Journal of Microbiological Methods 97 (2014) 68–73
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Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction Kalpana Sagar ⁎, Salam Pradeep Singh, Kapil Kumar Goutam, Bolin Kumar Konwar Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 4 November 2013 Accepted 13 November 2013 Available online 23 November 2013 Keywords: Humic substances Chimeric Metagenomic DNA
a b s t r a c t Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was N 23 kb and yield 0.5–5 μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction. © 2013 Elsevier B.V. All rights reserved.
1. Introduction DNA extraction from environmental samples has become an essential tool for constructing metagenomic DNA (mDNA) libraries to reveal the genotypic diversity which requires high quality DNA (Amann et al., 1995; Borneman and Triplett, 1997; Hugenholtz et al., 1998; Stackebrandt et al., 1993; Tiedje et al., 1997; Zhou et al., 1997). It is widely accepted that more than 99% of the microorganisms present in natural environments are not readily cultivable and therefore not accessible for biotechnology or basic research (Torsvik et al., 1990). Although laboratory enrichment culture bears only a limited biodiversity, to overcome the limitation of cultivation methods, several DNA based molecular approaches have been developed to explore the diversity and potential of the microbial communities. However, many workers have attempted to increase DNA quality and yield from soil samples by using severe chemical and physical treatments such as bead beating and sonication to lyse microbial cells. All such treatments caused shearing of DNA making it unsuitable for community analysis based on Taq DNA PCR analysis owing to the risk of forming chimeric products with smaller template DNAs (Liesack et al., 1991; Holben, 1994; Tsai and Olson, 1992; Smalla et al., 1993). Isolation of good quality DNA from contaminated environments is often complicated, as
⁎ Corresponding author. Fax: +91 3712 267005, +91 3712 267006. E-mail address: [email protected] (K. Sagar). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.11.008
polyphenols and polysaccharides present abundantly in such samples which become difficult to eliminate using standard DNA extraction protocols (Porteous and Armstrong, 1991). These compounds co-precipitate with DNA and interfere with subsequent analytical reactions such as enzymatic modification of DNA, PCR analysis and reduction of the transformation efficiency as well as DNA hybridization specificity (Steffen and Atlas, 1988; Tebbe and Vahjen, 1993; Yeates et al., 1998). The removal of humic substances is a critical step following DNA extraction (Rajendhran and Gunasekaran, 2008; Jackson et al., 1997; Rajendhran et al., 2011). However, there are reports of proteinase K and sodium dodecyl sulfate (SDS) reducing the humic acid contamination (Singh et al., 2013). Aromatic compounds such as humic substances and polyphenols from soil samples can be eliminated using cation-exchange resins and detergents (Jacobsen and Rasmussen, 1992), polyvinylpyrrolidone (PVP) (Koonjul et al., 1999), hydroxyapatite (Roh et al., 2005) and activated charcoal (Desai and Madamwar, 2007) but the purification compromises with the yield of quality DNA. For a successful mDNA library construction, humic substancefree cloneable DNA from environmental samples is a prerequisite (Rajendhran and Gunasekaran, 2008). The present study focused on isolation of pure and an optimized DNA yield isolated from soil samples using different methods. Based on comparative study of five different DNA extraction methods, a modified rapid method providing higher yield and quality of mDNA is presented for the extraction, purification and 16S rDNA based polymerase chain reaction and mDNA library construction.
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2. Materials and methods
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Table 2 New M5 method for quality soil DNA extraction modified from the protocol described by Porteous and Armstrong (1991).
2.1. Soil sample collection
Step Procedure
Soil samples were collected from a bakery industry in Tezpur town, Assam, India (26°42′3″N 92°49′49″E). Soil and sediments were homogenized by manual mixing, frozen in liquid nitrogen, transported on dry ice and stored at −20 °C.
1 2 3 4
2.2. Physical and chemical characterization of soil samples
5
Soil samples were air dried, weighed and physical and chemical characterizations were carried out. Soil samples used for particle size analysis were pre-treated with hydrogen peroxide to remove organic materials and then dispersed using sodium hexametaphosphate and sodium carbonate. Wet sieving was carried out to separate the soil particles of N0.060 mm in diameter. The pH of soil was determined in 1:1 (wt/wt) soil–water slurry. The total organic carbon was determined after removing inorganic carbon in 10% HCl followed by boiling and washing with distilled water.
6
7 8 9
10
Weigh 750 mg of soil sample in 2 ml microfuge tube. Add 1 ml of PBS buffer (pH 8.0) to the soil sample. Vortex for 5 min and centrifuge at 3000 × g for 10 min. Supernatant was transferred to 2 ml microfuge tube and 70 μl of lysis buffer (1.5 m NaCl, 0.1 M Na2EDTA, 4%SDS) was added followed by incubation at 72 °C for 45 min. Microfuge the sample at 13,000 × g for 5 min at 4 °C and the supernatant was transferred to a fresh 2 ml centrifuge tube. An aliquot of 100 μl of 6 M potassium acetate and 400 μl of 50% PEG were added to the supernatant and the mixture was allowed to precipitate for 20 min at −20 °C and centrifuged at 4 °C for 5 min. The supernatant was removed and the pellet was air dried. The pellet was dissolved in 500 μl TE buffer (pH:8.0) and then 500 μl of chloroform was added followed by centrifugation at 13,000 × g at 4 °C for 5 min. The chloroform extraction was repeated twice and 500 μl of isopropanol was added to the supernatant and then allowed to precipitate the aqueous DNA for 5 min at 4 °C and again centrifuged at 13,000 × g for 5 min. The DNA pellet was suspended in 100 μl of 1× TE (10 mM Tris–HCl, 1 mM EDTA). Each experiment was performed thrice.
2.3. mDNA extraction mDNA from the soil samples was extracted using five different methods viz. M1, M2, M3, M4 and M5. Methods M1, M2 and M3 were performed as outlined in Table 1. In M4, a commercial miniprep kit, was performed as per manufacturer's instruction (Mobio Ultraclean soil DNA isolation kit). Method M5 is a modification of the protocol described by Porteous and Armstrong (1991) as outlined in Table 2. 2.4. DNA isolation from gram positive and gram negative bacteria using the M5 method Method M5 was used to isolate genomic DNA from Bacillus subtilis and Escherichia coli as representative gram positive and gram negative DNA to validate the utility of the extraction method for cultivable bacteria.
the purified DNA sample was eluted in 100 μl of TE Buffer); iii) MP3: electroelution (Each DNA sample (50 μl) was loaded and resolved in 0.8% agarose. High molecular weight band of mDNA was cut and transferred into a dialysis bag containing 3 volumes of electrophoresis buffer. The DNA was eluted in to the dialysis bag by electrophoresis for 1.5 h. Then the DNA sample was precipitated with isopropanol and washed with 70% ethanol followed by air drying. The sample was suspended in 100 μl TE buffer); iv) MP4 and MP5: agarose gel electrophoresis (electroelution) (MP4) and agarose gel with PVP electrophoresis (electroelution) (MP5) (Humic acid co-migrates with nucleic acid under standard electrophoretic conditions. Addition of PVP to agarose gel halts the co-migration of humic compounds by retarding its electrophoretic mobility. Each DNA sample was loaded on 0.8 % agarose gel containing 2% of PVP). 2.6. Quantification of mDNA and humic acid
2.5. Methods of purification of crude DNA extract The mDNA extracted from soil samples using M3 and M5 was purified following five different methods: i) MP1: Sephadex column purification (Sephadex G-50 slurry was swollen overnight and packed in to spin columns to settle down. Each of the DNA sample (100 μl) to be purified was loaded into the column and kept at room temperature for 5 min and centrifuged at 3000 rpm for 5 min); ii) MP2: silica membrane based spin column purification (commercial kit) (The DNA sample was purified using silica membrane based commercial spin column. Each DNA sample (50 μl) was loaded into the column (Ultraclean soil DNA isolation kit, Mobio, USA). As per manufacturer's instructions the column was kept at room temperature for 5 min and Table 1 Methods used for the isolation of mDNA from soil samples. Method Extraction buffer
Cell lysis
Humic acid removal chemical
M1
SDS
CTAB
SDS, vortex
PVPP
M2 M3
M4 M5
EDTA, CTAB, Tris–HCl, NaCl, NaPO4 [Zhou et al., 1996] NaCl, Tris–HCl, EDTA [Gray and Herwig, 1996] EDTA, NaCl, Tris–HCl [Yeates et al., 1998]
Bead beating, SDS As per MO-Bio kit [Ultraclean soil DNA As per MOkit, MO-Bio, USA] Bio kit EDTA, SDS, NaCl [present study] Vortex, heating
PEG
As per MO-Bio kit PEG
DNA quantification (A260/A280) is commonly performed to determine the average DNA concentration and its purity in a solution. Quant iT Picogreen dsDNA kit (Molecular Probes, USA) was used for the quantification of mDNA as per manufacturer's standard protocol. Fluorescence was measured using Spectra Max fluorescence microplate reader (Molecular devices, USA) at an excitation of 480 nm and emission of 520 nm. Serially diluted λ Phage DNA (1.0–100 ng/ml) was used to prepare the standard curve. The quantification of humic acid was done by absorbance of DNA sample at 340 nm using a spectrophotometer (Thermo Scientific, UV-10, Japan). The concentration of humic acid was calculated based on the standard curve prepared with serial dilution (0.1–100 μg/ml) of commercial humic acid (Merck, India). Humic compounds absorb illumination at 230 nm, protein at 280 nm, and DNA at 260 nm. Therefore, the absorbance ratios at 260/230 nm (DNA/ humic acid) and 260/280 nm (DNA/protein) were used to evaluate the purity of the soil mDNA. 2.7. Polymerase chain reaction (PCR) 16S rRNA gene in the mDNA was amplified using the universal primers to confirm the suitability of mDNA for PCR, restriction digestion and cloning experiments. The forward primer B 27F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and the reverse primer U 1492R (5′ GGT TAC CTT GTT ACG ACT T 3′) were used for PCR amplification. PCR mixture contained 1× PCR buffer, 200 μM of each dNTP, 3.0 μM MgCl2, 0.2 μM of each forward and reverse primer and 2.5 U of Taq DNA polymerase (Sigma, USA) in 50 μl reaction volume. The positive control was taken
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for 5 ng of E. coli (MTCC40) and B. subtilis (MTCC121) genomic DNA and a sample without template was used as negative control. PCR was performed by subjecting a reaction mixture to initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 ° C for 2 min followed by the final extension at 72 °C for 10 min in a thermal cycler (Applied Biosystems). The amplification was determined by electrophoresis of reaction product in 1% agarose gel. 2.8. Restriction digestion by Eco RI, Hind III and Bam HI To examine the suitability of mDNA for restriction digestion, 0.25 μg of each DNA sample was digested separately with 2.5 U of Eco RI, Hind III and Bam HI (MBI Fermentas, Germany) restriction enzymes in a 25 μl reaction mixture. The mixtures were incubated at 37 °C for 4 h followed by inactivation of the restriction enzyme by heating at 70 °C for 10 min. The digested products were resolved on 0.8% agarose gel. 2.9. mDNA library construction Soil mDNA library was constructed using pUC19 as the cloning vector. Purified mDNA was partially digested with Bam HI (MBI Fermentas, Germany) restriction enzyme. The digested product was resolved in 0.8% agarose gel and DNA fragments ranging about 0.5–2.0 kb were fractionated by agarose gel purification using Qiaquick gel extraction kit (Qiagen, Germany). The purified mDNA fragments ranging about ~0.5–5 kb were separated and ligated to Bam HI digested and dephosphorylated pUC 19 cloning vector using T4 DNA ligase (MBI Fermentas, Germany) at 22 °C overnight. The ligated mixture was transformed to E. coli DH10B by electroporation (200 Ω, 25 μF and 2.5 kV) using gene pulser (Biorad, USA). The undigested pUC 19 cloning vector was transferred into DH10B competent cell as positive control to confirm the transformation efficiency. Transformed cells were cultured on LB agar plates supplemented with ampicillin (100 μg/ml), and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (20 μg/ml). The recombinants were scored by blue–white screening after overnight incubation at 37 °C. The resulting library was stored in 15% glycerol at −80 °C.
3.2. Extraction of mDNA from soil The highest DNA yield was obtained in the case of M5, followed by M3 and M2 and the lowest in the case of the M4 sample (Table 3). A260/A230 has been used to evaluate the purity of DNA with respect to the presence of humic substances. Due to the interference, the spectrophotometric quantification A260 values indicate the level of humic acid rather than the concentration of DNA (Jackson et al., 1997). The quantification of humic acid by A230 is also influenced by the concentration of nucleic acids and protein contaminants. Humic acid quantification at A340 could not be affected by the presence of DNA and protein and the measurements were reproducible with humic compounds (concentration 0.1–100 μg/ml). Therefore, in this study linearity of A340 values (range 0.1–100 μg/ml) was observed and the calibration curve was used to measure the humic acid in the mDNA samples. In another approach, the densitometric analysis was used to estimate the DNA concentration in ethidium bromide (EtBr)stained agarose gel. Many studies reported that the DNA concentration determined by EtBr stained agarose gel and A260 values failed to match each other (Steffen and Atlas, 1988). Therefore, spectrophotometric quantification of soil mDNA is challenging due to the presence of polyphenolic compounds. In the present study, the DNA extracted by method M2 showed higher level of fluorescence (Fig. 1) followed by staining with EtBr. However, the DNA concentration was lesser in M2; on other hand, humic acid concentration in M2 sample was high as evident from Table 3 and Fig. 2C. Therefore, the densitometric analysis might also be challenging due to the interference by the humic compounds. Alternatively, the fluorescent dye (PicoGreen and SYBRgreen) offers efficient quantification of mDNA by fluorometric analysis. PicoGreen specifically binds to double stranded DNA and forms DNA–PicoGreen complex. A fluorometer was used to quantify DNA from DNA–PicoGreen complex. The approach could quantify very low DNA concentration from 25 to 1.0 μg/ml. Humic acid concentration higher than 100 ng/μl interferes with PicoGreen fluorescence. However, humic acid concentration below 10 ng/μl does not interfere with DNA quantification and therefore mDNA samples are diluted to a level of 10 ng/μl of humic acid and then quantified using PicoGreen double stranded DNA quantification kit.
3. Results and discussion 3.1. Characterization of soil samples
3.3. DNA extraction with method M5 and comparative study of mDNA isolated by different methods
The physical and chemical properties of soil used in this study were diverse. The soil was classified as granular and sandy-clay on the basis of particle size. The percentages of carbon, hydrogen and nitrogen were 11.8%, 0.75% and 5.37%, respectively. The soil sample collected had a total moisture content of 21.3%. The elemental content of Ca, Fe, K, Mg and Na were 7117, 11978, 7589, 3073 and 4642 μg/g of dry soil, respectively. Soil pH and temperature were 6.3 °C and 28 °C, respectively.
The present laboratory based DNA extraction method i.e. M5 showed crude DNA yield of 3.8 ± 0.04 μg/g soil having a concentration of 36 ± 0.2 ng/μl. The method (M5) also gives a very low humic acid concentration of 8.0 ± 0.13 ng/μl which is the primary goal of the present investigation. Majority of the DNA extraction methods from soil samples resulted to humic acid contamination which hinders the PCR experiment and other processes such as restriction digestion. The present method might overcome the hindrance caused by humic acid
Table 3 Yield, purity and other useful parameters of crude DNA isolated by five different methods. Method Crude DNA yield (μg/g soil)
M1 M2 M3 M4 M5
2.8 3.2 3.8 0.6 3.8
± ± ± ± ±
0.02 0.08 0.02 0.06 0.04
Crude DNA concentration (ng/μl)
3.0 ± 0.04 6.8 ± 0.14 26 ± 0.08 3.0 ± 0.12 36 ± 0.2
Humic acid Optical density at Optical density at Processing concentration (ng/μl) A260/A280 A260/A230 time (h)
72 ± 3.2 86 ± 0.2 62 ± 3.0 4.0 ± 0.02 8.0 ± 0.13
+, methods are showing suitability for PCR and endonuclease activity. −, methods are not showing suitability for PCR and endonuclease activity.
1.3 1.2 1.5 1.6 1.6
0.5 0.2 0.6 0.9 0.8
5 3.5 7 1.5 2.5
16S rDNA amplification (PCR)
Endonuclease activity
− − − −
− − + +
Eco RI
Bam HI
Hind III
− − − + +
− − − + +
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Fig. 1. mDNA extracted from soil samples using different methods: lane M: 1 kb DNA ladder (MBI Fermentas, Germany); lanes 1 to 5: mDNA extracted using methods M1–M5 respectively; lane 6: genomic DNA extracted from E. coli (MTCC 40) using method M5; lane 7: genomic DNA extracted from B. subtilis (MTCC 121) using method M5.
contamination as this method reduces almost 85%–90% of humic acid present in the soil samples. Additionally, the comparative analysis on the crude DNA yield (μg/g soil) by the five different methods (M1–M5) is shown in the form of a bar graph in Fig. 2A. It indicated that method M4 gives the lowest crude DNA yield. Also, from Fig. 2B, it is observed that method M5 accounts for higher concentration of crude DNA concentration (36 ± 0.2 ng/μl) followed by method M3 (26 ± 0.08 ng/μl). Last, from Fig. 2C, method M4 and M5, it is observed that the DNA extraction method using methods M4 and M5 has lower concentration of humic acid, 4.0 ± 0.02 ng/μl and 8.0 ± 0.13 ng/μl for M4 and M5 respectively. Thus, method M5 is found to be the best method for the extraction of mDNA from soil samples with lesser humic acid contamination and higher DNA yield and concentration. Additionally, the authors have also presented the genomic DNA isolated from gram positive and gram negative bacteria using the method (M5) to confirm the suitability of this method in case of cultivable bacteria which is shown in Table 4. It is observed that the method also gives higher concentration in case of gram positive and gram negative bacteria too with 58.85 ng/μl for B. subtilis (MTCC 121) and 73.48 ng/μl for E. coli (MTCC 40). 3.4. Purification of isolated mDNA All extracted mDNA samples were used for 16S rDNA-based amplification and restriction digestion to determine the quality but none was suitable for PCR and/or restriction digestion (Table 5). The PCR amplification of 16S rDNA from mDNA after purification by five different methods is shown in Fig. 3. PCR and restriction digestion involve successive enzymatic reaction (Riesenfeld et al., 2004) and the enzymes require contamination-free sites. However, all extracted DNA samples obtained by using these methods were not pure enough for PCR and restriction digestion. The DNA sample extracted using M4 method (commercial kit) was digested separately with Eco RI, Bam HI and Hind III but none of them was found to be suitable for PCR. Therefore, the DNA extracted by using the commercial kit required further purification for the downstream application for mDNA library construction. In the present investigation various methods employed for mDNA extraction and purification were presented which is on the basis of 16S rDNA based amplification using PCR and restriction digestion by Eco RI, Bam HI and Hind III for further downstream processing
Fig. 2. Bar graph plots on the comparative analysis of the five different DNA extraction methods (M1–M5) showing impact on (A) crude DNA yield, (B) crude DNA concentration, and (C) humic acid concentration.
Table 4 Yield and quality of DNA isolated from gram positive and negative bacteria using the M5 method. Sample
DNA yield (μg/μl)
DNA concentration (ng/μl)
Optical density (A260/A280)
B. subtilis (MTCC 121) E. coli (MTCC 40)
0.14 0.44
58.85 73.48
1.58 1.71
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Table 5 DNA recovery, suitability for PCR and restriction digestion after purification by 5 different methods. Purification method (MP)
MP1 MP2 MP3 MP4 MP5
16S DNA recovery rDNA PCR (%)
Endonuclease activity M3
M5
M3 M5 M3 M5 Eco RI
Bam HI
Hind III
Eco RI
Bam HI
Hind III
96 80 81 40 13
+ − − + −
+ − − + −
+ + + + +
+ + + + +
+ + + + +
98 78 86 41 20
+ − − + −
+ − − + +
+ − − −
+, methods are showing suitability for PCR and endonuclease activity. −, methods are not showing suitability for PCR and endonuclease activity.
application as well as mDNA library construction. DNAs extracted using the methods M3 and M5 were subjected to further purification. The crude DNA extracts from soil samples are usually too impure for molecular analysis and need to be further purified. The purified DNA (method M5) was used for the construction of mDNA library. Several strategies like electroelution, Sephadex column purification and silica membrane based purification were reported to purify mDNA (Rajendhran et al., 2011). No single purification method could be considered as useful for the isolation and purification of mDNA from the different types of soil samples. Addition of polyvinylpyrrolidone (PVP), hexadecyltrimethylammonium bromide (CTAB) and polyethylene glycol (PEG) to the soil buffer slurry before cell lysis minimize co-precipitation of humic substances with DNA (Zhou et al., 1996). In the present study CTAB, PVP and PEG were included in the extraction methods M1, M2 and M3, respectively and among them, humic acid concentration was lesser in M3, where PEG was used in the extraction buffer. DNA samples extracted using these three methods were suitable neither for 16S rDNA-based amplification nor restriction digestion with Eco RI, Bam HI and Hind III. However, DNA extracted using the methods M4 and M5 was suitable for digestion by all 3 restriction enzymes but negative for PCR. Therefore, purification of DNA by these two methods was evaluated using the purification strategies of commercial spin column, gel filtration and electroelution. All these five mDNA extraction and purification methods were evaluated to confirm the suitability of the extracted DNA for subsequent molecular analyses. DNA purification using the method MP1 yielded pure DNA suitable for both PCR and restriction digestion and DNA recovery was also high in Sephadex (G-50) column-based purification. Dijkmans et al. (1993) reported that mDNA purification using Sephadex G-50 was very efficient with minimal loss and the purified DNA was suitable for PCR.
