*Author for correspondence E-mail: jrajendhran@gmail.com
Comparison of seven methods of DNA extraction from termitarium for functional metagenomic DNA library construction
A Manjula, S Sathyavathi, P Gunasekaran and J Rajendhran*
Department of Genetics, School of Biological Sciences, Madurai Kamaraj University (MKU), Madurai 625 021, India Received 18 February 2011; revised 12 September 2011; accepted 14 September 2011
This study evaluated seven methods, used to isolate metagenomic DNA from termitarium, on the basis of processing time, DNA yield, purity and suitability for PCR and restriction digestion. None of the extracted DNA sample was suitable for PCR and restriction digestion. Based on higher DNA yield and lower levels of humic acids, DNA extracted using three methods were further subjected to purification. Five methods using three approaches (electro elution, silica membrane based spin column purification and gel filtration) were evaluated for purification. The simple method, employing SDS to lyse cells in situ followed by ethanol precipitation (E7) and subsequent purification using Sephadex G-50 (P5) resin yielded high concentration of cloneable DNA from termitarium sample, has been successfully used for the construction of metagenomic DNA library.
Keywords: Cloneablity, DNA purification, Humic acid, Metagenomic DNA, Termitarium
Introduction
Termites are successful group of wood degrading organisms and considered as the potential sources of catalysts for converting lignocellulose into biofuels1. Termites feed on cellulosic materials in the form of wood, leaf litter, soil, etc. and rely on symbiotic bacteria that produce cellulases for digestion2,3. Termitarium is the nest of termites built using degraded wooden particles, saliva, faeces and soil. Microbial community present in termitarium, which is a combination of all these sources, can serve as potential source of lignocellulolytic genes.
Estimates based on rRNA sequence analysis indicate that only 1% or lesser than that of total microbial community from environmental sources could be cultured in the laboratory4. Though there are quite a few reports on insect gut metagenomics5, there are no reports on the metagenomics of termitarium. Isolation of DNA from environmental samples is termed as metagenomic DNA (mDNA), which is used to construct metagenomic library using suitable vector/host systems4. However, obtaining unbiased total community DNA from any environmental samples remains a challenge6. Several protocols have been reported for mDNA extraction and purification from soil and other environmental sources7. For a successful
metagenomic library construction, extraction of contaminant free cloneable DNA from environmental samples is a prerequisite8.
This study presents cloneable mDNA from termitarium sample. Based on comparative evaluation of seven different extraction methods, a modified rapid method that gives higher yield and purity of mDNA is presented for the extraction and purification of mDNA from termitarium.
Experimental Section
Termitarium samples were collected from MKU campus, Madurai, India. Each sample was sieved to remove solid wooden particles and stored at −20°C. For comparative analysis, same termitarium sample was used for mDNA extraction using seven methods.
Isolation of Metagenomic DNA (mDNA)
Methods 1 to 5 (Table 1) were performed as reported9-11. Method 6, a commercial miniprep kit, was performed as per manufacturer’s instruction (Mobio Ultraclean soil DNA isolation kit). In Method 7 (modified Yeates et al12 method), to termitarium sample (0.5 mg), 1 ml of extraction buffer (100 mM Tris-Hcl, 100 mM Na EDTA, 1.6 M NaCl) was added and vortexed vigorously for 2 min. To this slurry, 100 µl of 20% SDS and 100 µl proteinase K (10 mg/ml) were added and mixture was
Qiagen, Germany; Ultraclean soil DNA isolation kit, Mobio, USA) as per the manufacturer’s instructions.
Purified DNA sample was eluted in 50 µl of TE buffer.
Sephadex Column Purification
Sephadex G-50 slurry swollen overnight was packed into spin columns and allowed to settle for 5 min. DNA sample (50 µl) to be purified was loaded onto the column and kept at room temperature (RT) for 5 min and centrifuged at 2500 rpm for 2 min.
Quantification of DNA and Humic Acid
Quantification of mDNA was performed using Quant iTPicogreen dsDNA kit (Molecular Probes, USA) according to the manufacturer’s instruction.
Fluorescence was measured at an excitation of 480 nm and emission of 520 nm using SpectraMax fluorescence microplate reader (Molecular Devices, USA). Standard curve was prepared with serial dilutions (1 ng – 100 ng/
ml) of λ phage DNA (molecular probes).
Concentration of humic acid in DNA samples was quantified by absorbance at 340 nm in a spectrophotometer (Hitachi U-2900, Japan) as reported15, and then calculated based on standard curve prepared with serial dilutions (0·1–100 µg/ml) of commercial humic acid (Sigma Aldrich, USA).
PCR
Amplification of 16S rDNA was used to assess suitability of mDNA for PCR. PCR reaction mixture contained 1X PCR buffer, 200 µM of each dNTPs, 3.7 µM MgCl2, 0.2 µM of each primer, forward F27 (5′-AGAGTTTGATCMGGCTCAG-3′) and reverse incubated at 50°C for 10 min. Denatured proteins were
precipitated using equal vol of phenol: chloroform:
isoamylalcohol (25:24:1) and aqueous phase was collected after centrifugation at 10,000 X g for 10 min. DNA was precipitated using 0.6 vol of isopropanol and washed with 70% ethanol. DNA was finally resuspended in 50 µl of TE (10 mM Tris Hcl, 1 mM EDTA). Each experiment was performed in triplicate.
Purification of Metagenomic DNA (mDNA)
Methods used for purification of mDNA were: P1, Agarose gel electrophoresis (electroelution); P2, Agarose gel with PVP electrophoresis (electroelution); P3, Silica membrane based spin column purification (DNeasy, Qiagen); P4, Silica membrane based spin column purification (Ultraclean, Mobio); and P5, Sepahdex G- 50 gel filtration. All methods were performed in triplicate.
Electroelution
Each DNA sample (50 µl) was loaded and resolved on 0.7% agarose gel. High molecular weight mDNA band was cut and transferred to a dialysis bag containing 3 vol of electrophoresis buffer. DNA was eluted into dialysis bag by electrophoresis for 2 h. Sample was precipitated with isopropanol and washed with 70%
ethanol. Air-dried sample was suspended in 50 µl of TE buffer. The same procedure was followed for agarose gel with polyvinyl pyrrolidone (PVP), where gel contained 2% PVP14.
Silica Membrane based Spin Column Purification
Each DNA sample (50 µl) was purified using silica membrane based commercial spin columns (DNeasy,
Table 1—Methods used for isolation of metagenomic DNA from termitarium sample
S. No Extraction buffer Cell lysis Chemical used for humic acid Reference
removal
E1 Phosphate buffer, Bead beating, - 9
Tween-80 Thermal shock, SDS
E2 EDTA, Vortex, SDS PVPP 10
NaCl, Tris-HCl
E3 Phosphate buffer Lysozyme, SDS - 11
E4 EDTA, NaCl, Tris- SDS, Bead beating PEG 12
HCl
E5 CTAB, EDTA, Tris- SDS CTAB 13
HCl, NaCl, NaPO4,
E6 As per kit protocol As per kit protocol As per kit protocol Ultraclean soil DNA kit (MoBio, USA)
E7 EDTA, NaCl, Tris- Vortex, SDS, - This study
HCl Proteinase-K
R1525 (5′-AAGGAGGTGWTCCARCC-3′), and 5U of Taq DNA polymerase (MBI Fermentas, Germany) in a vol of 50 µl. PCR amplification was performed in a Mastergradient thermal cycler (Eppendorf, Germany) with 5 min of denaturation at 94°C followed by 35 cycles of denaturation (1 min at 94°C), annealing (1 min at 55°C) and extension (2 min at 72°C), with a final extension of 72°C for 10 min. A positive control using 5 ng of E. coli genomic DNA and no template negative control were included.
Restriction Analysis
To examine digestibility, 0.5 µg of each mDNA, sample was digested with 5U of BamHI (MBI Fermentas, Germany) restriction enzyme in a 50 µl reaction. After incubation at 37°C for 3 h, enzyme was inactivated by heating at 70°C for 10 min, and digested product was resolved on 0.7% agarose gel.
Construction of metagenomic DNA (mDNA) Library
Plasmid metagenomic library was constructed using pUC19 as cloning vector. Purified mDNA was partially digested with Sau3AI and 5-10 kb sized DNA fragments were fractionated by agarose gel purification using Qiaquick gel extraction kit (Qiagen, Germany). Purified DNA fragments (300 ng) were ligated to BamHI digested and dephosphorylated pUC19 vector (100 ng) using T4 DNA ligase (MBI Fermentas, Germany). Ligated mixture was transformed into E.coli DH10B by electroporation using BioRad gene Xcell Electroporator (USA) with the preset program for E. coli (capacitance, 25uFD;
resistance, 200 ohms; and voltage, 2.5 kV). Transformed cells were plated on LB agar plates supplemented with X-gal (20 µg/ml), IPTG (40 µg/ml) and ampicillin (100 mg/ml) and recombinant clones were scored by blue white screening after overnight incubation at 37°C.
Results & Discussion
Extraction of Metagenomic DNA (mDNA) from Termitarium
To isolate mDNA from termitarium, seven different extraction methods were compared with respect to: i) DNA yield; ii) humic acid contamination; iii) processing time; iv) digestibility by restriction enzyme; and v) suitability for PCR. Highest yield (mg/g) of DNA of termitarium sample was observed in the method E4 followed by E7 and E1 and lowest in E6 (Table 2). In earlier studies16, A260 and A230values have been used to quantify DNA and humic substances respectively.
