EFFICIENT RANDOM SUBCLONING OF DNA SHEARED IN A RECIRCULATING POINT-SINK FLOW SYSTEM

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-mu m orifice w ith ensuing fractionation to a minimum fragment size of -300 base pair s, Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow ext ension of the DNA molecules into a highly extended conformation and, h ence, breakage to occur at midpoint, In most instances, 30 passages su fficed to obtain a narrow size distribution, with >90% of the fragment s lying within a 2-fold size distribution, The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 pow er of the molecular weight, Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal impr ovements in cloning efficiency having been observed upon prior end rep air with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.