DNA recovery before purification was considered as 100%. The recovery of DNA in MP4 and MP5 was less than 40%. DNA purification using electroelution in the presence of 2% polyvinylpyrrolidone (PVP) was not sufficient to purify DNA due to the low rate of DNA recovery. DNA extracted using commercial kit was not efficient for PCR. Harry et al. (1999) reported that the humic substances might compete with DNA for binding sites during purification using the commercial purification columns. Tiedje et al. (1997) reported that the commercial gel filtration resins failed to revive the inhibitors that came along with soil DNA. Jackson et al. (1997) and Miller (2001) reported superior separation of DNA from humic substances with the use of Sepharose resins. According to Tsai and Olson (1991, 1992) polyacrylamide and dextran gel separated DNA on the basis of size could not remove humic substances from the complex. Hilger and Myrold (1991) reported agarose gel electrophoresis to be more efficient in separating DNA from the humic substances. Jackson et al. (1997) reported a comparison of effectiveness of mDNA isolation using Sepharose 4B, Sephadex G-200 and Sephadex G-50 in the case of different soil types and found Sepharose 4 B to be more effective in providing good separation. Therefore, it could be stated that the purification of mDNA does not depend only on the type of purification strategy used but also on the type of humic substances present in the soil sample. Many of the protocols could be efficient on soil types for which they were developed. 3.5. mDNA library construction A small mDNA library was constructed successfully using the plasmid pUC 19 cloning vector. The transformation efficiency was calculated to be ~106 cells/μg DNA. Here we have calculated the transformation efficiency to confirm the cloning. Further, the recombinants were selected on the basis of blue–white screening after overnight incubation at 37 °C temperature. Therefore, mDNA library was constructed successfully using DNA extracted by method M5 followed by purification using the Sephadex G-50 purification method [MP1]. 4. Conclusion mDNA extraction using methods M4 and M5 were suitable for restriction digestion by Eco RI, Bam HI and Hind III but not for PCR. The extraction methods M3 and M5 followed by Sephadex G-50 purification (M1) were suitable for both restriction digestion and 16S rDNA amplification. In the present investigation, a simple and suitable method was reported with respect to processing time, purity and DNA yield, employing NaCl, SDS and heating at 72 °C temperature to lyse the bacterial cells followed by isopropanol precipitation without using phenol cleanup. Purification of the extracted mDNA using Sephadex G-50 column (MP1 method) yielded higher amount of DNA suitable for further molecular analyses. Therefore, DNA extracted using method M5 (reduction in humic acid), followed by purification (MP1) was successfully used for the construction of mDNA library. Acknowledgements The authors acknowledge Tezpur University and Department of Molecular Biology and Biotechnology, Tezpur University, Assam, India for the necessary support. References
Fig. 3. PCR amplification of 16S rDNA from mDNA after purification by five different methods. Lane M: 1 kb DNA ladder (MBI Fermentas, Germany); lanes 1 and 2, 3 and 4, 5 and 6, and 7 and 8 represent purification by MP1, MP2, MP3 and MP4, respectively. In each pair, M3 and M5 are the respective DNA extraction methods.
Amann, R.I., Lu dwig, W., Schleifer, K., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169. Borneman, J., Triplett, E.W., 1997. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol. 63, 2647–2653. Desai, C., Madamwar, D., 2007. Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments. Bioresour. Technol. 98, 761–768.
K. Sagar et al. / Journal of Microbiological Methods 97 (2014) 68–73 Dijkmans, R., Jagers, A., Kreps, S., Collard, J.M., Mergeay, M., 1993. Rapid method for purification of soil DNA for hybridization and PCR analysis. Microb. Releases 2, 29–34. Gray, J.P., Herwig, R.P., 1996. Phylogenetic analysis of the bacterial communities in marine sediments. Appl. Environ. Microbiol. 62, 4049–4059. Harry, M., Gambier, B., Bourezgui, Y., Garnier-Sillam, E., 1999. Evaluation of purification procedures for DNA extracted from organic rich samples: interference with humic substances. Analusis 27, 439–442. Hilger, A.B., Myrold, D.D., 1991. Method for extraction of Frankia DNA from soil. Agric. Ecosyst. Environ. 34, 107–113. Holben, W.E., 1994. Isolation and purification of bacterial DNA from soil. Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, pp. 727–751. Hugenholtz, P., Goebel, B.M., Pace, N.R., 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 4765–4774. Jackson, C.R., Harper, J.P., Willoughby, D., Rodin, E.E., Churchill, P.F., 1997. A simple, efficient method for the separation of humic substances and DNA from environmental samples. Appl. Environ. Microbiol. 63, 4993–4995. Jacobsen, C.S., Rasmussen, O.F., 1992. Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin. Appl. Environ. Microbiol. 58, 2458–2462. Koonjul, P.K., Brandt, W.F., Farrant, J.M., Lindsey, G.G., 1999. Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA. Nucleic Acids Res. 27, 915–916. Liesack, W., Wryland, H., Stackebrandt, E., 1991. Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed culture of strict barophilic bacteria. Microb. Ecol. 21, 191–198. Miller, D.N., 2001. Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. J. Microbiol. Methods 44, 49–58. Porteous, L.A., Armstrong, J.L., 1991. Recovery of bulk DNA from soil by a rapid, smallscale extraction method. Curr. Microbiol. 22, 345–348. Rajendhran, J., Gunasekaran, P., 2008. Strategies for accessing soil metagenome for desired applications. Biotechnol. Adv. 26, 576–590. Rajendhran, J., Gunasekaran, P., Manjula, A., Sathyavathi, S., 2011. Comparison of seven methods of DNA extraction from termitarium for functional metagenomic DNA library construction. Sci. Ind. Res. 70, 945–951.
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Riesenfeld, C.S., Schloss, P.D., Handelsman, J., 2004. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38, 525–552. Roh, C., Villatte, F., Kim, B.G., Schmid, R.D., 2005. Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples. Electrophoresis 26, 3055–3061. Singh, S.P., Sagar, K., Konwar, B.K., 2013. Strategy in metagenomic DNA isolation and computational studies of humic acid. Curr. Res. Microbiol. Biotechnol. 1, 9–11. Smalla, K., Cresswell, N., Mendonca-Hagler, L.C., Wolters, A., van Elsas, J.D., 1993. Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification. J. Appl. Bacteriol. 74, 78–85. Stackebrandt, E., Liesack, W., Goebel, B.M., 1993. Bacterial diversity in a soil sample from a subtropical Australian environment as determined by SSU rDNA analysis. FASEB J. 7, 232–236. Steffen, R.J., Atlas, R.M., 1988. DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl. Environ. Microbiol. 54, 2185–2191. Tebbe, C.C., Vahjen, W., 1993. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast. Appl. Environ. Microbiol. 59, 2657–2665. Tiedje, J.M., Zhou, J.Z., Nusslein, K., Moyer, C.L., Fulthorpe, R.R., 1997. Extent and patterns of soil microbial diversity. In: Martin, M.T., et al. (Eds.), Progress in Microbial Ecology. Brazilian Society for Microbiology, Sao Paulo, Brazil, pp. 35–41. Torsvik, V., Goksoyr, J., Daae, F.L., 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782–787. Tsai, Y.L., Olson, B.H., 1992. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl. Environ. Microbiol. 58, 2292–2295. Tsai, Y., Olson, B.H., 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57, 1070–1074. Yeates, C., Gillings, M.R., Davison, A.D., Altavilla, N., Veal, D.A., 1998. Methods for microbial DNA extraction from soil for PCR amplification. Biol. Proced. Online 1, 40–47. Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soil of diverse composition. Appl. Environ. Microbiol. 62, 316–322. Zhou, J.Z., Davey, M.E., Figueras, J.B., Rivkina, E., Gilichinsky, D., Tiedje, J.M., 1997. Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA. Microbiology 143, 3913–3919.
