Biotechnol Lett DOI 10.1007/s10529-014-1591-5
ORIGINAL RESEARCH PAPER
A method suitable for DNA extraction from humus-rich soil Tianjin Miao • Song Gao • Shengwei Jiang • Guoshi Kan • Pengju Liu • Xianming Wu • Yingfeng An • Shuo Yao
Received: 17 April 2014 / Accepted: 12 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTABbased method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient,
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1591-5) contains supplementary material, which is available to authorized users. T. Miao S. Gao G. Kan P. Liu X. Wu Y. An (&) S. Yao College of Biosciences and Biotechnology, Shenyang Agricultural University, No. 120 Dongling Road, Shenyang 110161, People’s Republic of China e-mail: [email protected] S. Jiang State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, People’s Republic of China
providing an efficient choice especially for DNA extraction from humus-rich soils. Keywords DNA extraction Humic substances Metagenomic DNA Soil DNA Soil microbiology
Introduction Isolation of pure, non-sheared, and high molecular weight DNA is important because further steps of metagenomics rely upon it. However, it is difficult to provide pure DNA extracts from humus-rich soils because humic substances can co-precipitate with DNA and interfere with its downstream processing. The presence of trace amounts of humic compounds can significantly affect downstream steps of PCR amplification, restriction digestion and transformation by binding with the enzymes and chelate Mg2? ions (Gabor et al. 2003). In some cases, extensive dilution of the crude DNA extract will allow for direct PCR amplification (Schneegurt et al. 2003), but cannot fundamentally solve the problem. Therefore, further purification of DNA extracted from soil sample is mandatory for downstream processing. To solve this problem, several methods have been developed that can significantly improve the purity of soil DNA (Desai and Madamwar 2007; Engel et al. 2012; LaMontagne et al. 2002; Li et al. 2011; Liu et al. 2010; Martin-Laurent et al. 2001; Seo and Ohgaki
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2001; Verma and Satyanarayana 2011). For example, PEG, powdered activated charcoal (PAC), polyvinylpolypyrrolidone (PVPP), and silica-based columns have been used for soil DNA purification (MartinLaurent et al. 2001; Seo and Ohgaki 2001; Robe et al. 2003). PEG 8000 instead of 2-propanol was used to decrease humic substances without decreasing DNA yields (LaMontagne et al. 2002). PAC was used by Desai and Madamwar (2007) for extracting the inhibitor-free metagenome from polluted sediments. Nonlinear electrophoresis with the synchronous coefficient of drag alteration (SCODA) instrument (Boreal Genomics) was used to selectively concentrate highmolecular soil DNA and remove contaminants, including humic acids (Engel et al. 2012). Although very efficient, these methods either have relatively long protocols or require infrequently used reagents or equipment. More importantly, some of the methods may have bias towards certain kinds of soil types, among which the humus-rich soils should be the most challenging ones (Inceoglu et al. 2010; Islam et al. 2012). Therefore, to develop a convenient, efficient and time-saving method for high purity DNA extraction from humus-rich soils still has obvious necessity. Nowadays a number of commercial kits have also been developed for extraction of DNA from humus-rich soil but no kit is efficient for all kinds of soil types, especially for humus-rich soils. Here we present a convenient DNA purification kit and DNA gel extraction kit-based method, providing a valuable choice especially for DNA extraction from humus-rich soils.