Therefore, absorbance ratio at 260/230 nm (DNA/humic acid) had been commonly used to evaluate purity of
mDNA. However, due to the interference of humic substances, spectrophotometric quantification of mDNA indicates the levels of humic substances rather than DNA17. Therefore, spectrophotometric quantification of DNA containing humic substances is challenging.
Fluorometric analysis using fluorescent dyes (picogreen and SYBRgreen) offers efficient quantification of mDNA. Picogreen specifically binds to double-stranded DNA and DNA-picogreen complex can be quantified using a fluorometer. Using this approach, DNA (conc., 25 pg/ml - 1 µg/ml) can be quantified. Humic acid (conc., > 100 ng/µl) interfere with the picogreen fluorescence. However, humic acids (conc., < 10 ng/µl) do not interfere DNA quantitation and therefore, DNA sample can be suitably diluted to minimize interference by humic compounds18. In this study, DNA samples were diluted to a level of 10 ng/µl of humic acids and then quantified using picogreen double stranded DNA quantification kit. Similarly, estimation of humic acids by A230 is also influenced by the concentration of nucleic acids and protein contaminants. Howeler et al15 found humic acid measurement by measuring A340 was not affected by the presence of DNA and protein and measurements were reproducible with humic acid (conc. range, 0.1 - 100 µg/ml). Similarly, in this study, linearity of A340 values (range, 0.1 - 100 µg/ml) was observed and this calibration curve was used to measure humic acids in mDNA samples.
Estimation of DNA concentration by densitometric analysis of ethidium bromide (EtBr) stained agarose gel is another major approach. In most of the studies19, DNA concentrations determined by EtBr stained agarose gels and spectrophotometric values did not match with each other. In this study, EtBr stained fluorescence was also not comparable with picogreen based quantification. For example, DNA extracted by method E3 showed higher level of fluorescence after EtBr staining (Fig. 1).
However, DNA concentration was lesser than the other samples, whereas concentration of humic acid in this sample was very high (Table 2). Therefore, densitometric analysis might also be interfered by humic substances.
Purification of Isolated Metagenomic DNA (mDNA)
All extracted DNA samples were used for PCR amplification and restriction digestion, but none of the sample was suitable for both (Table 2). DNA extracted using commercial kit (E6) could be digested with restriction enzyme but PCR was negative, indicating necessity of pur ification for further downstream
application (mDNA library construction). Humic acids affect almost all molecular biological methods (hybridization, restriction digestions of DNA, PCR and bacterial transformation)20. Humic substances interfere with PCR by inhibiting DNA and Taq polymerase interaction21. Therefore, successful PCR amplification is generally used as an indicator of mDNA purity. In addition, a more suitable purity marker would be
cloneability of mDNA, which can be studied by restriction digestion and ligation efficiencies. In this study, PCR amplification using broad range 16S rDNA specific primers and restriction digestion by BamHI were used to evaluate purity of termitarium DNA.
Based on the higher DNA yields and lower humic concentrations, extracted DNA using methods E1, E4 and E7 were subjected to further purification. Several strategies (preprocessing of soil samples, electroelution and chromatographical separations) have been reported for purification of mDNA. Similar to extraction methods, no single purification strategy is universally applicable for all types of samples. Addition of hexadecyltrimethylammonium bromide (CTAB), polyvinylpolypyrrolidone (PVPP) or polyethylene glycol (PEG) to soil-buffer slurry before cell lysis minimizes co-precipitation of humic substances13. In this study, PVPP, PEG and CTAB were included in extraction methods E2, E4 and E5 respectively. Among these, humic acid concentration was lesser in E4, where PEG was included in extraction buffer. However, extracted DNA using these methods were suitable for neither PCR nor restriction digestion. Therefore, additional strategies (electroelution, ion exchange chromatography using commercial spin columns and gel filtration) were evaluated for purification of extracted DNA (Table 3).
