Abstract
Currently, there are several protocols to extract bacterial DNA based on different principles. However, the quantity and the quality of the DNA obtained by each method are highly variable and microorganism dependent. In most of these classical crude methods, highly toxic and hazardous organic solvents such as phenol and chloroform are used for deproteinization, whereas in certain protocols, expensive enzymes including RNases and Proteinases are used. This study was designed to introduce a simple, rapid, inexpensive and effective genomic DNA isolation procedure for Gram-negative bacteria, without the usage of toxic chemicals and costly enzymes. This novel method was compared with another classical method known as the salting-out method, which uses proteinase-K. Concentration and yield of the extracted DNA were determined by gel electrophoresis by comparing the gel band intensity of the sample DNA to that of a DNA quantitation standard and by the Quantus™ fluorometer. According to the results, the yield of extracted DNA was higher in the novel method compared to the salting-out method. Moreover, the entire process was accomplished in less than 2 h with the novel method. Purity and integrity of extracted genomic DNA by both methods were similar. In addition, the quality of DNA was determined using Multicopy Associated Filamentation (MAF) gene amplification by polymerase chain reaction (PCR). Thus, the described technique is non-toxic, less time and fund consuming, efficient and a well-suited method for routine DNA isolation from Gram negative bacteria.
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Authors would like to acknowledge the academic and nonacademic staff of Department of Biochemistry and Molecular Biology of Faculty of Medicine, University of Colombo, Sri Lanka.
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LRW—conception and design, conducting the research, specifically performing the experiment, methodology, data analysis and interpretation, drafting the original article, material preparation. CW—supervision, conceptualization, review and the final approval of the draft to be submitted. All authors read and approved the final manuscript.
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Weerakkody, L.R., Witharana, C. A rapid, inexpensive and effective method for the efficient isolation of genomic DNA from Gram-negative bacteria. Mol Genet Genomics 299, 26 (2024). https://doi.org/10.1007/s00438-024-02120-x
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DOI: https://doi.org/10.1007/s00438-024-02120-x