The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method.
The biodiversity of microbes within soil is significant for the maintenance of healthy soil because these microbes are involved in many vital functions like crucial cycles of C, N, P, formation of soil, toxin removal, and so on. Previously, studies on the development of microbial communities required the isolation of these microbes from soil sample by culture dependent techniques followed by a series test for phenotypic evaluation and their identification. However, the microbial diversity studies conducted in soil have been biased essentially due to the unculturability of many microbes. Specific media, which are used to culture microbes, are selective in nature and only subpopulations of microbes from environment sample that will grow mainly depend on the particular conditions. It is reported that only 1% of microbes can be cultured in the laboratory using traditional culture techniques [
To study the microbial community, microbiologists have adopted culture-independent techniques. These techniques employ molecular biology based methods, in which soil extracted nucleic acid is subjected to PCR amplification [
The analysis of microbial diversity in the soil DNA extracts is then based on ARDRA-amplified ribosomal DNA restriction analysis [
In the present study, four DNA extraction methods and a commercial
Five DNA extraction methods were evaluated in this study with respect to the quality and purity of extracted DNA using single type of rhizospheric soil. Three modified mannitol-based methods [
One gram of soil sample was mixed with 10 mL of DNA extraction buffer (120 mM Na2HPO4 (pH 7.4), 5% SDS (w/v) and 0.02 g PVPP) in centrifuge tubes and incubated for 1 h at 65°C with occasional stirring. The supernatant was collected after centrifugation at 8,000 rpm for 10 min at 4°C and mixed with half volume of PEG and 1 volume of NaCl and incubated at 4°C for overnight. Further, 1 volume of chloroform : isoamyl alcohol (24 : 1) was added and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant obtained was precipitated by addition of 1/10th volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. Finally, the pellet was recovered by centrifugation at 12,000 rpm at 4°C and dissolved in 25
DNA was extracted from soil sample (1 gm) according to the specifications of the supplier (EPICENTRE, Madison, WI, USA). The method involved direct cell lysis with prewarmed 5 mL solution A at 65°C and vortexing for 10 min. This mixture was incubated for 15 min at 65°C. The supernatants were collected after centrifugation at 8,000 rpm at 4°C for 10 min and mixed with equal volume of solution B that led to precipitation. The steps were repeated two times when the color of supernatant changed to yellow. Finally the pellet was recovered by centrifugation at 12,000 rpm for 10 min at 4°C and dissolved in 25
One gram of soil sample was ground using liquid nitrogen. This was followed by addition of 5 mL of 120 mM phosphate buffer saline (pH 7.4) and shaking at 150 rpm for 10 min at 4°C. The soil suspension was centrifuged at 7,000 rpm for 10 min. The pellet was rewashed with PBS buffer and suspended in 10 mL of DNA extraction buffer containing 1 M Tris-HCl (pH 8.0), 5 M NaCl, 0.5 M EDTA (pH 8.0), 10% CTAB, 10% SDS, and 0.2 M mannitol. The suspension was incubated for 1 h at 65°C with occasional stirring of 150 rpm and subjected to three different treatments as indicated.
The DNA concentration of the soil sample was measured by examining the absorbance of the sample at 260 nm and the amount of DNA was calculated (1.0 A260 unit = 50
Soil DNA was amplified by PCR using a PCR BIORAD Thermal Cycler (United Kingdom). Each 25
Soil DNA was submitted for PCR amplification by using PCR BIORAD Thermal Cycler (United Kingdom). A region from 18S rRNA gene was amplified using internal transcribed spacer (ITS) primers, namely, ITS 5: (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS 4 (5′-TCCTCCGCTTATTGATATGC-3′). Each 25
To test the efficiency of soil DNA extraction methods, RAPD was performed on community DNA. Four decameric RAPD primers, namely, OPA 3, OPA 13, OPA 15, and OPA 20 (Operon Technologies), were investigated (Table
Random primers used for RAPD analysis and their annealing temperature.
Random primer | Primer sequences | Annealing temperature |
---|---|---|
OPA 3 | 5′-AGTCAGCCAC | 32°C |
OPA 13 | 5′-CAGCACCCAC | 32°C |
OPA 15 | 5′-TTCCGAACCC | 32°C |
OPA 20 | 5′-GTTGCGATCC | 32°C |
For visualizing PCR products, 5
For soil microbial analysis, it is essential to design protocols which yield high quality soil DNA of appropriate yield and purity for PCR amplifications. Besides, the selected methods for soil DNA extraction should be cost-effective and time-saving. Effectiveness of soil DNA extraction procedures may be influenced by various parameters such as incomplete cell lysis, DNA sorption to soil surfaces, extraction of humic contaminants, and DNA degradation. Thus, extraction of high molecular weight DNA, proper lysis of microbes, and inhibitor-free DNA are the major requirements for any protocol used for metagenomic study [
For cell lysis to be effective, mechanical treatment should be followed rather than chemical ones [
Soil DNA extraction procedures should therefore be free from PCR inhibitors or their concentration must be low enough so that they do not interfere with the enzymatic reactions. Usually organic matter is the major source of inhibitors that may be coextracted with the microbial DNA present with in the soil. Majorly, humic acids create considerable problem like interference in activity of DNA polymerase used for PCR reactions [
The present study involved comparison of five methods for isolation of soil DNA. Three methods having mannitol in their extraction buffer yielded an amount of DNA that was significantly higher than that obtained with the
Comparison of amount, purity of DNA, and humic acid contamination extracted from various isolation protocols.