Materials and methods A dark brown forest soil sample was collected from Botanical Garden of Shenyang Agricultural University in Liaoning Province, China. The three steps in the soil DNA extraction process were as follows: the first step is preparation of crude soil DNA. five gram soil was suspended with 10 ml extraction buffer containing 125 mM EDTA, 5 % (w/v) SDS, 100 mM Tris/ HCl (pH 8.0) and 10 ml phenol/chloroform/isoamyl alcohol (3-methyl-1-butanol) (25:24:1, by volume), and ground manually with a mortar and pestle for 2 min. The mixture was centrifuged at 10,0009g for 5 min, and the supernatant was gently mixed with 0.6 volume 2-propanol, and cooled on ice for 20 min
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followed by centrifugation at 10,0009g for 5 min. The precipitates were dissolved in 100 ll TE buffer containing 10 mM Tris/HCl and 1 mM EDTA (pH 8.0) to provide brown slurries which should be crude DNA mixed with humic substances and other contaminants. The second and third steps are DNA purification with modified AxyPrep DNA Purification Kit and AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Inc., California, USA), respectively. The slurries obtained in the first step were centrifuged and the supernatants were mixed with binding buffer (Buffer A) solution (1:3, v/v) from DNA Purification Kit. The mixtures were cooled on ice for 5 min followed by centrifugation at 10,0009g for 5 min, and the supernatants were purified according to the instructions of the kit. Then DNA samples eluted from columns of DNA purification kit were submitted to agarose gel electrophoresis on a 0.8 % agarose gel at 120 V for 20 min, and further purified by DNA gel extraction kit. The DNA samples were finally eluted from columns and suspended in 100 ll TE buffer (pH 8.0) for further analysis. In the process of DNA purification with both kits, the columns loaded with elution buffer (TE buffer) were incubated in 65 °C for 5 min before DNA elution from columns with centrifugation. The same soil samples were also performed DNA extraction using SoilGen DNA Kit (CoWin Biotech Co., Beijing, China) and the classic CTAB-based soil DNA extraction method (Zhou et al. 1996; Jiang et al. 2013; Jin et al. 2014). The full details of the kit-based protocols are shown as Supplementary Information. DNAs extracted by different methods were measured and the yields were calculated from the A260/ A230, A260/A280, A260/A465, A260/A665 ratios. To further estimate the efficiency of the DNA extraction methods, the extracted soil DNAs were used as templates for PCR amplification of 16S rRNA using primers 16S rRNA-For (50 -AGAGT TTGAT CCTGG CTCAG-30 ) and 16S rRNA-Rev (50 -AAGGA GGTGA TCCAG CCGCA-30 ) (Yao et al. 2006). PCR was performed using 2 U Pfu DNA polymerase, Pfu buffer, 0.5 ng template DNA/ll, 0.2 mM dNTP and 0.5 lM both primers in 20 ll. The reactions were carried out at 94 °C for 2 min, 25 cycles of 94 °C for 30 s, 55 °C for 40 s and 72 °C for 4 min, and a final incubation at 72 °C for 10 min. The purity of the extracted DNAs was further determined by restriction digestion with BamHI. One micro gram of soil DNA in 20 ll reaction
Biotechnol Lett Fig. 1 Outline of the twokit purification method for DNA extraction from humus-rich soil. Microbial cells in soil are ground manually with a mortar and pestle for crude DNA extraction with the presence of SDS and phenol/ chloroform/isoamyl alcohol mixture (first step). Then crude DNA sample is further purified with modified DNA purification kit (second step) and DNA gel extraction kit (third step)
mixture was digested with 5 U BamHI and incubated at 37 °C for 10 min. The PCR products and digested DNAs were visualized on 0.8 % (w/v) agarose gels.
Results In this study, the method for soil DNA isolation consists of three steps (Fig. 1): microbial cells breaking and crude DNA extraction with the presence of SDS and phenol/chloroform/isoamyl alcohol mixture (first step) followed by purification with modified DNA purification kit (second step) and DNA gel extraction kit (third step).