Table 2—Yield and purity of extracted metagenomic DNA by seven methods
Method Processing DNA yield DNA conc. Humic acid conc. 16S rDNA PCR Restriction
time, h µg/g soil ng/µl ng/µl amplification digestion
E1 15.5 1.8±.0.08 10± 0.02 86±1.3 − −
E2 3.0 1.5±0.22 5±0.18 90±0.1 − −
E3 5.5 0.8±0.05 1.8±0.06 558±2.8 − −
E4 7.0 2.4±0.03 22±0.09 50±2.0 − −
E5 4.5 1.4±0.02 1.9±0.05 124±1.0 − −
E6 0.5 0.4±0.09 2±0.16 2±0.04 − +
E7 1.0 2.0±0.03 17±0.12 10±0.12 − −
Table 3—DNA recovery, purity and suitability for PCR and restriction digestion after purification by five different methods Method DNA recovery Purification factor 16S rDNA PCR Restriction digestion
% amplification
E1 E4 E7 E1 E4 E7 E1 E4 E7 E1 E4 E7
P1 27 41 35 3.7 4.54 3.3 + + + + + +
P2 10 25 15 3.1 1.6 2 + + + - - -
P3 77 75 85 8.6 10 20 - - - - - -
P4 77 83 80 - - - - - - - - -
P5 94 97 96 86 77 100 + + + + + +
Fig. 1—Metagenomic DNA extracted from termitarium sample using different methods (Lane M - Lambda DNA digested with HindIII; Lane E1 to E7 - metagenomic DNA extracted using
methods E1 to E7 respectively)
An ideal purification method should efficiently remove humic acids without much DNA loss. In this study, among tested strategies, gel filtration using
Sephadex G-50 (method P5) yielded pure DNA suitable for both restriction digestion (Fig. 2) and PCR (Fig. 3).
DNA recovery after purification and purification factor
Fig. 2—Restriction digestion of metagenomic DNA with BamHI before (top row) and after (bottom row) purification by five methods (Lane M - Lambda DNA digested with HindIII; Lane 1 to 3 - purification of DNA extracted by E1, E4 and E7 respectively by method P1; 4 to 6 - purification of DNA extracted by E1, E4 and E7 respectively by method P2; 7 to 9 - purification of DNA extracted by E1,
E4 and E7 respectively by method P3; 10 to 12 - purification of DNA extracted by E1, E4 and E7 respectively by method P4;
13 to 15 - purification of DNA extracted by E1, E4 and E7 respectively by method P5)
Fig. 3—PCR amplification of 16S rDNA from metagenomic DNA after purification by five methods (Lane M – 1 kb ladder (MBI Fermentas, Germany); Lane 1 to 3 - after purification by P1 of DNA extracted by E1, E4 and E7 respectively; Lane 4 to 6 - after purification by P2 of DNA extracted by E1, E4 and E7 respectively; Lane 7 to 9 - after purification by P3 of DNA extracted by
E1, E4 and E7 respectively; Lane 10 to 12 - after purification by P4 of DNA extracted by E1, E4 and E7 respectively; Lane 13 to 15 - after purification by P5 of DNA extracted by E1, E4 and E7 respectively; Lane 16 - positive using E. coli genomic
DNA; Lane 17 - no template negative control)
were also higher in gel filtration with all tested samples (Table 3). DNA recovery rate was lowest in the case of electroelution. DNA recovery in methods P1 and P2 was considerably low (20-40%). Similarly, purification factor was also considerably lesser. Therefore, electroelution even after the electrophoresis in the presence of 2% PVP did not sufficiently purify the DNA.
DNA recovery and purification factor were comparatively higher in silica based column purification.
However, PCR was negative after purification using both Qiagen and Mobio columns. Certain kinds of humic compounds might compete with DNA for binding site during purification using these minicolumns22. Sagova- Mareckova et al23 reported that manually optimized protocol was more efficient than commercial kits in yielding PCR-compatible mDNA from tested soil samples. Gel filtration resins have been widely reported for purification of mDNA extracted from soil. Dijkmans et al24 reported efficient purification of mDNA using Sephadex G-50 with minimal DNA loss and purified DNA was suitable for PCR and hybridization. Jackson et al17 and Miller6 reported that Sepharose resins (Sepharose 2B and 4B) gave superior separation of DNA from humic substances. However, few studies25 have reported that gel filtration resins failed to remove inhibitors from soil DNA. Therefore, obtaining pure DNA depends not only based on the type of strategy used but also the type of humic substances present in the sample.
Conclusions
Extraction methods E1, E4 and E7 followed by Sephadex G-50 purification (P5) were suitable for mDNA extraction from termitarium. When considering the processing time as a factor, E7 is the most suitable method. Thus, the simple method reported in this study, employing SDS to lyse cells in situ followed by ethanol precipitation (E7) and subsequent purification using Sephadex G-50 (P5) resin yielded high concentration of cloneable DNA from termitarium sample, which has been successfully used for the construction of mDNA library.
Acknowledgements
One of the authors (AM) thanks MKU for Research Fellowship (USRF). Authors also thank Centre for Advanced Studies in Functional Genomics, Centre for Excellence in Genomic Sciences & Networking Resource Centre in Biological Sciences at School of Biological Sciences, MKU, Madurai, India.
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