Post-hybridization recovery of membrane filter-bound DNA for enzymatic DNA amplification.
We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymerase chain reaction amplification. The source of membrane-bound DNA successfully tested includes plasmid and human leukocyte DNA and DNA immobilized on bacterial colony filters and plaque lifts. The sensitivity of the procedure is such that DNA recovered from 0.5 microgram of filter-bound total human DNA was enough for PCR amplification of a 0.3-kb fragment. Our protocol will be useful for recycling of scarce DNA samples for cloning and sequencing purposes.
Chong
K Y
KY
Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China.
Chen
C M
CM
Choo
K B
KB
D001709:Biotechnology; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D004121:Dimethyl Sulfoxide; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D005374:Filtration; D005784:Gene Amplification; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial; D004121:Dimethyl Sulfoxide