Rapid and simple method for purification of nucleic acids.
We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.
Department of Virology, Academic Medical Center, Amsterdam, The Netherlands.
R
R
Boom
C J
CJ
Sol
M M
MM
Salimans
C L
CL
Jansen
P M
PM
Wertheim-van Dillen
J
J
van der Noordaa
D004247:DNA / Q000097:blood / Q000302:isolation & purification* / Q000652:urine; D004270:DNA, Circular / Q000097:blood / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000097:blood / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D056890:Eukaryota; D005898:Glass; D006801:Humans; D008863:Microspheres; D012313:RNA / Q000097:blood / Q000302:isolation & purification* / Q000652:urine; D012335:RNA, Ribosomal / Q000302:isolation & purification / Q000652:urine; D012822:Silicon Dioxide
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D012335:RNA, Ribosomal; D012313:RNA; D012822:Silicon Dioxide; D004247:DNA