A method for assessing efficiency of bacterial cell disruption and DNA release
| Autor: | H. C. Birnboim, Olle M. de Bruin |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
DNA
Bacterial 0301 basic medicine Microbiology (medical) Lysis Microorganism 030106 microbiology Bacillus subtilis Microbiology Bacterial cell structure 03 medical and health sciences chemistry.chemical_compound Cell Wall DNA Fungal Molecular Biology Chromatography High Pressure Liquid Spores Bacterial Bacteriological Techniques Bacteria biology Methodology Article Mycobacterium smegmatis Ribonucleotides biology.organism_classification DNA extraction RNA Bacterial 030104 developmental biology Biochemistry chemistry Purines DNA |
| Zdroj: | BMC Microbiology |
| ISSN: | 1471-2180 |
| DOI: | 10.1186/s12866-016-0815-3 |
| Popis: | Background DNA-based testing is becoming the preferred method both for identifying microorganisms and for characterizing microbial communities. However, no single DNA extraction method exists that is suitable for all types of microorganisms because bacteria are variable in their susceptibility to lysis by available extraction procedures. To develop and test new DNA extraction procedures, it would be helpful to determine their efficiencies. While the amount of extracted DNA can readily be measured by different methods, calculation of true efficiency requires knowledge of the initial amount of DNA in the starting bacterial sample, which cannot be done with precision by any existing method. In the process of developing a new extraction procedure, we developed a method that can be used to determine the total amount of both DNA and RNA in bacteria. The amount of DNA can be calculated from the amount of purines released after mild acid and alkali treatment. The amount of RNA in the same extract can also be calculated from the amount of ribonucleoside monophosphates. The released purines and ribonucleoside monophosphates can be quantified by absorbance using HPLC, with reference to appropriate standards. Results The acid/HPLC method was used to measure the efficiency of commonly used bead-beating and chemical protocols for releasing DNA from a particularly hardy organism, Mycobacterium smegmatis as well as several other species (Bacillus subtilis vegetative cells and spores; Francisella philomiragia; Pseudomonas aeruginosa; Moraxella catarrhalis; Bacillus thuringiensis; Staphylococcus aureus). Surprisingly large differences in efficiency between methods were found. Conclusions The acid/HPLC method is a new tool to determine DNA extraction efficiencies and should aid in the development of improved protocols for releasing DNA from a broad range of microorganisms. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0815-3) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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