Degradation of DNA by DNase is a critical factor that may cause failure during DNA extraction from soil. With the reported methods and commercial kits, EDTA and SDS are commonly used as inhibitors to suppress DNase activity. In the subsequent step, the supernatant obtained by centrifugation is extracted by phenol/chloroform/isoamyl alcohol mixture to remove DNase. Although EDTA and SDS can strongly inhibit DNase, the residual DNase activity can be still high enough to degrade DNA and often cause failure during DNA extraction from soil, which may account for the poor reproducibility of some methods and kits. For our method, phenol/chloroform/isoamyl alcohol is directly mixed with soil and DNase can be directly inactivated during the release of DNA from microbial
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cells by grinding with a mortar and pestle. Therefore, degradation of DNA can be effectively prevented and this method has satisfactory yield and repeatability. After breaking the microbial cells in soil, the extracted DNA was purified by modified DNA purification kit and DNA gel extraction kit. According to producer’s instructions of the DNA purification kit, the DNA sample to be purified should be mixed with binding buffer (Buffer A) and directly loaded on to a column followed by centrifugation. However, when crude soil DNA was mixed with binding buffer, precipitates occurred which block column during centrifugation. Therefore, centrifugation prior to loading the mixture on to the column was necessary to remove the precipitate. After removal of the precipitate, the supernatant changed from dark brown to light brown, indicating that this step had removed the contaminations. Both the DNA purification kit and DNA gel extraction kit were not originally designed for purification of genomic DNAs and this may be the first report of isolating genomic DNAs of soil microorganisms with these two kits. The satisfactory yield (about 30 lg DNA/g soil) indicated that these two kits were suitable for this application. In addition, the sizes of metagenomic DNA fragments obtained by this method were larger than 10 kb and no obvious DNA degradation and low molecular weight contaminants were visible. A commercial soil DNA isolation kit (SoilGen DNA Kit) and a classic CTAB-based soil DNA extraction method were also used to extract DNA from the same soil sample to compare the efficiency of different methods. As a result, no DNA could be extracted with the SoilGen DNA Kit for an unknown reason, maybe this soil type is outside the scope of the kit, because different commercial soil DNA isolation kits (e.g., UltraClean, Powersoil, and SoilMaster) may have a bias towards certain kinds of soil types (e.g., clay, silt, sand, and gravel) (Whitehouse and Hottel 2007). In this study, high levels of humic substances and other contaminants in soils might affect the stability of the soil DNA isolation kit. Although DNA could be isolated by using CTAB-based soil DNA extraction method, the obtained DNA sample was dark brown indicating that it was not pure. The A260/A230 and A260/A280 values of DNA extracted by this two-kit purification method were close to the theoretical values of pure DNA indicating that this two-kit
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purification method was efficient to remove contaminations of soil DNA in this study. As these values of DNA purified by CTAB-based method were much lower than the theoretical values of pure DNA, this method is not efficient for removing contamination (e.g., carbohydrates, phenols, peptides, and aromatic compounds) from this humus-rich soil sample. Additionally, the A260/A465 and A260/A665 values of DNA extracted by the two-kit purification method were the highest ones, which were much higher than the value measured from DNA extracted by the CTAB-based method. These results indicated that the two-kit purification method was more efficient than the CTAB-based method in removing humic substances. The absorption ratios also revealed the necessity of the DNA purification steps with the modified DNA purification kit and DNA gel extraction kit. In addition, both restriction digestion and PCR amplification using DNA extracted by two-kit purification method were successful, while neither restriction digestion nor PCR amplification using DNA extracted by CTAB-based method was successful (Fig. 2b, c). These results further indicated that only DNA extracted by two-kit purification method had satisfactory purity. Additionally, both restriction digestion and PCR amplification were performed using DNA only after purification with the modified DNA purification kit. As a result, PCR amplification was successful but restriction digestion was not complete, indicating that both humic materials and DNA bound to the silica gel of the DNA purification kit, and some humic acids could be sequentially eluted from the matrix. But the silica gel protocols alone were not enough to remove all the humic materials from crude DNA. As the electrical charges and molecular sizes of humic components and other small molecular impurities are different from that of soil DNA, therefore the remaining impurities can be removed by agarose gel electrophoresis and DNA gel extraction kit, which is verified by high purity of soil DNA obtained after purification with two kits.
