Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990

Rapid and simple method for purification of nucleic acids.

R Boom 1, C J Sol 1, M M Salimans 1, C L Jansen 1, P M Wertheim-van Dillen 1, J van der Noordaa 1
PMCID: PMC269651  PMID: 1691208

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.

Full text

PDF
495

Images in this article

497Image
on p.497
498Image
on p.498
498Image
on p.498
499Image
on p.499
499Image
on p.499
500Image
on p.500
501Image
on p.501

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aaij C., Borst P. The gel electrophoresis of DNA. Biochim Biophys Acta. 1972 May 10;269(2):192–200. doi: 10.1016/0005-2787(72)90426-1. [DOI] [PubMed] [Google Scholar]
  2. Boros I., Pósfai G., Venetianer P. High-copy-number derivatives of the plasmid cloning vector pBR322. Gene. 1984 Oct;30(1-3):257–260. doi: 10.1016/0378-1119(84)90130-6. [DOI] [PubMed] [Google Scholar]
  3. Bowtell D. D. Rapid isolation of eukaryotic DNA. Anal Biochem. 1987 May 1;162(2):463–465. doi: 10.1016/0003-2697(87)90421-0. [DOI] [PubMed] [Google Scholar]
  4. 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. doi: 10.1016/0003-2697(84)90339-7. [DOI] [PubMed] [Google Scholar]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. 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. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Ciulla T. A., Sklar R. M., Hauser S. L. A simple method for DNA purification from peripheral blood. Anal Biochem. 1988 Nov 1;174(2):485–488. doi: 10.1016/0003-2697(88)90047-4. [DOI] [PubMed] [Google Scholar]
  8. Firestein G. S., Gardner S. M., Roeder W. D. Quantitative molecular hybridization with unfractionated, solubilized cells using RNA probes and polyacrylamide gel electrophoresis. Anal Biochem. 1987 Dec;167(2):381–386. doi: 10.1016/0003-2697(87)90180-1. [DOI] [PubMed] [Google Scholar]
  9. Hatefi Y., Hanstein W. G. Solubilization of particulate proteins and nonelectrolytes by chaotropic agents. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1129–1136. doi: 10.1073/pnas.62.4.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kwoh D. Y., Davis G. R., Whitfield K. M., Chappelle H. L., DiMichele L. J., Gingeras T. R. 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. doi: 10.1073/pnas.86.4.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kwok S., Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. doi: 10.1038/339237a0. [DOI] [PubMed] [Google Scholar]
  13. Marko M. A., Chipperfield R., Birnboim H. C. 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. doi: 10.1016/0003-2697(82)90497-3. [DOI] [PubMed] [Google Scholar]
  14. Miller R. H., Kaneko S., Chung C. T., Girones R., Purcell R. H. Compact organization of the hepatitis B virus genome. Hepatology. 1989 Feb;9(2):322–327. doi: 10.1002/hep.1840090226. [DOI] [PubMed] [Google Scholar]
  15. Norval M., Bingham R. W. 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. doi: 10.1007/BF01314418. [DOI] [PubMed] [Google Scholar]
  16. Raptis L., Menard H. A. Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus. J Clin Invest. 1980 Dec;66(6):1391–1399. doi: 10.1172/JCI109992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rigby P. W., 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. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  18. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  19. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., 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. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  20. Schochetman G., Ou C. Y., Jones W. K. Polymerase chain reaction. J Infect Dis. 1988 Dec;158(6):1154–1157. doi: 10.1093/infdis/158.6.1154. [DOI] [PubMed] [Google Scholar]
  21. Tenover F. C. Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin Microbiol Rev. 1988 Jan;1(1):82–101. doi: 10.1128/cmr.1.1.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thompson J., Gillespie D. Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate. Anal Biochem. 1987 Jun;163(2):281–291. doi: 10.1016/0003-2697(87)90225-9. [DOI] [PubMed] [Google Scholar]
  23. VONHIPPEL P. H., WONG K. Y. NEUTRAL SALTS: THE GENERALITY OF THEIR EFFECTS ON THE STABILITY OF MACROMOLECULAR CONFORMATIONS. Science. 1964 Aug 7;145(3632):577–580. doi: 10.1126/science.145.3632.577. [DOI] [PubMed] [Google Scholar]
  24. 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. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yang R., Lis J., Wu R. Elution of DNA from agarose gels after electrophoresis. Methods Enzymol. 1979;68:176–182. doi: 10.1016/0076-6879(79)68012-6. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES