Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil

Abstract

In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

A modified method for genomic DNA extraction from the fish intestinal microflora

“…To compensate for the low DNA extraction efficiency of lysozyme for most Gram-negative bacteria, a treatment step with proper ultrasonic disruption was used in the CLU method, which could break down the cell walls of Gram-negative bacteria and allowed good liberation of the DNA. In addition, the incubation temperature during the process of lysozyme lysis was usually maintained at 37 °C in previous studies (Kathiravan et al 2015 ; Bag et al 2016 ). However, some investigators noted that the activity of lysozyme from egg whites increased gradually with increasing temperature within the range of 25–65 °C (Liu et al 2008 ).…”

“…In early studies, conventional culture-dependent techniques were used; however, only a small percentage of the bacterial flora was identified (Kathiravan et al 2015 ). Recently, molecular techniques, including but not limited to denatured gradient gel electrophoresis (Li et al 2011 ), fluorescence in situ hybridization (Hoffmann et al 2006 ) and 16S rDNA high-throughput sequencing (Gajardo et al 2016 ), have been used successfully to analyze the complex microbial community from fish intestines.…”

Biases from different DNA extraction methods in intestine microbiome research based on 16S rDNA sequencing: a case in the koi carp, Cyprinus carpio var. Koi

“…Over the last decade, an increasing number of studies have focused on the gut microbiota of fish (Narrowe et al., ; Xia et al., ). In early studies, conventional culture‐dependent techniques were used; however, only a small percentage of the resulting bacterial flora was identified (Kathiravan et al., ). Recently, new technologies based on meta‐genomics/high‐throughput sequencing have been developed and successfully applied to analyzing the complex bacterial ecosystem of the gut.…”

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

“… 5 Accordingly, the isolation of high-quality DNA that covers the whole microbial diversity in the original sample was phrased as a limiting step for the construction of metagenomic libraries and other DNA based metagenomic projects. 6 …”

“…In these regards, many studies have reported the development of methods and even commercial kits for mDNA extraction. 5 , 6 However, still, there is a surge in developing, improving, and optimizing new or current protocols for mDNA extraction from different types of samples. A metagenomic DNA extraction must be followed by a quality control of the extracted DNA.…”