Discussion In a typical experiment, the full process of DNA isolation with two-kit purification method can be finished within 70 min, which is faster than with the CTAB-based method, commercial soil DNA isolation
Biotechnol Lett
Fig. 2 Analysis of the purity of soil DNAs extracted with different methods and treatments. a The OD260/OD230, OD260/OD280, OD260/OD465 and OD260/OD665 values of soil DNAs isolated by different methods and treatments. Lanes 1–4 soil DNAs isolated by the introduced method but without further purification, purification with one kit only, purification with two kits, and by CTAB-based method, respectively. Error bars represent the standard deviation; n = 3. b Restriction digestions of soil DNAs produced by different methods and treatments. Lane M DNA ladder. The soil DNAs isolated by the introduced method but without further purification, purified by one kit only, purified by two kits, and isolated by the CTAB-
based method are shown in lanes 1, 4, 7 and 10, respectively. Restriction digestions of the four DNA samples but without the presence of BamHI are shown in lanes 2, 5, 8 and 11, respectively. Restriction digestions of the four DNA samples with the presence of BamHI are shown in lines 3, 6, 9 and 12, respectively. c DNA fragments obtained by PCR amplification of 16s rRNA genes using templates of soil DNAs produced by different methods and treatments. Lane M DNA ladder; lanes 1–4 PCR with template of soil DNAs isolated by the introduced method but without further purification, purification with one kit only, purification with two kits, and by CTAB-based method, respectively; lane 5 PCR without template
kits (e.g., SoilGen, UltraClean, Powersoil, and SoilMaster), and other commonly used methods (Lakay et al. 2007; Islam et al. 2012). Different DNA purification kits (e.g., GenElute, UltraClean, and SpinPrep) and DNA gel extraction kits (e.g., QIAquick, GeneJET, and StrataPrep) would affect the efficiency of the two-kit purification method. Therefore, further studies would be necessary to investigate the efficiency of the method and its scope of application. However, at least the two-kit purification method could provide a novel and valuable choice especially for DNA extraction from humus-rich soils. To sum up, this two-kit purification method has at least three distinctive characteristics. Firstly, during the stage of crude DNA preparation, phenol/
chloroform/isoamyl alcohol mixture is directly mixed with soil for grinding with a mortar and pestle, which can effectively prevent the degradation of DNA. This special design is essential for satisfactory repeatability and DNA yield, which should also have important reference value for other methods. Secondly, this may be the first report of isolating high quality genomic DNAs of soil microorganisms with a DNA purification kit and DNA gel extraction kit. These two kits are widely used in nearly all the molecular biology laboratories, which makes the isolation of soil DNAs very convenient. Finally, the full process of DNA isolation with this method can be finished within only 70 min, which is shorter than the currently used kits and methods.
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In conclusion, we have developed a rapid and convenient two-kit purification method to extract high quality soil DNA with good reproducibility and high yield, which has some obvious advantages over widely used CTAB-based method and a commercial soil DNA isolation kit especially for DNA extraction from humus-rich soils. Therefore, this method may play an important role in the studies of microbial diversity, metagenomic analysis, etc. Acknowledgments This work was supported by National Natural Science Foundations of China (No. 31100045 and No. 31270114) and Program for Liaoning Excellent Talents in University (No. LJQ2011067).
References Desai C, Madamwar D (2007) Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ionexchange treatments. Bioresour Technol 98:761–768 Engel K, Pinnell L, Cheng J, Charles TC, Neufeld JD (2012) Nonlinear electrophoresis for purification of soil DNA for metagenomics. J Microbiol Methods 88:35–40 Gabor EM, de Vries EJ, Janssen DB (2003) Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. FEMS Microbiol Ecol 44:153–163 Inceoglu O, Hoogwout EF, Hill P, van Elsas JD (2010) Effect of DNA extraction method on the apparent microbial diversity of soil. Appl Environ Microbiol 76:3378–3382 Islam MR, Sultana T, Melvin Joe M, Cho JC, Sa T (2012) Comparisons of direct extraction methods of microbial DNA from different paddy soils. Saudi J Biol Sci 19:337–342 Jiang L, Lin M, Zhang Y, Li Y, Xu X, Li S, Huang H (2013) Identification and characterization of a novel trehalose synthase gene derived from saline-alkali soil metagenomes. PLoS One 8:e77437 Jin H, Li B, Peng X, Chen L (2014) Metagenomic analyses reveal phylogenetic diversity of carboxypeptidase gene