DNA extraction protocol | Amount of DNA ( |
|
|
---|---|---|---|
DNA extraction using PEG/NaCl method [ |
0.73 | 1.12 | 1.26 |
DNA extraction using commercial soil DNA extraction kit ( |
0.79 | 1.21 | 1.32 |
DNA extraction by mannitol-PBS-PEG/NaCl method | 2.20 | 1.84 | 1.81 |
DNA extraction by mannitol-PBS-PCI method | 2.36 | 1.93 | 1.84 |
DNA extraction by mannitol-PBS-CTAB method | 2.67 | 2.07 | 1.85 |
The three modified mannitol-based methods led to the recovery of high molecular weight soil DNA (Figure
Visualization of soil DNA extracted by various methods. Lane 1: PEG/NaCl method without liquid nitrogen; lane 2:
High quality PCR amplicons with higher yields were observed in case of these three methods using 16S rRNA-specific and ITS-specific primers (ITS1/ITS4) for bacterial and fungal analysis, respectively. In each case, amplified products corresponded to expected sizes according to primers used. A single amplification product of ~1.2 Kbp for bacteria (Figure
Visualization of PCR amplification products of soil DNA isolated by five different methods using 16S rRNA by different methods. Lane M:
Visualization of PCR amplification products of soil DNA isolated by five different methods using 18S rRNA by different methods. Lane M:
Thus, these methods proved to be a low-cost and practical alternative to accessing metagenomic content by addition of phosphate buffer (PBS) and mannitol within the soil sample.
Varying patterns of RAPD bands were found when soil community DNA samples were amplified using random primers. This indicated that the reported soil DNA extraction methods were quite feasible and reproducible for microbial diversity analysis (Figures
RAPD analysis of soil DNA samples isolated by five methods using random decameric primers. (a) OPA 3 and (b) OPA 13. Lane M:
RAPD analysis of soil DNA samples isolated by five methods using random decameric primers. (a) OPA 15 and (b) OPA 20. Lane M:
Thus, the procedure presented here proves to be an inexpensive procedure, which not only prevents the loss of DNA but also reduces the risk of contamination by laboratory DNA source. The protocols involved the usage of mannitol within the lysis buffer to isolate DNA from bacterial and fungal mycelia. The methods used in the present study exhibited sufficient quality and integrity to amplify the genetic regions, which provided a complete information and understanding of microbial biota. An inclusion of mannitol and sodium chloride promoted cell disruption and extracted humic acid and other organic contaminants, the presence of which would have otherwise inhibited PCR reaction. Hereby, it is demonstrated that a molecular approach using culture independent study can be used to complement more traditional methods used for the survey of microbial communities and provides an expanding toolbox, which helps the soil ecologists and taxonomists to explore microbial communities, which are still unidentified. The modified protocols can also contribute to
In the present study, efficient soil DNA extraction procedures have been reported, which are simple and efficient and do not require elaborate instrumentation and yield good quality DNA suitable for the study of bacterial and fungal genes. It has been demonstrated that an additional step of using phosphate buffer saline with inclusion of mannitol was useful to achieve these objectives. The PCR amplification procedures involve several enzymatic reactions where the enzyme DNA polymerase requires sites, which should be contamination-free. It is suggested that the initial washing with PBS buffer led to removal of unwanted impurities such as humic acid present in the soil. Mannitol, having high salt nature, led the recovery of high molecular weight DNA. It probably interacted with cell wall resulting in cell disruption and extraction of humic acid near the beginning of the isolation procedure. Thus, these modified mannitol-based protocols help not only in improving the yield and quality of extracted soil DNA but also in exploitation of large-scale preparations which provide greater possibility for detecting genes present in low abundance within the soil environment. When combined with the methods developed for normalization of total metagenomic DNA, these modified protocols may offer an easy method for monitoring the population dynamics of the total microbial population in soils over time.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The financial support in the form of research grant from UP State Biodiversity Board, India, is highly acknowledged. The grant of UGC-Maulana Azad National Fellowship is gratefully acknowledged.