GENOMIC DNA EXTRACTION METHODS: A COMPARATIVE CASE STUDY WITH GRAM-NEGATIVE ORGANISMS

DOI: 10.7904/2068–4738–VI(11)–61

Sanjida JAHAN, Sayeeda Fahmee CHOWDHURY, Shahida Akter MITU, Mohammad SHAHRIAR, Mohiuddin Ahmed BHUIYAN*

Department of Pharmacy, University of Asia Pacific, Department of Pharmacy. House No. 73, Road No. 5A, Dhanmondi, Dhaka-1209, BANGLADESH
E-mail: mohiuddin@uap-bd.edu

Abstract. The cell wall structure of gram–negative bacteria is more complex than that of gram–positive bacteria because of the presence of two layers external to the cytoplasmic membrane. Purpose of this study is to compare the four different methods for genomic DNA extraction from three gram negative strains–Escherichia coli, Klebsiella species, and Salmonella typhi. The genomic DNA was extracted from gram–negative bacteria cultures using enzymatic lysis with Proteinase–K, chemical lysis with cetyl trimethyl ammonium bromide (CTAB), physical lysis with Triton X–100 and Beads beating methods. The yield and quality of DNA were compared by spectrophotometry and agarose gel electrophoresis. The genomic DNA purity ratio was found within 1.1–1.9 for all four methods. This study revealed that the chemical lysis with CTAB was the most efficient method for extracting genomic DNA from gram negative strains. In terms of chemical lysis with CTAB for Salmonella typhi and Klebsiella species the highest purity ratio were 1.9 and 1.8 respectively. However Escherichia coli also showed the highest purity ratio (1.7) for CTAB extraction. Enzymatic lysis with Proteinase–K was also very effective and purity ratio for Salmonella typhi and Klebsiella species were 1.8 and 1.7 respectively.

Keyword: Bead beating, CTAB, Genomic DNA, gram negative organism, Proteinase–K, Triton X–100

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