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sequences in activated sludge of a wastewater treatment plant in Shanghai, China. Ann Microbiol 64:689–697 Lakay FM, Botha A, Prior BA (2007) Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils. J Appl Microbiol 102:265–273 LaMontagne MG Jr, Michel FC, Holden PA, Reddy CA (2002) Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. J Microbiol Methods 49:255–264 Li J, Li B, Zhou Y, Xu J, Zhao J (2011) A rapid DNA extraction method for PCR amplification from wetland soils. Lett Appl Microbiol 52:626–633 Liu J, Li J, Feng L, Cao H, Cui Z (2010) An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction. J Microbiol 48:728–733 Martin-Laurent F, Philippot L, Hallet S, Chaussod R (2001) DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol 67:2354–2359 Robe P, Nalin R, Capellano C, Vogel TM, Simonet P (2003) Extraction of DNA from soil. Eur J Soil Biol 39:183–190 Schneegurt MA, Dore SY Jr, Kulpa CF (2003) Direct extraction of DNA from soils for studies in microbial ecology. Curr Issues Mol Biol 5:1–8 Seo GT, Ohgaki S (2001) Evaluation of refractory organic removal in combined biological powdered activated carbon-microfiltration for advanced wastewater treatment. Water Sci Technol 43:67–74 Verma D, Satyanarayana T (2011) An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries. Appl Biochem Biotechnol 165:454–464 Whitehouse CA, Hottel HE (2007) Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples. Mol Cell Probes 21:92–96 Yao XH, Min H, Yuan HP (2006) Microbial diversity in ann acetamiprid-polluted upland soil. Acta Ecol Sin 26:3074–3080 Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322
ORIGINAL RESEARCH PAPER
A method suitable for DNA extraction from humus-rich soil Tianjin Miao • Song Gao • Shengwei Jiang • Guoshi Kan • Pengju Liu • Xianming Wu • Yingfeng An • Shuo Yao
Received: 17 April 2014 / Accepted: 12 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTABbased method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient,
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1591-5) contains supplementary material, which is available to authorized users. T. Miao S. Gao G. Kan P. Liu X. Wu Y. An (&) S. Yao College of Biosciences and Biotechnology, Shenyang Agricultural University, No. 120 Dongling Road, Shenyang 110161, People’s Republic of China e-mail: [email protected] S. Jiang State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, People’s Republic of China
providing an efficient choice especially for DNA extraction from humus-rich soils. Keywords DNA extraction Humic substances Metagenomic DNA Soil DNA Soil microbiology
Introduction Isolation of pure, non-sheared, and high molecular weight DNA is important because further steps of metagenomics rely upon it. However, it is difficult to provide pure DNA extracts from humus-rich soils because humic substances can co-precipitate with DNA and interfere with its downstream processing. The presence of trace amounts of humic compounds can significantly affect downstream steps of PCR amplification, restriction digestion and transformation by binding with the enzymes and chelate Mg2? ions (Gabor et al. 2003). In some cases, extensive dilution of the crude DNA extract will allow for direct PCR amplification (Schneegurt et al. 2003), but cannot fundamentally solve the problem. Therefore, further purification of DNA extracted from soil sample is mandatory for downstream processing. To solve this problem, several methods have been developed that can significantly improve the purity of soil DNA (Desai and Madamwar 2007; Engel et al. 2012; LaMontagne et al. 2002; Li et al. 2011; Liu et al. 2010; Martin-Laurent et al. 2001; Seo and Ohgaki
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2001; Verma and Satyanarayana 2011). For example, PEG, powdered activated charcoal (PAC), polyvinylpolypyrrolidone (PVPP), and silica-based columns have been used for soil DNA purification (MartinLaurent et al. 2001; Seo and Ohgaki 2001; Robe et al. 2003). PEG 8000 instead of 2-propanol was used to decrease humic substances without decreasing DNA yields (LaMontagne et al. 2002). PAC was used by Desai and Madamwar (2007) for extracting the inhibitor-free metagenome from polluted sediments. Nonlinear electrophoresis with the synchronous coefficient of drag alteration (SCODA) instrument (Boreal Genomics) was used to selectively concentrate highmolecular soil DNA and remove contaminants, including humic acids (Engel et al. 2012). Although very efficient, these methods either have relatively long protocols or require infrequently used reagents or equipment. More importantly, some of the methods may have bias towards certain kinds of soil types, among which the humus-rich soils should be the most challenging ones (Inceoglu et al. 2010; Islam et al. 2012). Therefore, to develop a convenient, efficient and time-saving method for high purity DNA extraction from humus-rich soils still has obvious necessity. Nowadays a number of commercial kits have also been developed for extraction of DNA from humus-rich soil but no kit is efficient for all kinds of soil types, especially for humus-rich soils. Here we present a convenient DNA purification kit and DNA gel extraction kit-based method, providing a valuable choice especially for DNA extraction from humus-rich soils.
