Rapid and simple method for purification of nucleic acids.
R. Boom,C. J. A. Sol,M. M. M. Salimans,C. L. Jansen,P.M.E. Wertheim-van Dillen,J. van der Noordaa +5 more
TLDR
A simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine, based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent.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.read more
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Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia
Menno D. de Jong,Cameron P. Simmons,Tran Tan Thanh,Vo Minh Hien,Gavin J. D. Smith,Tran Nguyen Bich Chau,Dang Minh Hoang,Nguyen Van Vinh Chau,Truong Huu Khanh,Vo Cong Dong,Phan Tu Qui,Bach Van Cam,Do Quang Ha,Yi Guan,J. S. Malik Peiris,Nguyen Tran Chinh,Tran Tinh Hien,Jeremy Farrar +17 more
TL;DR: The observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis and the focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.
Journal ArticleDOI
Identification of a new human coronavirus
Lia van der Hoek,Krzysztof Pyrc,Maarten F. Jebbink,Wilma Vermeulen-Oost,Ron J. M. Berkhout,Katja C. Wolthers,Pauline M. E. Wertheim-van Dillen,Jos Kaandorp,Joke Spaargaren,Ben Berkhout +9 more
TL;DR: A new group 1 coronavirus, HCoV-NL63, was identified in a 7-month-old child suffering from bronchiolitis and conjunctivitis as discussed by the authors.
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Reliable Genotyping of Samples with Very Low DNA Quantities Using PCR
Pierre Taberlet,Sally Griffin,Benoit Goossens,Sophie Questiau,Valérie Manceau,Nathalie Escaravage,Lisette P. Waits,Jean Bouvet +7 more
TL;DR: An experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA is identified and should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.
Journal ArticleDOI
An inexpensive, automation-friendly protocol for recovering high-quality DNA
TL;DR: This study presents a silica-based method that is sensitive, inexpensive and compliant with automation that has now been tested on more than 5000 animal specimens with highly positive results.
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Noninvasive genetic sampling: look before you leap
TL;DR: The need for a more cautious approach to noninvasive sampling, which includes quantifying the genotyping error rate is indicated, which will probably be overcome with improved methodology.
References
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Journal ArticleDOI
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
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Journal ArticleDOI
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Journal ArticleDOI
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TL;DR: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
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Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I
TL;DR: Labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.