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction Kalpana Sagar ⁎, Salam Pradeep Singh, Kapil Kumar Goutam, Bolin Kumar Konwar Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India
a r t i c l e
i n f o
Article history: Received 27 June 2013 Received in revised form 4 November 2013 Accepted 13 November 2013 Available online 23 November 2013 Keywords: Humic substances Chimeric Metagenomic DNA
a b s t r a c t Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was N 23 kb and yield 0.5–5 μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction. © 2013 Elsevier B.V. All rights reserved.
1. Introduction DNA extraction from environmental samples has become an essential tool for constructing metagenomic DNA (mDNA) libraries to reveal the genotypic diversity which requires high quality DNA (Amann et al., 1995; Borneman and Triplett, 1997; Hugenholtz et al., 1998; Stackebrandt et al., 1993; Tiedje et al., 1997; Zhou et al., 1997). It is widely accepted that more than 99% of the microorganisms present in natural environments are not readily cultivable and therefore not accessible for biotechnology or basic research (Torsvik et al., 1990). Although laboratory enrichment culture bears only a limited biodiversity, to overcome the limitation of cultivation methods, several DNA based molecular approaches have been developed to explore the diversity and potential of the microbial communities. However, many workers have attempted to increase DNA quality and yield from soil samples by using severe chemical and physical treatments such as bead beating and sonication to lyse microbial cells. All such treatments caused shearing of DNA making it unsuitable for community analysis based on Taq DNA PCR analysis owing to the risk of forming chimeric products with smaller template DNAs (Liesack et al., 1991; Holben, 1994; Tsai and Olson, 1992; Smalla et al., 1993). Isolation of good quality DNA from contaminated environments is often complicated, as
⁎ Corresponding author. Fax: +91 3712 267005, +91 3712 267006. E-mail address: [email protected] (K. Sagar). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.11.008
polyphenols and polysaccharides present abundantly in such samples which become difficult to eliminate using standard DNA extraction protocols (Porteous and Armstrong, 1991). These compounds co-precipitate with DNA and interfere with subsequent analytical reactions such as enzymatic modification of DNA, PCR analysis and reduction of the transformation efficiency as well as DNA hybridization specificity (Steffen and Atlas, 1988; Tebbe and Vahjen, 1993; Yeates et al., 1998). The removal of humic substances is a critical step following DNA extraction (Rajendhran and Gunasekaran, 2008; Jackson et al., 1997; Rajendhran et al., 2011). However, there are reports of proteinase K and sodium dodecyl sulfate (SDS) reducing the humic acid contamination (Singh et al., 2013). Aromatic compounds such as humic substances and polyphenols from soil samples can be eliminated using cation-exchange resins and detergents (Jacobsen and Rasmussen, 1992), polyvinylpyrrolidone (PVP) (Koonjul et al., 1999), hydroxyapatite (Roh et al., 2005) and activated charcoal (Desai and Madamwar, 2007) but the purification compromises with the yield of quality DNA. For a successful mDNA library construction, humic substancefree cloneable DNA from environmental samples is a prerequisite (Rajendhran and Gunasekaran, 2008). The present study focused on isolation of pure and an optimized DNA yield isolated from soil samples using different methods. Based on comparative study of five different DNA extraction methods, a modified rapid method providing higher yield and quality of mDNA is presented for the extraction, purification and 16S rDNA based polymerase chain reaction and mDNA library construction.
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2. Materials and methods
69
Table 2 New M5 method for quality soil DNA extraction modified from the protocol described by Porteous and Armstrong (1991).
2.1. Soil sample collection
Step Procedure
Soil samples were collected from a bakery industry in Tezpur town, Assam, India (26°42′3″N 92°49′49″E). Soil and sediments were homogenized by manual mixing, frozen in liquid nitrogen, transported on dry ice and stored at −20 °C.
1 2 3 4
2.2. Physical and chemical characterization of soil samples
5
Soil samples were air dried, weighed and physical and chemical characterizations were carried out. Soil samples used for particle size analysis were pre-treated with hydrogen peroxide to remove organic materials and then dispersed using sodium hexametaphosphate and sodium carbonate. Wet sieving was carried out to separate the soil particles of N0.060 mm in diameter. The pH of soil was determined in 1:1 (wt/wt) soil–water slurry. The total organic carbon was determined after removing inorganic carbon in 10% HCl followed by boiling and washing with distilled water.
6
7 8 9
10
Weigh 750 mg of soil sample in 2 ml microfuge tube. Add 1 ml of PBS buffer (pH 8.0) to the soil sample. Vortex for 5 min and centrifuge at 3000 × g for 10 min. Supernatant was transferred to 2 ml microfuge tube and 70 μl of lysis buffer (1.5 m NaCl, 0.1 M Na2EDTA, 4%SDS) was added followed by incubation at 72 °C for 45 min. Microfuge the sample at 13,000 × g for 5 min at 4 °C and the supernatant was transferred to a fresh 2 ml centrifuge tube. An aliquot of 100 μl of 6 M potassium acetate and 400 μl of 50% PEG were added to the supernatant and the mixture was allowed to precipitate for 20 min at −20 °C and centrifuged at 4 °C for 5 min. The supernatant was removed and the pellet was air dried. The pellet was dissolved in 500 μl TE buffer (pH:8.0) and then 500 μl of chloroform was added followed by centrifugation at 13,000 × g at 4 °C for 5 min. The chloroform extraction was repeated twice and 500 μl of isopropanol was added to the supernatant and then allowed to precipitate the aqueous DNA for 5 min at 4 °C and again centrifuged at 13,000 × g for 5 min. The DNA pellet was suspended in 100 μl of 1× TE (10 mM Tris–HCl, 1 mM EDTA). Each experiment was performed thrice.
2.3. mDNA extraction mDNA from the soil samples was extracted using five different methods viz. M1, M2, M3, M4 and M5. Methods M1, M2 and M3 were performed as outlined in Table 1. In M4, a commercial miniprep kit, was performed as per manufacturer's instruction (Mobio Ultraclean soil DNA isolation kit). Method M5 is a modification of the protocol described by Porteous and Armstrong (1991) as outlined in Table 2. 2.4. DNA isolation from gram positive and gram negative bacteria using the M5 method Method M5 was used to isolate genomic DNA from Bacillus subtilis and Escherichia coli as representative gram positive and gram negative DNA to validate the utility of the extraction method for cultivable bacteria.
the purified DNA sample was eluted in 100 μl of TE Buffer); iii) MP3: electroelution (Each DNA sample (50 μl) was loaded and resolved in 0.8% agarose. High molecular weight band of mDNA was cut and transferred into a dialysis bag containing 3 volumes of electrophoresis buffer. The DNA was eluted in to the dialysis bag by electrophoresis for 1.5 h. Then the DNA sample was precipitated with isopropanol and washed with 70% ethanol followed by air drying. The sample was suspended in 100 μl TE buffer); iv) MP4 and MP5: agarose gel electrophoresis (electroelution) (MP4) and agarose gel with PVP electrophoresis (electroelution) (MP5) (Humic acid co-migrates with nucleic acid under standard electrophoretic conditions. Addition of PVP to agarose gel halts the co-migration of humic compounds by retarding its electrophoretic mobility. Each DNA sample was loaded on 0.8 % agarose gel containing 2% of PVP). 2.6. Quantification of mDNA and humic acid
2.5. Methods of purification of crude DNA extract The mDNA extracted from soil samples using M3 and M5 was purified following five different methods: i) MP1: Sephadex column purification (Sephadex G-50 slurry was swollen overnight and packed in to spin columns to settle down. Each of the DNA sample (100 μl) to be purified was loaded into the column and kept at room temperature for 5 min and centrifuged at 3000 rpm for 5 min); ii) MP2: silica membrane based spin column purification (commercial kit) (The DNA sample was purified using silica membrane based commercial spin column. Each DNA sample (50 μl) was loaded into the column (Ultraclean soil DNA isolation kit, Mobio, USA). As per manufacturer's instructions the column was kept at room temperature for 5 min and Table 1 Methods used for the isolation of mDNA from soil samples. Method Extraction buffer
Cell lysis
Humic acid removal chemical
M1
SDS
CTAB
SDS, vortex
PVPP
M2 M3
M4 M5
EDTA, CTAB, Tris–HCl, NaCl, NaPO4 [Zhou et al., 1996] NaCl, Tris–HCl, EDTA [Gray and Herwig, 1996] EDTA, NaCl, Tris–HCl [Yeates et al., 1998]
Bead beating, SDS As per MO-Bio kit [Ultraclean soil DNA As per MOkit, MO-Bio, USA] Bio kit EDTA, SDS, NaCl [present study] Vortex, heating
PEG
As per MO-Bio kit PEG
DNA quantification (A260/A280) is commonly performed to determine the average DNA concentration and its purity in a solution. Quant iT Picogreen dsDNA kit (Molecular Probes, USA) was used for the quantification of mDNA as per manufacturer's standard protocol. Fluorescence was measured using Spectra Max fluorescence microplate reader (Molecular devices, USA) at an excitation of 480 nm and emission of 520 nm. Serially diluted λ Phage DNA (1.0–100 ng/ml) was used to prepare the standard curve. The quantification of humic acid was done by absorbance of DNA sample at 340 nm using a spectrophotometer (Thermo Scientific, UV-10, Japan). The concentration of humic acid was calculated based on the standard curve prepared with serial dilution (0.1–100 μg/ml) of commercial humic acid (Merck, India). Humic compounds absorb illumination at 230 nm, protein at 280 nm, and DNA at 260 nm. Therefore, the absorbance ratios at 260/230 nm (DNA/ humic acid) and 260/280 nm (DNA/protein) were used to evaluate the purity of the soil mDNA. 2.7. Polymerase chain reaction (PCR) 16S rRNA gene in the mDNA was amplified using the universal primers to confirm the suitability of mDNA for PCR, restriction digestion and cloning experiments. The forward primer B 27F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and the reverse primer U 1492R (5′ GGT TAC CTT GTT ACG ACT T 3′) were used for PCR amplification. PCR mixture contained 1× PCR buffer, 200 μM of each dNTP, 3.0 μM MgCl2, 0.2 μM of each forward and reverse primer and 2.5 U of Taq DNA polymerase (Sigma, USA) in 50 μl reaction volume. The positive control was taken
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for 5 ng of E. coli (MTCC40) and B. subtilis (MTCC121) genomic DNA and a sample without template was used as negative control. PCR was performed by subjecting a reaction mixture to initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 ° C for 2 min followed by the final extension at 72 °C for 10 min in a thermal cycler (Applied Biosystems). The amplification was determined by electrophoresis of reaction product in 1% agarose gel. 2.8. Restriction digestion by Eco RI, Hind III and Bam HI To examine the suitability of mDNA for restriction digestion, 0.25 μg of each DNA sample was digested separately with 2.5 U of Eco RI, Hind III and Bam HI (MBI Fermentas, Germany) restriction enzymes in a 25 μl reaction mixture. The mixtures were incubated at 37 °C for 4 h followed by inactivation of the restriction enzyme by heating at 70 °C for 10 min. The digested products were resolved on 0.8% agarose gel. 2.9. mDNA library construction Soil mDNA library was constructed using pUC19 as the cloning vector. Purified mDNA was partially digested with Bam HI (MBI Fermentas, Germany) restriction enzyme. The digested product was resolved in 0.8% agarose gel and DNA fragments ranging about 0.5–2.0 kb were fractionated by agarose gel purification using Qiaquick gel extraction kit (Qiagen, Germany). The purified mDNA fragments ranging about ~0.5–5 kb were separated and ligated to Bam HI digested and dephosphorylated pUC 19 cloning vector using T4 DNA ligase (MBI Fermentas, Germany) at 22 °C overnight. The ligated mixture was transformed to E. coli DH10B by electroporation (200 Ω, 25 μF and 2.5 kV) using gene pulser (Biorad, USA). The undigested pUC 19 cloning vector was transferred into DH10B competent cell as positive control to confirm the transformation efficiency. Transformed cells were cultured on LB agar plates supplemented with ampicillin (100 μg/ml), and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (20 μg/ml). The recombinants were scored by blue–white screening after overnight incubation at 37 °C. The resulting library was stored in 15% glycerol at −80 °C.
3.2. Extraction of mDNA from soil The highest DNA yield was obtained in the case of M5, followed by M3 and M2 and the lowest in the case of the M4 sample (Table 3). A260/A230 has been used to evaluate the purity of DNA with respect to the presence of humic substances. Due to the interference, the spectrophotometric quantification A260 values indicate the level of humic acid rather than the concentration of DNA (Jackson et al., 1997). The quantification of humic acid by A230 is also influenced by the concentration of nucleic acids and protein contaminants. Humic acid quantification at A340 could not be affected by the presence of DNA and protein and the measurements were reproducible with humic compounds (concentration 0.1–100 μg/ml). Therefore, in this study linearity of A340 values (range 0.1–100 μg/ml) was observed and the calibration curve was used to measure the humic acid in the mDNA samples. In another approach, the densitometric analysis was used to estimate the DNA concentration in ethidium bromide (EtBr)stained agarose gel. Many studies reported that the DNA concentration determined by EtBr stained agarose gel and A260 values failed to match each other (Steffen and Atlas, 1988). Therefore, spectrophotometric quantification of soil mDNA is challenging due to the presence of polyphenolic compounds. In the present study, the DNA extracted by method M2 showed higher level of fluorescence (Fig. 1) followed by staining with EtBr. However, the DNA concentration was lesser in M2; on other hand, humic acid concentration in M2 sample was high as evident from Table 3 and Fig. 2C. Therefore, the densitometric analysis might also be challenging due to the interference by the humic compounds. Alternatively, the fluorescent dye (PicoGreen and SYBRgreen) offers efficient quantification of mDNA by fluorometric analysis. PicoGreen specifically binds to double stranded DNA and forms DNA–PicoGreen complex. A fluorometer was used to quantify DNA from DNA–PicoGreen complex. The approach could quantify very low DNA concentration from 25 to 1.0 μg/ml. Humic acid concentration higher than 100 ng/μl interferes with PicoGreen fluorescence. However, humic acid concentration below 10 ng/μl does not interfere with DNA quantification and therefore mDNA samples are diluted to a level of 10 ng/μl of humic acid and then quantified using PicoGreen double stranded DNA quantification kit.
3. Results and discussion 3.1. Characterization of soil samples
3.3. DNA extraction with method M5 and comparative study of mDNA isolated by different methods
The physical and chemical properties of soil used in this study were diverse. The soil was classified as granular and sandy-clay on the basis of particle size. The percentages of carbon, hydrogen and nitrogen were 11.8%, 0.75% and 5.37%, respectively. The soil sample collected had a total moisture content of 21.3%. The elemental content of Ca, Fe, K, Mg and Na were 7117, 11978, 7589, 3073 and 4642 μg/g of dry soil, respectively. Soil pH and temperature were 6.3 °C and 28 °C, respectively.