Materials and methods A dark brown forest soil sample was collected from Botanical Garden of Shenyang Agricultural University in Liaoning Province, China. The three steps in the soil DNA extraction process were as follows: the first step is preparation of crude soil DNA. five gram soil was suspended with 10 ml extraction buffer containing 125 mM EDTA, 5 % (w/v) SDS, 100 mM Tris/ HCl (pH 8.0) and 10 ml phenol/chloroform/isoamyl alcohol (3-methyl-1-butanol) (25:24:1, by volume), and ground manually with a mortar and pestle for 2 min. The mixture was centrifuged at 10,0009g for 5 min, and the supernatant was gently mixed with 0.6 volume 2-propanol, and cooled on ice for 20 min
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followed by centrifugation at 10,0009g for 5 min. The precipitates were dissolved in 100 ll TE buffer containing 10 mM Tris/HCl and 1 mM EDTA (pH 8.0) to provide brown slurries which should be crude DNA mixed with humic substances and other contaminants. The second and third steps are DNA purification with modified AxyPrep DNA Purification Kit and AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Inc., California, USA), respectively. The slurries obtained in the first step were centrifuged and the supernatants were mixed with binding buffer (Buffer A) solution (1:3, v/v) from DNA Purification Kit. The mixtures were cooled on ice for 5 min followed by centrifugation at 10,0009g for 5 min, and the supernatants were purified according to the instructions of the kit. Then DNA samples eluted from columns of DNA purification kit were submitted to agarose gel electrophoresis on a 0.8 % agarose gel at 120 V for 20 min, and further purified by DNA gel extraction kit. The DNA samples were finally eluted from columns and suspended in 100 ll TE buffer (pH 8.0) for further analysis. In the process of DNA purification with both kits, the columns loaded with elution buffer (TE buffer) were incubated in 65 °C for 5 min before DNA elution from columns with centrifugation. The same soil samples were also performed DNA extraction using SoilGen DNA Kit (CoWin Biotech Co., Beijing, China) and the classic CTAB-based soil DNA extraction method (Zhou et al. 1996; Jiang et al. 2013; Jin et al. 2014). The full details of the kit-based protocols are shown as Supplementary Information. DNAs extracted by different methods were measured and the yields were calculated from the A260/ A230, A260/A280, A260/A465, A260/A665 ratios. To further estimate the efficiency of the DNA extraction methods, the extracted soil DNAs were used as templates for PCR amplification of 16S rRNA using primers 16S rRNA-For (50 -AGAGT TTGAT CCTGG CTCAG-30 ) and 16S rRNA-Rev (50 -AAGGA GGTGA TCCAG CCGCA-30 ) (Yao et al. 2006). PCR was performed using 2 U Pfu DNA polymerase, Pfu buffer, 0.5 ng template DNA/ll, 0.2 mM dNTP and 0.5 lM both primers in 20 ll. The reactions were carried out at 94 °C for 2 min, 25 cycles of 94 °C for 30 s, 55 °C for 40 s and 72 °C for 4 min, and a final incubation at 72 °C for 10 min. The purity of the extracted DNAs was further determined by restriction digestion with BamHI. One micro gram of soil DNA in 20 ll reaction
Biotechnol Lett Fig. 1 Outline of the twokit purification method for DNA extraction from humus-rich soil. Microbial cells in soil are ground manually with a mortar and pestle for crude DNA extraction with the presence of SDS and phenol/ chloroform/isoamyl alcohol mixture (first step). Then crude DNA sample is further purified with modified DNA purification kit (second step) and DNA gel extraction kit (third step)
mixture was digested with 5 U BamHI and incubated at 37 °C for 10 min. The PCR products and digested DNAs were visualized on 0.8 % (w/v) agarose gels.
