الفهرس 
AbstractOnly 14 pages are availabe for public view
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Abstract
To establish an effective and improved protocol for soil environmental DNA extraction and purification. That makes the extracted DNA available to different molecular analysis such as: restriction endonucleases digestion, PCR performance and metagenomic library construction. Methods and Results: Different soil DNA extraction and purification methods were compared according to agarose gel electrophoresis and spectrophotometry measurements with variable rates of success. Four literature based methods were selected for soil DNA extraction evaluation. The DNA extraction method from Hurt et al., (2001) was modified and it showed better results. Among the different denaturing solutions tested during DNA extraction, 6M guanidine hydrochloride (6M GuHCl) was the best denaturing solution and suitable for different tested environmental samples with different characters. Skimmed milk addition showed a strong positive influence on the extracted soil DNA quantity and quality. Different physical and chemical cell disruption methods were tested during cell chemical lysis; vortexing the sample with glass beads additions during extraction buffer procedure gave significantly higher DNA yields than the other tested physical and mechanical cell disruption methods. Soil pre-lysis washing decreased the amount of proteins and humic substances contaminants. Successive soil pre-lysis washings using more than washing solution (CuSO4 - EDTA.2Na - PVP - Skimmed milk) showed the best results and applied for different environmental samples. For soil DNA purification, some classical electrophoretical procedures and other minicoloum purification methods were performed; base washed polyvinylpyrrolidone (PVPP) minicolumn combined with 30% polyethylene glycol (PEG) 8000 prepared in 1.6M NaCl showed the best purification result for both DNA purity and recovery. An optimized DNA extraction and purification protocol performed on four different environmental samples. Environmental DNA was amplified with universal bacterial-specific primers: B27 F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and U1492 R (5’-GGT TAC CTT GTT ACG ACT T-3’) in a successful PCR in addition to 5%DMSO. Environmental DNA was successfully partially digested by Hae III restriction endonuclease and cloned to pUC19 vector using T4 DNA ligase. The recombinant DNA was successfully transformed to competent cells (EPI100-T1R and DH5α). Conclusions: Optimizations of soil DNA extraction and purification were carried out after testing different combinations of previously described as well as new tested methods, aiming to provide high molecular weight (HMW) and pure environmental soil DNA. There are some important factors have been taken into account such as total processing time for DNA extraction and purification, cost and being suitable for different types of environmental samples. Different molecular analyses were performed on the extracted soil DNA such as restriction endonucleases digestion, PCR and metagenomic library construction. Significance and Impact of the Study: This study developed an optimized protocol for DNA extraction and purification, suitable for different environmental samples and valid for different molecular biology approaches.