Nucleic Acids Research, Vol 24, Issue 20 3879-3886, Copyright © 1996 by Oxford University Press
PJ Oefner, SP Hunicke-Smith, L Chiang, F Dietrich, J Mulligan and RW Davis
Based on a high-performance liquid chromatographic pump, we have built a
device that allows recirculation of DNA through a 63-microm orifice with
ensuing fractionation to a minimum fragment size of approximately 300 base
pairs. Residence time of the DNA fragments in the converging flow created
by a sudden contraction was found to be sufficiently long to allow
extension of the DNA molecules into a highly extended conformation and,
hence, breakage to occur at midpoint. In most instances, 30 passages
sufficed to obtain a narrow size distribution, with >90% of the
fragments lying within a 2-fold size distribution. The shear rate required
to achieve breakage was found to be inversely proportional to the 1.0 power
of the molecular weight. Compared with a restriction digest, up to 40% of
all fragments could be cloned directly, with only marginal improvements in
cloning efficiency having been observed upon prior end repair with Klenow,
T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly
random distribution of the fragments.
ARTICLES
Efficient random subcloning of DNA sheared in a recirculating point- sink flow system
Department of Biochemistry, Stanford University, CA, USA. oefner@genome.stanford.edu
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