Results In this study, the method for soil DNA isolation consists of three steps (Fig. 1): microbial cells breaking and crude DNA extraction with the presence of SDS and phenol/chloroform/isoamyl alcohol mixture (first step) followed by purification with modified DNA purification kit (second step) and DNA gel extraction kit (third step).
Degradation of DNA by DNase is a critical factor that may cause failure during DNA extraction from soil. With the reported methods and commercial kits, EDTA and SDS are commonly used as inhibitors to suppress DNase activity. In the subsequent step, the supernatant obtained by centrifugation is extracted by phenol/chloroform/isoamyl alcohol mixture to remove DNase. Although EDTA and SDS can strongly inhibit DNase, the residual DNase activity can be still high enough to degrade DNA and often cause failure during DNA extraction from soil, which may account for the poor reproducibility of some methods and kits. For our method, phenol/chloroform/isoamyl alcohol is directly mixed with soil and DNase can be directly inactivated during the release of DNA from microbial
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cells by grinding with a mortar and pestle. Therefore, degradation of DNA can be effectively prevented and this method has satisfactory yield and repeatability. After breaking the microbial cells in soil, the extracted DNA was purified by modified DNA purification kit and DNA gel extraction kit. According to producer’s instructions of the DNA purification kit, the DNA sample to be purified should be mixed with binding buffer (Buffer A) and directly loaded on to a column followed by centrifugation. However, when crude soil DNA was mixed with binding buffer, precipitates occurred which block column during centrifugation. Therefore, centrifugation prior to loading the mixture on to the column was necessary to remove the precipitate. After removal of the precipitate, the supernatant changed from dark brown to light brown, indicating that this step had removed the contaminations. Both the DNA purification kit and DNA gel extraction kit were not originally designed for purification of genomic DNAs and this may be the first report of isolating genomic DNAs of soil microorganisms with these two kits. The satisfactory yield (about 30 lg DNA/g soil) indicated that these two kits were suitable for this application. In addition, the sizes of metagenomic DNA fragments obtained by this method were larger than 10 kb and no obvious DNA degradation and low molecular weight contaminants were visible. A commercial soil DNA isolation kit (SoilGen DNA Kit) and a classic CTAB-based soil DNA extraction method were also used to extract DNA from the same soil sample to compare the efficiency of different methods. As a result, no DNA could be extracted with the SoilGen DNA Kit for an unknown reason, maybe this soil type is outside the scope of the kit, because different commercial soil DNA isolation kits (e.g., UltraClean, Powersoil, and SoilMaster) may have a bias towards certain kinds of soil types (e.g., clay, silt, sand, and gravel) (Whitehouse and Hottel 2007). In this study, high levels of humic substances and other contaminants in soils might affect the stability of the soil DNA isolation kit. Although DNA could be isolated by using CTAB-based soil DNA extraction method, the obtained DNA sample was dark brown indicating that it was not pure. The A260/A230 and A260/A280 values of DNA extracted by this two-kit purification method were close to the theoretical values of pure DNA indicating that this two-kit
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purification method was efficient to remove contaminations of soil DNA in this study. As these values of DNA purified by CTAB-based method were much lower than the theoretical values of pure DNA, this method is not efficient for removing contamination (e.g., carbohydrates, phenols, peptides, and aromatic compounds) from this humus-rich soil sample. Additionally, the A260/A465 and A260/A665 values of DNA extracted by the two-kit purification method were the highest ones, which were much higher than the value measured from DNA extracted by the CTAB-based method. These results indicated that the two-kit purification method was more efficient than the CTAB-based method in removing humic substances. The absorption ratios also revealed the necessity of the DNA purification steps with the modified DNA purification kit and DNA gel extraction kit. In addition, both restriction digestion and PCR amplification using DNA extracted by two-kit purification method were successful, while neither restriction digestion nor PCR amplification using DNA extracted by CTAB-based method was successful (Fig. 2b, c). These results further indicated that only DNA extracted by two-kit purification method had satisfactory purity. Additionally, both restriction digestion and PCR amplification were performed using DNA only after purification with the modified DNA purification kit. As a result, PCR amplification was successful but restriction digestion was not complete, indicating that both humic materials and DNA bound to the silica gel of the DNA purification kit, and some humic acids could be sequentially eluted from the matrix. But the silica gel protocols alone were not enough to remove all the humic materials from crude DNA. As the electrical charges and molecular sizes of humic components and other small molecular impurities are different from that of soil DNA, therefore the remaining impurities can be removed by agarose gel electrophoresis and DNA gel extraction kit, which is verified by high purity of soil DNA obtained after purification with two kits.
