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 meta-genomic 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.
Papaloapan Basin is the common name to refer to the Papaloapan River watershed and its tributaries. It is located in the southeast of Mexico encompassing 3 states (north of Oaxaca, south of Puebla and Veracruz center). It has fertile land and a warm and humid climate. These factors have made it a mega-diverse area, which has led to the development of agriculture, livestock, fisheries and the sugar industry. One of the most representative crops of the region is sugarcane, which occupies 30% of the arable surface [
Soil is considered as a complex environment, which appears to be a major reservoir of microbial genetic diversity. The complexity of microbial diversity results from multiple interacting parameters, which include pH, water content, soil structure, climatic variations and biotic activity [
Samples were collected from four sites. The four sites belong to the same plot (lat. 18.00˚N, 96.07˚W and alt. 32 m.a.s.l.) and samples were collected in April 2013. This soil is used to grow sugar cane and is classified as rhizosphere and composed of clay: 51.96%, silt: 30%, sand: 18.04% and pH: moderately acid. About 4 kg total soils were collected from the upper 0 to 15 and 15 to 30cm using a small spade and shovel. Samples were placed in plastic bags and stored at 4˚C [
We used DNA extraction protocol that involves bead beating: a method described previously Griffiths et al. (2000) [
PCR assay was designed to amplify a 1.6 kb region of the 16S gene. PCR amplification from 70 ng of extracted soil DNA was conducted with a total volume of 50 µl by using 0.5 - 1 µl (10 mM) concentrations of the fD1 primers (5’-CCG AAT TCG TCG ACA ACA GAG TTT GAT CCT GGC TCA G-3’) and rD1 (5’- CCC GGG ATC CAA GCT TAA GGA GGT GAT CCA GCC-3’) and 1.4 µl (5 U/µl) of Platinum Taq DNA polymerase (Invitrogen cat. 10966-026) under the following conditions: 3 minutes at 94˚C, 30 cycles of 30 seconds at 94˚C, 30 seconds at 56˚C, and 2 minutes at 72˚C, plus an additional 5 minutes cycle at 72˚C. The original reaction mixture was modified for the PCR assay. Three reaction mixtures were made. In each reaction, the following reagents were added: Reaction mixture 1. Were added 3 µl of MgCl2 (3 mM), Reaction mixture 2. 10X BSA (1 µg/µl) was added and Reaction mixture 3. Were added 3 µl of MgCl2 (3 mM) and BSA 10X (1 µg/µl).
Following extraction protocol described by the supplier very poor quality ground DNA was observed. The DNA was loaded onto an agarose gel, DNA degradation was observed. The analysis on the nanodrop showed that DNA had a low yield (20 ng/μl on average) and low purity (260/280: approximately 1.2) (
With these purified DNA the PCR was repeated and it was possible to obtain an amplification product of 1.6 Kb, however, the amount of amplified was not optimal (
PCR amplification. DNA samples of soil obtained in the extraction protocol (modified) were used in PCR assays occupying the original reaction mixture. Expected amplification was obtained, however, the amount of ampli-
Agarose gel electrophoresis (0.8%) of Soil DNA. Lane 1: Molecular weight 1 Kb (Fermentas cat. SM0314). Lanes 2 - 3: Soil DNA samples (30ng). DNA degradation was obser- ved
Agarose gel electrophoresis (0.8%) of Soil DNA puri- fied by column. Lane 1: Molecular weight 1 Kb (Fermentas cat. SM0314). Lanes 2 - 5: Soil DNA samples (30 ng). No degrada- tion was observed in the samples.
Agarose gel electrophoresis (0.8%) of PCR products (Soil DNA purified by colum). Lane 1: Molecular weight 1 Kb (Fermentas cat. SM0314). Lanes 2 - 8: 1600 bp amplified pro- duct using fD1 and rD1 primers (16S rDNA gene). Amplified observed, however, the intensity is low
fied was very weak. The PCR protocol was also modified. To the reaction mixture were added contaminants inhibitors (BSA and MgCl2) at different final concentrations each. In the first reaction mixture will be added only
Agarose gel electrophoresis (0.8%) of Soil DNA (Modified Protocol). Lane 1: Molecular weight 1 Kb (Fer- mentas cat. SM0314). Lane 2: Empty. Lanes 3 - 5: Soil DNA samples (30 ng). DNA is observed without degradation or presence of RNA
MgCl2. The second reaction mixture was added BSA, while the third reaction mixture was a combination of both inhibitors (BSA/MgCl2). In all three PCR assays were obtained the expected amplified. However, when loaded on an agarose gel, the most intense amplification was observed in the reaction mixture number 1 (MgCl2 only) (
The biosphere is dominated by microorganisms, yet most microbes in nature have not been studied. Traditional methods for culturing microorganisms limit analysis to those that grow under laboratory conditions [
Agarose gel electrophoresis (0.8%) of PCR products (Modified Protocol). Lane 1: Molecular weight 1 Kb (Fermentas cat. SM0314). Lane 2: Amplified product (1600 bp) (Without BSA/MgCl2). Lane 3: Negative control. Lane 4: Amplified product (1600 bp) (BSA). Lane 5: Amplified product (1600 bp) (MgCl2). Lane 6: Amplified product (1600 bp) (BSA/ MgCl2). 1600 bp amplified product using fD1 and rD1 primers (16S rDNA gene) in all lanes
nor proportion of soil microorganisms are culturable on standard media, analysis of directly extracted DNA has the potential to detect specific genes of otherwise cryptic microorganisms or monitor changes in the genotypic diversity of soil microbial communities [
Currently, there are no studies about soil metagenomics of sugarcane cultivation in the region of the Papaloapan Basin in Mexico. It is very important to develop metagenomic studies on these soils that are rich in agriculture and the diversity of microorganisms. In this work, it is the first time the extraction of DNA from soil in this region is performed, and the PCR assays to amplification 16S rDNA gene as quality control soil DNA. These results open the way to continue performing metagenomics studies in this region and then identify bacteria with cellulase or amylase activity that are of interest to our research group.
This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) 154683 and Programa de Mejoramiento para el Profesorado (PROMEP) 2009-02 103.5/11/6149 projects. Gutierrez-Lucas LR, Cortés- López NG, Montor-Antonio JJ thanks CONACyT by the grants 171724, 20254 and 28094, respectively. The authors declare that they have no conflict of interest.