The objective of this study was to compare the efficiency of three methods of nucleic acids extraction from gram-positive bacteria by evaluating the quantity and purity of DNA extracts. Nucleic acids extraction of gram-positive bacteria is normally hampered by a thick and resistant cell wall. Gram positive bacteria usually have a thick cell wall consisting mainly of many layers of peptidoglycan, which is not easily destroyed. This paper compares the different procedures based on mechanical and enzymatic cell breakage to extract DNA from Rhodoccocus pyridinivorans using GES method, Ultraclean Microbial DNA isolation Kit, and Prepman Microbial DNA isolation kit. DNA extracts were analyzed by agarose gel electrophoresis and UV spectroscopy. Yield and quality of DNA obtained by the GES method were higher than the other methods. Nucleic acids extracts with the highest yield and purity were amplified by Polymerase Chain Reaction (PCR) using various primers targeted on gene encoding nitrilase gene such as BLITF and PNITR, a NH1 and a NH2, ß NH1 and ß NH2, Amd1 and Amd 2. The gene encoding for nitrilase were amplified which was confirmed by sequencing analyses. However, the targeted gene length from the primes was not obtained. Therefore further amplification optimization may be needed.
Keywords: extraction, DNA isolation Rhodococcus pyridinovorans, nitrilase gene