The present laboratory based DNA extraction method i.e. M5 showed crude DNA yield of 3.8 ± 0.04 μg/g soil having a concentration of 36 ± 0.2 ng/μl. The method (M5) also gives a very low humic acid concentration of 8.0 ± 0.13 ng/μl which is the primary goal of the present investigation. Majority of the DNA extraction methods from soil samples resulted to humic acid contamination which hinders the PCR experiment and other processes such as restriction digestion. The present method might overcome the hindrance caused by humic acid
Table 3 Yield, purity and other useful parameters of crude DNA isolated by five different methods. Method Crude DNA yield (μg/g soil)
M1 M2 M3 M4 M5
2.8 3.2 3.8 0.6 3.8
± ± ± ± ±
0.02 0.08 0.02 0.06 0.04
Crude DNA concentration (ng/μl)
3.0 ± 0.04 6.8 ± 0.14 26 ± 0.08 3.0 ± 0.12 36 ± 0.2
Humic acid Optical density at Optical density at Processing concentration (ng/μl) A260/A280 A260/A230 time (h)
72 ± 3.2 86 ± 0.2 62 ± 3.0 4.0 ± 0.02 8.0 ± 0.13
+, methods are showing suitability for PCR and endonuclease activity. −, methods are not showing suitability for PCR and endonuclease activity.
1.3 1.2 1.5 1.6 1.6
0.5 0.2 0.6 0.9 0.8
5 3.5 7 1.5 2.5
16S rDNA amplification (PCR)
Endonuclease activity
− − − −
− − + +
Eco RI
Bam HI
Hind III
− − − + +
− − − + +
K. Sagar et al. / Journal of Microbiological Methods 97 (2014) 68–73
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Fig. 1. mDNA extracted from soil samples using different methods: lane M: 1 kb DNA ladder (MBI Fermentas, Germany); lanes 1 to 5: mDNA extracted using methods M1–M5 respectively; lane 6: genomic DNA extracted from E. coli (MTCC 40) using method M5; lane 7: genomic DNA extracted from B. subtilis (MTCC 121) using method M5.
contamination as this method reduces almost 85%–90% of humic acid present in the soil samples. Additionally, the comparative analysis on the crude DNA yield (μg/g soil) by the five different methods (M1–M5) is shown in the form of a bar graph in Fig. 2A. It indicated that method M4 gives the lowest crude DNA yield. Also, from Fig. 2B, it is observed that method M5 accounts for higher concentration of crude DNA concentration (36 ± 0.2 ng/μl) followed by method M3 (26 ± 0.08 ng/μl). Last, from Fig. 2C, method M4 and M5, it is observed that the DNA extraction method using methods M4 and M5 has lower concentration of humic acid, 4.0 ± 0.02 ng/μl and 8.0 ± 0.13 ng/μl for M4 and M5 respectively. Thus, method M5 is found to be the best method for the extraction of mDNA from soil samples with lesser humic acid contamination and higher DNA yield and concentration. Additionally, the authors have also presented the genomic DNA isolated from gram positive and gram negative bacteria using the method (M5) to confirm the suitability of this method in case of cultivable bacteria which is shown in Table 4. It is observed that the method also gives higher concentration in case of gram positive and gram negative bacteria too with 58.85 ng/μl for B. subtilis (MTCC 121) and 73.48 ng/μl for E. coli (MTCC 40). 3.4. Purification of isolated mDNA All extracted mDNA samples were used for 16S rDNA-based amplification and restriction digestion to determine the quality but none was suitable for PCR and/or restriction digestion (Table 5). The PCR amplification of 16S rDNA from mDNA after purification by five different methods is shown in Fig. 3. PCR and restriction digestion involve successive enzymatic reaction (Riesenfeld et al., 2004) and the enzymes require contamination-free sites. However, all extracted DNA samples obtained by using these methods were not pure enough for PCR and restriction digestion. The DNA sample extracted using M4 method (commercial kit) was digested separately with Eco RI, Bam HI and Hind III but none of them was found to be suitable for PCR. Therefore, the DNA extracted by using the commercial kit required further purification for the downstream application for mDNA library construction. In the present investigation various methods employed for mDNA extraction and purification were presented which is on the basis of 16S rDNA based amplification using PCR and restriction digestion by Eco RI, Bam HI and Hind III for further downstream processing
Fig. 2. Bar graph plots on the comparative analysis of the five different DNA extraction methods (M1–M5) showing impact on (A) crude DNA yield, (B) crude DNA concentration, and (C) humic acid concentration.
Table 4 Yield and quality of DNA isolated from gram positive and negative bacteria using the M5 method. Sample
DNA yield (μg/μl)
DNA concentration (ng/μl)
Optical density (A260/A280)
B. subtilis (MTCC 121) E. coli (MTCC 40)
0.14 0.44
58.85 73.48
1.58 1.71
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Table 5 DNA recovery, suitability for PCR and restriction digestion after purification by 5 different methods. Purification method (MP)
MP1 MP2 MP3 MP4 MP5
16S DNA recovery rDNA PCR (%)
Endonuclease activity M3
M5
M3 M5 M3 M5 Eco RI
Bam HI
Hind III
Eco RI
Bam HI
Hind III
96 80 81 40 13
+ − − + −
+ − − + −
+ + + + +
+ + + + +
+ + + + +
98 78 86 41 20
+ − − + −
+ − − + +
+ − − −
+, methods are showing suitability for PCR and endonuclease activity. −, methods are not showing suitability for PCR and endonuclease activity.
application as well as mDNA library construction. DNAs extracted using the methods M3 and M5 were subjected to further purification. The crude DNA extracts from soil samples are usually too impure for molecular analysis and need to be further purified. The purified DNA (method M5) was used for the construction of mDNA library. Several strategies like electroelution, Sephadex column purification and silica membrane based purification were reported to purify mDNA (Rajendhran et al., 2011). No single purification method could be considered as useful for the isolation and purification of mDNA from the different types of soil samples. Addition of polyvinylpyrrolidone (PVP), hexadecyltrimethylammonium bromide (CTAB) and polyethylene glycol (PEG) to the soil buffer slurry before cell lysis minimize co-precipitation of humic substances with DNA (Zhou et al., 1996). In the present study CTAB, PVP and PEG were included in the extraction methods M1, M2 and M3, respectively and among them, humic acid concentration was lesser in M3, where PEG was used in the extraction buffer. DNA samples extracted using these three methods were suitable neither for 16S rDNA-based amplification nor restriction digestion with Eco RI, Bam HI and Hind III. However, DNA extracted using the methods M4 and M5 was suitable for digestion by all 3 restriction enzymes but negative for PCR. Therefore, purification of DNA by these two methods was evaluated using the purification strategies of commercial spin column, gel filtration and electroelution. All these five mDNA extraction and purification methods were evaluated to confirm the suitability of the extracted DNA for subsequent molecular analyses. DNA purification using the method MP1 yielded pure DNA suitable for both PCR and restriction digestion and DNA recovery was also high in Sephadex (G-50) column-based purification. Dijkmans et al. (1993) reported that mDNA purification using Sephadex G-50 was very efficient with minimal loss and the purified DNA was suitable for PCR.
