Efficient random subcloning of DNA sheared in a recirculating point-sink flow system.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Oefner, PJ; Hunicke-Smith, SP; Chiang, L; Dietrich, F; Mulligan, J; Davis, RW

Published Date

  • October 15, 1996

Published In

Volume / Issue

  • 24 / 20

Start / End Page

  • 3879 - 3886

PubMed ID

  • 8918787

International Standard Serial Number (ISSN)

  • 0305-1048

Language

  • eng

Conference Location

  • England