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.
Boom
R
R
Department of Virology, Academic Medical Center, Amsterdam, The Netherlands.
Sol
C J
CJ
Salimans
M M
MM
Jansen
C L
CL
Wertheim-van Dillen
P M
PM
van der Noordaa
J
J
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