KnE Life Sciences
ISSN: 2413-0877
The latest conference proceedings on life sciences, medicine and pharmacology.
COMPARISON OF THREE DNA ISOLATION AND PURIFICATION METHODS OF BACTERIAL DNA
Published date: Sep 20 2015
Journal Title: KnE Life Sciences
Issue title: International Conference on Biological Sciences (ICBS-2013)
Pages: 491-494
Authors:
Abstract:
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
References:
Fujimoto, S., Y. Nakagami, and F. Kojima. 2004. Optimal Bacterial DNA Isolation Method using Bead-Beating Technique. Medical Sch. 3:33-38
Kaisu, R.J., and J. Jalava. 2002. Optimal DNA Isolation Method for Detectioan of Bacteria in Clinical Specimens by Broad-Range PCR. J. Clin. Microbiol. Vol 40 11:4211-4217
Niemi, R.M., I. Heiskanen, K. Wallenius, and K. Lindstroms. 2001. Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia. J. Microbiol Methods48: 155=165
O’Reilly, C., P.D. Turner. 2003. The nitrilase of CN hydolysing enzyme a comparative study. J.Appl. Microbiol 95 1161-1174
Picher, D.G., N.A. Saunders, and R.J. Owen. 1989. Rapid Extraction of Bacterial Genomic DNA with Guanidium Thiocyanate. Lett Appl Microbiol. 8:151-156
Sambrook, K., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A laboratory Manual 2nd edition. Cold Spring Harbor Laboratory, New York.