Amplifiable DNA from gram-negative and gram-positive bacteria by a low strength pulsed electric field method.
An efficient electric field-based procedure for cell disruption and DNA isolation is described. Isoosmotic suspensions of Gram-negative and Gram-positive bacteria were treated with pulsed electric fields of <60 V/cm. Pulses had an exponential decay waveform with a time constant of 3.4 micros. DNA yield was linearly dependent on time or pulse number, with several thousand pulses needed. Electrochemical side-effects and electrophoresis were minimal. The lysates contained non-fragmented DNA which was readily amplifiable by PCR. As the method was not limited to samples of high specific resistance, it should be applicable to physiological fluids and be useful for genomic and DNA diagnostic applications.
Vitzthum
F
F
Laboratory of Biochemistry, Institute for Interfacial Engineering, University of Stuttgart and Fraunhofer-IGB, Nobelstrasse 12, D-70569 Stuttgart, Germany.
Geiger
G
G
Bisswanger
H
H
Elkine
B
B
Brunner
H
H
Bernhagen
J
J
D002458:Cell Fractionation / Q000379:methods; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004560:Electricity; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000235:genetics; D016982:Micrococcus luteus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013696:Temperature
D004269:DNA, Bacterial; D008263:Macrophage Migration-Inhibitory Factors