Rapid and simple method for purification of nucleic acids. (original) (raw)

J Clin Microbiol. 1990 Mar; 28(3): 495–503.

Department of Virology, Academic Medical Center, Amsterdam, The Netherlands.

Abstract

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.

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Aaij C, Borst P. The gel electrophoresis of DNA. Biochim Biophys Acta. 1972 May 10;269(2):192–200. [PubMed] [Google Scholar]
Boros I, Pósfai G, Venetianer P. High-copy-number derivatives of the plasmid cloning vector pBR322. Gene. 1984 Oct;30(1-3):257–260. [PubMed] [Google Scholar]
Bowtell DD. Rapid isolation of eukaryotic DNA. Anal Biochem. 1987 May 1;162(2):463–465. [PubMed] [Google Scholar]
Cheley S, Anderson R. A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization. Anal Biochem. 1984 Feb;137(1):15–19. [PubMed] [Google Scholar]
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. [PubMed] [Google Scholar]
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed] [Google Scholar]
Ciulla TA, Sklar RM, Hauser SL. A simple method for DNA purification from peripheral blood. Anal Biochem. 1988 Nov 1;174(2):485–488. [PubMed] [Google Scholar]
Firestein GS, Gardner SM, Roeder WD. Quantitative molecular hybridization with unfractionated, solubilized cells using RNA probes and polyacrylamide gel electrophoresis. Anal Biochem. 1987 Dec;167(2):381–386. [PubMed] [Google Scholar]
Hatefi Y, Hanstein WG. Solubilization of particulate proteins and nonelectrolytes by chaotropic agents. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1129–1136. [PMC free article] [PubMed] [Google Scholar]
Ish-Horowicz D, Burke JF. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. [PMC free article] [PubMed] [Google Scholar]
Kwoh DY, Davis GR, Whitfield KM, Chappelle HL, DiMichele LJ, Gingeras TR. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1173–1177. [PMC free article] [PubMed] [Google Scholar]
Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. [PubMed] [Google Scholar]
Marko MA, Chipperfield R, Birnboim HC. A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem. 1982 Apr;121(2):382–387. [PubMed] [Google Scholar]
Miller RH, Kaneko S, Chung CT, Girones R, Purcell RH. Compact organization of the hepatitis B virus genome. Hepatology. 1989 Feb;9(2):322–327. [PubMed] [Google Scholar]
Norval M, Bingham RW. Advances in the use of nucleic acid probes in diagnosis of viral diseases of man. Brief review. Arch Virol. 1987;97(3-4):151–165. [PubMed] [Google Scholar]
Raptis L, Menard HA. Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus. J Clin Invest. 1980 Dec;66(6):1391–1399. [PMC free article] [PubMed] [Google Scholar]
Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. [PubMed] [Google Scholar]
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed] [Google Scholar]
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed] [Google Scholar]
Schochetman G, Ou CY, Jones WK. Polymerase chain reaction. J Infect Dis. 1988 Dec;158(6):1154–1157. [PubMed] [Google Scholar]
Tenover FC. Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin Microbiol Rev. 1988 Jan;1(1):82–101. [PMC free article] [PubMed] [Google Scholar]
Thompson J, Gillespie D. Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate. Anal Biochem. 1987 Jun;163(2):281–291. [PubMed] [Google Scholar]
Vogelstein B, Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. [PMC free article] [PubMed] [Google Scholar]
VONHIPPEL PH, WONG KY. NEUTRAL SALTS: THE GENERALITY OF THEIR EFFECTS ON THE STABILITY OF MACROMOLECULAR CONFORMATIONS. Science. 1964 Aug 7;145(3632):577–580. [PubMed] [Google Scholar]
Yang R, Lis J, Wu R. Elution of DNA from agarose gels after electrophoresis. Methods Enzymol. 1979;68:176–182. [PubMed] [Google Scholar]

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