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-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.
Oefner
P J
PJ
Department of Biochemistry, Stanford University, CA, USA. oefner@genome.stanford.edu
Hunicke-Smith
S P
SP
Chiang
L
L
Dietrich
F
F
Mulligan
J
J
Davis
R W
RW
D017104:Bacteriophage M13 / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D002689:Chlamydia / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification; D009994:Osmolar Concentration; D010316:Particle Size; D014998:Y Chromosome / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D004247:DNA