Strategies for the Extraction, Purification and Amplification of Metagenomic DNA from Soil Growing Sugarcane

Abstract

Recently, studies were initiated to investigate the metagenome, which represents the genomes of cultured and uncultured microbes, as a rich source for isolation of many novel genes. The metagenomic approach originated from the molecular analysis of microbial communities, which revealed that the majority of microorganisms in nature were not cultivable by standard culturing techniques. Therefore, most microorganisms in nature have not been characterized. Although numerous methods have been reported for direct DNA isolation and purification from microorganisms in soil, the sample preparation procedures and experimental conditions used in different studies vary widely. Soils are therefore one of the most challenging environmental matrices from which to obtain microbial DNA that will support PCR. The Papaloapan River is the second largest river basin in México. For the climatic conditions of this region, there is great diversity in plants, animals and microorganisms. In the Papaloapan region different fruits are grown, however, the main crops are sugarcane and pineapple. In this work the extraction of DNA from soils of sugarcane cultivation was performed. We used PCR tests to assess the quality of DNA extracted from soil by amplifying the 16S rDNA gene. Changes in both protocols were performed; satisfactory results were obtained as to the quality of DNA and gene amplification. These results will allow continuing the metagenomic studies, such as sequencing, library construction and identification of enzymes cellulase and amylase activity. It is the first time these studies were performed in the Papaloapan region.

Metagenomic applications in exploration and development of novel enzymes from nature: a review

“…Each gel concentration profile shows the optimal state of the length of the nucleic acid fragment used as a sample when running gel electrophoresis. Gutiérrez-lucas et al [39] have used a 0.8% gel concentration for samples originating from the soil. The choice of 0.8% agarose gel concentration is a strategy for electrophoresis from metagenomic samples because environmental DNA fragments (eDNA) have an extended size.…”

“…Gutiérrez-lucas et al . [ 39 ] have used a 0.8% gel concentration for samples originating from the soil. The choice of 0.8% agarose gel concentration is a strategy for electrophoresis from metagenomic samples because environmental DNA fragments (eDNA) have an extended size.…”

Starch- and cellulose-related microbial diversity of soil sown with sugarcane crops in the Papaloapan Basin, a megadiverse region of Mexico

“…cycles of 30 seconds at 94˚C, 30 seconds at 56˚C, and 2 min at 72˚C, plus an additional 5 min cycle at 72˚C (Gutiérrez-Lucas et al, 2014). PCR products were analyzed on 1% agarose gels.…”

Comparative assessment of methods for metagenomic DNA isolation from soils of different crop growing fields

“…The isolation of metagenomic DNA from soil is an important consideration and needs standardization. Since soil is rich in organic matter like humic acid and other inhibitory substances, which hinder downstream applications like PCR amplification and cloning, several protocols have been reported in literature (Berthelet et al 1996 ; Gutiérrez-Lucas et al 2014 ; Nair et al 2014 ; Picard et al 1992 ).…”