Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from aged refuse for microbial community analysis

Abstract

In order to develop and establish a method for isolation of PCR-amplifiable DNA from aged refuse, the orthogonal experiment was conducted to evaluate systematically the effects of various steps within each method in terms of DNA yield, purity, fragment size, humus contamination, PCR amplifiability using universal eubacterial and archaeal 16S rDNA prime pairs, and genetic diversity estimate from denaturing gradient gel electrophoresis. The inclusion of the pretreatment step was demonstrated to be crucial for the recovery of high-quality DNA preparations from the aged refuse and the pre-washing with TENP-PBS buffer was recommended. Satisfactory DNA yields and unbiased DNA extraction required the introduction of lysis by bead-beating treatment. The modified Sephadex G-200 spin column was effective in obtaining relatively stable DNA preparations from the aged refuse. It was established that a combination of pre-washes with TENP-PBS buffer plus gentle bead lysis and proteinase K treatment followed by SDS-based extraction and subsequent modified Sephadex G-200 spin column purification could ensure the acquisition of PCR-amplifiable DNA from the aged refuse expediently and cheaply.