Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments.
PCR inhibitor-free metagenomic DNA of high quality and high yield was extracted from highly polluted sediments using a simple remediation strategy of adsorption and ion-exchange chromatography. Extraction procedure was optimized with series of steps, which involved gentle mechanical lysis, treatment with powdered activated charcoal (PAC) and ion-exchange chromatography with amberlite resin. Quality of the extracted DNA for molecular diversity analysis was tested by amplifying bacterial 16S rDNA (16S rRNA gene) with eubacterial specific universal primers (8f and 1492r), cloning of the amplified 16S rDNA and ARDRA (amplified rDNA restriction analysis) of the 16S rDNA clones. The presence of discrete differences in ARDRA banding profiles provided evidence for expediency of the DNA extraction protocol in molecular diversity studies. A comparison of the optimized protocol with commercial Ultraclean Soil DNA isolation kit suggested that method described in this report would be more efficient in removing metallic and organic inhibitors, from polluted sediment samples.
Desai
Chirayu
C
Postgraduate Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India.
Madamwar
Datta
D
D000327:Adsorption; D001483:Base Sequence; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000302:isolation & purification*; D023281:Genomics; D019015:Geologic Sediments / Q000737:chemistry*; D007475:Ion Exchange Resins
D004275:DNA, Ribosomal; D007475:Ion Exchange Resins