Discussion In a typical experiment, the full process of DNA isolation with two-kit purification method can be finished within 70 min, which is faster than with the CTAB-based method, commercial soil DNA isolation
Biotechnol Lett
Fig. 2 Analysis of the purity of soil DNAs extracted with different methods and treatments. a The OD260/OD230, OD260/OD280, OD260/OD465 and OD260/OD665 values of soil DNAs isolated by different methods and treatments. Lanes 1–4 soil DNAs isolated by the introduced method but without further purification, purification with one kit only, purification with two kits, and by CTAB-based method, respectively. Error bars represent the standard deviation; n = 3. b Restriction digestions of soil DNAs produced by different methods and treatments. Lane M DNA ladder. The soil DNAs isolated by the introduced method but without further purification, purified by one kit only, purified by two kits, and isolated by the CTAB-
based method are shown in lanes 1, 4, 7 and 10, respectively. Restriction digestions of the four DNA samples but without the presence of BamHI are shown in lanes 2, 5, 8 and 11, respectively. Restriction digestions of the four DNA samples with the presence of BamHI are shown in lines 3, 6, 9 and 12, respectively. c DNA fragments obtained by PCR amplification of 16s rRNA genes using templates of soil DNAs produced by different methods and treatments. Lane M DNA ladder; lanes 1–4 PCR with template of soil DNAs isolated by the introduced method but without further purification, purification with one kit only, purification with two kits, and by CTAB-based method, respectively; lane 5 PCR without template
kits (e.g., SoilGen, UltraClean, Powersoil, and SoilMaster), and other commonly used methods (Lakay et al. 2007; Islam et al. 2012). Different DNA purification kits (e.g., GenElute, UltraClean, and SpinPrep) and DNA gel extraction kits (e.g., QIAquick, GeneJET, and StrataPrep) would affect the efficiency of the two-kit purification method. Therefore, further studies would be necessary to investigate the efficiency of the method and its scope of application. However, at least the two-kit purification method could provide a novel and valuable choice especially for DNA extraction from humus-rich soils. To sum up, this two-kit purification method has at least three distinctive characteristics. Firstly, during the stage of crude DNA preparation, phenol/
chloroform/isoamyl alcohol mixture is directly mixed with soil for grinding with a mortar and pestle, which can effectively prevent the degradation of DNA. This special design is essential for satisfactory repeatability and DNA yield, which should also have important reference value for other methods. Secondly, this may be the first report of isolating high quality genomic DNAs of soil microorganisms with a DNA purification kit and DNA gel extraction kit. These two kits are widely used in nearly all the molecular biology laboratories, which makes the isolation of soil DNAs very convenient. Finally, the full process of DNA isolation with this method can be finished within only 70 min, which is shorter than the currently used kits and methods.
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In conclusion, we have developed a rapid and convenient two-kit purification method to extract high quality soil DNA with good reproducibility and high yield, which has some obvious advantages over widely used CTAB-based method and a commercial soil DNA isolation kit especially for DNA extraction from humus-rich soils. Therefore, this method may play an important role in the studies of microbial diversity, metagenomic analysis, etc. Acknowledgments This work was supported by National Natural Science Foundations of China (No. 31100045 and No. 31270114) and Program for Liaoning Excellent Talents in University (No. LJQ2011067).
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