DNA recovery before purification was considered as 100%. The recovery of DNA in MP4 and MP5 was less than 40%. DNA purification using electroelution in the presence of 2% polyvinylpyrrolidone (PVP) was not sufficient to purify DNA due to the low rate of DNA recovery. DNA extracted using commercial kit was not efficient for PCR. Harry et al. (1999) reported that the humic substances might compete with DNA for binding sites during purification using the commercial purification columns. Tiedje et al. (1997) reported that the commercial gel filtration resins failed to revive the inhibitors that came along with soil DNA. Jackson et al. (1997) and Miller (2001) reported superior separation of DNA from humic substances with the use of Sepharose resins. According to Tsai and Olson (1991, 1992) polyacrylamide and dextran gel separated DNA on the basis of size could not remove humic substances from the complex. Hilger and Myrold (1991) reported agarose gel electrophoresis to be more efficient in separating DNA from the humic substances. Jackson et al. (1997) reported a comparison of effectiveness of mDNA isolation using Sepharose 4B, Sephadex G-200 and Sephadex G-50 in the case of different soil types and found Sepharose 4 B to be more effective in providing good separation. Therefore, it could be stated that the purification of mDNA does not depend only on the type of purification strategy used but also on the type of humic substances present in the soil sample. Many of the protocols could be efficient on soil types for which they were developed. 3.5. mDNA library construction A small mDNA library was constructed successfully using the plasmid pUC 19 cloning vector. The transformation efficiency was calculated to be ~106 cells/μg DNA. Here we have calculated the transformation efficiency to confirm the cloning. Further, the recombinants were selected on the basis of blue–white screening after overnight incubation at 37 °C temperature. Therefore, mDNA library was constructed successfully using DNA extracted by method M5 followed by purification using the Sephadex G-50 purification method [MP1]. 4. Conclusion mDNA extraction using methods M4 and M5 were suitable for restriction digestion by Eco RI, Bam HI and Hind III but not for PCR. The extraction methods M3 and M5 followed by Sephadex G-50 purification (M1) were suitable for both restriction digestion and 16S rDNA amplification. In the present investigation, a simple and suitable method was reported with respect to processing time, purity and DNA yield, employing NaCl, SDS and heating at 72 °C temperature to lyse the bacterial cells followed by isopropanol precipitation without using phenol cleanup. Purification of the extracted mDNA using Sephadex G-50 column (MP1 method) yielded higher amount of DNA suitable for further molecular analyses. Therefore, DNA extracted using method M5 (reduction in humic acid), followed by purification (MP1) was successfully used for the construction of mDNA library. Acknowledgements The authors acknowledge Tezpur University and Department of Molecular Biology and Biotechnology, Tezpur University, Assam, India for the necessary support. References
Fig. 3. PCR amplification of 16S rDNA from mDNA after purification by five different methods. Lane M: 1 kb DNA ladder (MBI Fermentas, Germany); lanes 1 and 2, 3 and 4, 5 and 6, and 7 and 8 represent purification by MP1, MP2, MP3 and MP4, respectively. In each pair, M3 and M5 are the respective DNA extraction methods.
Amann, R.I., Lu dwig, W., Schleifer, K., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169. Borneman, J., Triplett, E.W., 1997. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol. 63, 2647–2653. Desai, C., Madamwar, D., 2007. Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments. Bioresour. Technol. 98, 761–768.
K. Sagar et al. / Journal of Microbiological Methods 97 (2014) 68–73 Dijkmans, R., Jagers, A., Kreps, S., Collard, J.M., Mergeay, M., 1993. Rapid method for purification of soil DNA for hybridization and PCR analysis. Microb. Releases 2, 29–34. Gray, J.P., Herwig, R.P., 1996. Phylogenetic analysis of the bacterial communities in marine sediments. Appl. Environ. Microbiol. 62, 4049–4059. Harry, M., Gambier, B., Bourezgui, Y., Garnier-Sillam, E., 1999. Evaluation of purification procedures for DNA extracted from organic rich samples: interference with humic substances. Analusis 27, 439–442. Hilger, A.B., Myrold, D.D., 1991. Method for extraction of Frankia DNA from soil. Agric. Ecosyst. Environ. 34, 107–113. Holben, W.E., 1994. Isolation and purification of bacterial DNA from soil. Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, pp. 727–751. Hugenholtz, P., Goebel, B.M., Pace, N.R., 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 4765–4774. Jackson, C.R., Harper, J.P., Willoughby, D., Rodin, E.E., Churchill, P.F., 1997. A simple, efficient method for the separation of humic substances and DNA from environmental samples. Appl. Environ. Microbiol. 63, 4993–4995. Jacobsen, C.S., Rasmussen, O.F., 1992. Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin. Appl. Environ. Microbiol. 58, 2458–2462. Koonjul, P.K., Brandt, W.F., Farrant, J.M., Lindsey, G.G., 1999. Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA. Nucleic Acids Res. 27, 915–916. Liesack, W., Wryland, H., Stackebrandt, E., 1991. Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed culture of strict barophilic bacteria. Microb. Ecol. 21, 191–198. Miller, D.N., 2001. Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments. J. Microbiol. Methods 44, 49–58. Porteous, L.A., Armstrong, J.L., 1991. Recovery of bulk DNA from soil by a rapid, smallscale extraction method. Curr. Microbiol. 22, 345–348. Rajendhran, J., Gunasekaran, P., 2008. Strategies for accessing soil metagenome for desired applications. Biotechnol. Adv. 26, 576–590. Rajendhran, J., Gunasekaran, P., Manjula, A., Sathyavathi, S., 2011. Comparison of seven methods of DNA extraction from termitarium for functional metagenomic DNA library construction. Sci. Ind. Res. 70, 945–951.
73
Riesenfeld, C.S., Schloss, P.D., Handelsman, J., 2004. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38, 525–552. Roh, C., Villatte, F., Kim, B.G., Schmid, R.D., 2005. Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples. Electrophoresis 26, 3055–3061. Singh, S.P., Sagar, K., Konwar, B.K., 2013. Strategy in metagenomic DNA isolation and computational studies of humic acid. Curr. Res. Microbiol. Biotechnol. 1, 9–11. Smalla, K., Cresswell, N., Mendonca-Hagler, L.C., Wolters, A., van Elsas, J.D., 1993. Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification. J. Appl. Bacteriol. 74, 78–85. Stackebrandt, E., Liesack, W., Goebel, B.M., 1993. Bacterial diversity in a soil sample from a subtropical Australian environment as determined by SSU rDNA analysis. FASEB J. 7, 232–236. Steffen, R.J., Atlas, R.M., 1988. DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl. Environ. Microbiol. 54, 2185–2191. Tebbe, C.C., Vahjen, W., 1993. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast. Appl. Environ. Microbiol. 59, 2657–2665. Tiedje, J.M., Zhou, J.Z., Nusslein, K., Moyer, C.L., Fulthorpe, R.R., 1997. Extent and patterns of soil microbial diversity. In: Martin, M.T., et al. (Eds.), Progress in Microbial Ecology. Brazilian Society for Microbiology, Sao Paulo, Brazil, pp. 35–41. Torsvik, V., Goksoyr, J., Daae, F.L., 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56, 782–787. Tsai, Y.L., Olson, B.H., 1992. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl. Environ. Microbiol. 58, 2292–2295. Tsai, Y., Olson, B.H., 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57, 1070–1074. Yeates, C., Gillings, M.R., Davison, A.D., Altavilla, N., Veal, D.A., 1998. Methods for microbial DNA extraction from soil for PCR amplification. Biol. Proced. Online 1, 40–47. Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soil of diverse composition. Appl. Environ. Microbiol. 62, 316–322. Zhou, J.Z., Davey, M.E., Figueras, J.B., Rivkina, E., Gilichinsky, D., Tiedje, J.M., 1997. Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA. Microbiology 143, 3913–3919.
