Content area
Full Text
Many attempts have been made to adapt molecular biology methods for the study of microbial community composition. However, nucleic acid extraction with desirable quality and quantity is one of the most critical steps especially in the case of environmental samples like activated sludge. The bacteria present in activated sludge are surrounded by a mucilaginous envelope and produces large quantities of polysaccharides. This causes difficulties in DNA isolation and subsequent molecular approaches. The aim of the present study was to develop a strategy for extraction of high-quality DNA from activated sludge, and compare it with the traditional methods. The protocols were based on the traditional phenol-chloroform DNA extraction methodology, utilization of CTAB buffer and Xanthogenate buffer for DNA extraction. All the three methods were successful for isolation of DNA. Different cell lysis methods were employed for these methods. CTAB, and phenol-chloroform extraction method employed mechanical agitation with glass beads in combination with chemical lysis. Both the methods produced the satisfactory yields of DNA, however, the DNA obtained was sheared. Xanthogenate method required no extra cell lysis step and proved to be the best method to isolate high quality, pure DNA. The quality of DNA was further tested by submitting these DNA for successful PCR amplification using 16S rDNA primers. Thus, it can be concluded that Xanthogenate method is the simplest method omitting phenol-chloroform steps, quick, and most effective for dealing with activated sludge samples.
Key Words: Activated sludge, DNA extraction, Cell lysis, PCR.
INTRODUCTION
DNA isolation is a routine procedure to collect DNA for subsequent molecular analysis. The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Many different protocols and commercially available kits have been developed for DNA extractions from environmental samples. However, the problem of DNA extraction is still an important issue in the case of environmental samples like activated sludge, (Gabor et al. 2003). Since samples differ in their physical and chemical characteristics, no single protocol is suitable for all types of samples. Presence of some inhibitors in extracted DNA solution causes a reduction in Taqpolymerase activities in PCR and hinders the function of cutter enzymes as well, (Bushra et al. 1999). Most approaches require primarily high molecular weight DNA, which...