
SP
Hunicke-Smith
P J
PJ
Oefner
R W
RW
Davis
D001331:Automation; D001482:Base Composition; D003001:Cloning, Molecular / Q000379:methods*; D003360:Cosmids; D053938:DNA Fragmentation; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D004587:Electrophoresis, Agar Gel; D005821:Genetic Techniques / Q000295:instrumentation*; D016681:Genome, Fungal; D015698:Genomic Library; D008970:Molecular Weight; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D013696:Temperature
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase