A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications

RESEARCH ARTICLE

A Rapid and Economical Method for Efficient

DNA Extraction from Diverse Soils Suitable

for Metagenomic Applications

Selvaraju Gayathri Devi

, Anwar Aliya Fathima

, Sudhakar Radha

, Rex Arunraj

, Wayne

R. Curtis

, Mohandass Ramya

1 Department of Genetic Engineering, SRM University, Kattankulathur, Tamilnadu, India, 2 Department of

Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, United States of

America

* [email protected]

Abstract

A rapid, cost effective method of metagenomic DNA extraction from soil is a useful tool for

environmental microbiology. The present work describes an improved method of DNA

extraction namely “powdered glass method” from diverse soils. The method involves the

use of sterile glass powder for cell lysis followed by addition of 1% powdered activated char-

coal (PAC) as purifying agent to remove humic substances. The method yielded substantial

DNA (5.87 ± 0.04 μg/g of soil) with high purity (A

260/280

: 1.76 ± 0.05) and reduced humic sub-

stances (A

340

: 0.047 ± 0.03). The quality of the extracted DNA was compared against five

different methods based on 16S rDNA PCR amp lification, BamHI digestion and validated

using quantitative PCR. The digested DNA was used for a metagenomic library construction

with the transformation efficiency of 4 X 10

CFU mL

-1

. Besides providing rapid, efficient

and economical extraction of metgenomic DNA from diverse soils, this method’s applicabil-

ity is also demonstrated for cultivated organisms (Gram positive B. subtilis NRRL-B-201,

Gram negative E. coli MTCC40, and a micro algae C. sorokiniana UTEX#1666).

Introduction

Current estimates of microbial diversity reveal that 99% of the microorganisms present in

nature are not cultivatable by standard techniques. The genetic information and biotechnologi-

cal potential of the majority of organisms is therefore not revealed by conventional microbio-

logical approaches [1]. Metagenomics, which involves direct cloning of environmental DNA,

can obviate the need for cell cultivation, and thereby capture the genetic information from the

total microbial community [2]. Since the abiotic and biotic factors of each habitat vary both

spatially and temporally, a metagenomic DNA extraction method is needed that is broadly

applicable, and yet standardized to permit relative comparisons.

The two important requirements for metagenomic DNA extraction are efficient cell lysis

and purification of DNA from the complex milieu of an environmental sample. Cell lysis in

PLOS ONE | DOI:10.1371/journal.pone.0132441 July 13, 2015 1/16

OPEN ACCESS

Citation: Devi SG, Fathima AA, Radha S, Arunraj R,

Curtis WR, Ramya M (2015) A Rapid and

Economical Method for Efficient DNA Extraction from

Diverse Soils Suitable for Metagenomic Applications.

PLoS ONE 10(7): e0132441. doi:10.1371/journal.

pone.0132441

Editor: Vishal Shah, West Chester University of

Pennsylvania, UNITED STATES

Received: September 22, 2014

Accepted: June 15, 2015

Published: July 13, 2015

transmitted, modified, built upon, or otherwise used

by anyone for any lawful purpose. The work is made

available under the Creative Commons CC0 public

domain dedication.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information files.

Funding: The authors have no support or funding to

report.

Competing Interests: The authors have declared

that no competing interests exist.

soil samples has been accomplished by many methods that include chemicals such as sodium

dodecyl sulphate (SDS) [3], chelex 100 [4], and guanidine thiocyanate [5], and physical meth-

ods such as bead beating [6], sonication [7], liquid nitrogen [8] and freeze thawing [9]. A tradi-

tional method dating back over 20 years uses a combination of freeze thawing and lysozyme

[10]. However, it has been shown that these methods are often not sufficient to achieve com-

plete cell lysis, or require the use of sophisticated equipment [11]. These methods are often

time consuming and usually require an additional purification step before being subjected to

molecular analysis. For soil samples in particular, purification to remove humic substances is

necessary. Previously utilized purification agents include Polyvinylpolypyrrolidone (PVPP)

[12], Sephadex G 200 [13], Q-Sepharose [14], electroelution [15] and silica gel [16]. Commer-

cial kits are available for purification of metagenomic DNA; however, most are quite expensive.

Upon considering the limitations of previous methods (variable efficiency, time consuming

and high cost), the current study focused on developing a rapid inexpensive method for extrac-

tion of metagenomic DNA with sufficient quantity and purity to be broadly suitable for meta-

genomic applications. Since, cell lysis and purification are the key steps in metagenomic DNA

extraction; this study includes a particular focus on these two factors. Cell lysis is accomplished

by homogenizing with glass powder that is obtained from laboratory waste glassware. Silica,

the major component of ground glass powder, has been widely used for DNA extraction from

various sources including soils and sediments [16], tissues and blood of transgenic animals

[17] and plasmid from E.coli [18]. Autoclaved silica-based sand has been reported for extrac-

tion of fungal DNA [19], and glass powder along with skim milk was used for detection of Phy-

tophthora infestans [20]. In a recent study by Radha et al. 2013 [21], a glass grinding step was

included for direct colony PCR of various microalgae. However, a comparable glass powder

based DNA extraction is not yet reported for metagenomic DNA from soils. Purification is

accomplished with powdered activated charcoal (PAC), because it absorbs humic substances

and allows the release of pure DNA [22, 11]. Hence, PAC was included in the extraction buffer

of this study with the goal of eliminating the need for subsequent purification steps. An overall

goal for the development of this method is to provide a procedure that is faster and less expen-

sive than the method of Yeates et al 1998 [6] which utilize bead-beating, and multiple extrac-

tions for purification.

Diverse soils are tested along with cultivated examples for Gram positive bacteria, Gram

negative bacteria and microalgae. The method is also compared with several alternative meth-

ods that utilize different homogenizing agent or equipment. We report the improved perfor-

mance of this “powdered glass” method for 16S rDNA PCR amplification, quantitative PCR

(qPCR) analysis, BamHI restriction assay, and metagenomic library construct ion.

Materials and Methods

Chemicals, strains and plasmids

All chemicals of analytical or molecular biology grade were purchased from Sigma Aldrich,

India. pUC19 plasmid and E.coli TOP 10 cells were purchased from Invitrogen Bio Services

India Pvt. Ltd.

Sample collection and analysis

Four different soil samples were collected for the study namely: (1) Garden soil from Nagesh-

waraRao public park (13°2’11”N80°15’7”E) Chennai, India, (2) Sewage sludge from the Com-

mon effluent treatment plant (CETP) for tanneries, located at Pallavaram (12°57'44"N 80°8'8"E),

Chennai, India, (3) Lake soil from the SRM University campus (12°49'25"N 80°2'39"E), Chen-

nai, India, and (4) Compost sample from local house kitchen wastes. Samples were sieved

Metagenomic DNA Extraction

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using a 2 mm mesh and stored at -20°C for further analysis. No specific permissions were

required to collect the samples from these locations and the field studies did not involve any

endangered or protected species. Soil characteristics including texture, pH, electrical conduc-

tivity, organic matter, carbon, iron, cadmium and chromium were estimated as per APHA,

(2005) [ 23](S1 Table).

Preparation of glass powder

A clean piece of broken borosilicate-based laboratory glass was ground finely using a mortar

and pestle until it becomes a fine powder. The fine powder was autoclaved and stored in a ster-

ile container, but may alternatively be pre-aliquoted and sterilized in amounts that correspond

to a standardized procedure. The approximate size of the glass particles were measured by

Field Emission-Scanning Electron Microscopy (Quanta FEI 200). The sample was examined

on accelerating beam at a voltage of 10 kV at 10,000X magnification (S1 Fig).

Metagenomic DNA extraction

Metagenomic DNA was extracted from all the four soil samples using six different methods.

Method M1, M2, M3 and M4 were performed based on the previous studies (Table 1). Method

5 was performed using a commercially available kit (Fast DNA spin kit for soil; MP Biomedi-

cals, Santa Ana, CA). M6 (powdered glass method) is the currently developed method, based

largely on a prior method of extracting DNA from soil for subsequent PCR [6] as outlined in

Table 2 . In order to validate the superiority of the powdered glass method, the individual com-

ponents of method M6 (glass powder and glass powder + powdered activated charcoal) we re

tested with method M2 (Glass beads). The other steps of method M2 were kept constant. The

method M6 was also tested for genomic DNA extraction from Gram negative E. coli MTCC40,

Gram positive B. subtilis NRRL-B-201 and eukaryotic microalgae Chlorella sorokiniana

UTEX#1666 in order to confirm the suitability of the method for cultivatable organisms.

Assessment of yield and purity of the metagenomic DNA

Equal volume (3 μL) of the extracted metagenomic DNA from all the samples for methods M1

to M6 were loaded in 0.8% agarose gel along with 3 μL of 1Kb DNA ladder (50 μg/mL) and the

bands were visualized using UVP-Multidoc-It digital imaging system, CA, USA (Fig 1, S2 Fig).

Purity and concentration of metagenomic DNA was determined by spectrophotometry analy-

sis (UV-vis spectrophotometer, Eppendorf, NY). Concentration of the DNA (ng/μL) allowed

Table 1. Different methods used for extraction of metagenomic DNA from four different soils.

Method Homogenizing agent Homogenizing equipment Processing time

(h)

DNA Yield(μg/g of

soil)

M1 [39] Extraction buffer Nil 5 1.29 ± 0.02

M2 [6] Glass beads Bead beater 7 3.42 ± 0.04

M3 [6] Nil Sonicator 7 1.47 ± 0.04

M4 [10] Lysozyme -70°C deep freezer and dryice ethanol

bath

8 3.38 ± 0.05

M5 (Fast DNA SPIN

kit)

As per the manufacturer’s

protocol

Bead beater 1.5 3.51 ± 0.03

M6 (present study) Glass Powder Nil 1.5 5.87 ± 0.04

DNA yield represents the Average ± SD values of four different soils.

Nil represents there is no usage of homogenizing equipment.

doi:10.1371/journal.pone.0132441.t001

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calculation of Yield in μg per gram of soil = concentration of DNA (μg/μL)•volume used to sus-

pend DNA (μL) /weight of soil (g) [24]. The absorbance A

260/280

was used to determine protein

contamination, and A

340

as an indication of humic acid contamination.

PCR amplification and quantitative PCR analysis

PCR amplification of the 16S rDNA was performed for all the methods (one sample per tripli-

cate) using the universal forward primer B 27F (5

-AGAGTTTGATCCTGGCTCAG-3

) and

the reverse primer U 1492R (5

-GGTTACCTTGTTACGACTT-3

). The reaction mix consisted

of 50 ng of the metagenomic DNA as the template, 5 pmoles of each primer, 0.5 U Taq poly-

merase (GeNet Bio, Korea), 5 μL of 10X Taq buffer [10X buffer composition: Tris-HCl pH 9.0;

PCR enhancers; (NH

)

; 20 mM MgCl

] and 10 mM dNTP mix (NEB, USA). The final

mixture was adjusted to 50 μL by addition of sterile, purified water. The amplification steps

includes initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 1 min,

annealing at 55°C for 30 s and extension at 72°C for 1.5 min with the final extension of 72°C

for 7 min. 50 ng of genomic DNA from Gram positive and Gram negative bacteria was also

amplified under similar conditions. 50 ng of the genomic DNA from the algae Chlorella soro-

kiniana UTEX#1666 was amplified using universal internal transcribed spacer 2 (ITS-2) prim-

ers: forward (5

-AGGAGAAGTCGTAACAAGGT-3

) and reverse (5

-TCCTCCGCTTATT

GATATGC-3

). The conditions used for PCR were initial denaturation at 95°C for 5 min,

Table 2. Metagenomic DNA extraction from soils using the currently developed powdered glass

method M6 modified from [6].

Step Procedure

1. Take a clean, broken laboratory glass ware (borosilicate) and grind it using a pestle and mortar until

it becomes a ﬁne powder (Wear gloves and facemask). Transfer the ﬁnely ground powder to a

container and sterilize by autoclaving at 121°C for 15 to 20 min or pre-aliquot and sterilize in

amounts that correspond to a standardized procedure.

2. Weigh 1 g of soil sample and 1 g of sterile glass powder, transfer in an autoclaved mortar and

pestle and grind ﬁnely for about 5 min.

3. Prepare enough DNA extraction buffer [100 mM Tris, 100 mM EDTA, 1.5 M NaCl (pH 8)]. Sterilize it

by autoclaving at 121°C for 15 to 20 min or ﬁlter sterilization and store at room temperature. Weigh

10mg (1%) of powdered activated charcoal.

4. Add 1 mL of DNA extraction buffer and 10mg of powdered activated charcoal to the soil- glass

powder mixture and mix it by pipetting several times. Slowly transfer the contents into a 2 mL

eppendorf tube.

5. Incubate the tube at 65°C for 10 min in a water bath and then centrifuge at 12000g for 5 min at 4°C.

Transfer 500 μL of the supernatant to a fresh 2 mL microfuge tube.

6. Prepare enough quantity of 3M sodium acetate (pH 5.2) and 30% PEG (MW-8000). Sterilize by

autoclaving at 121°C for 15 to 20 min or ﬁlter sterilization and store at room temperature.

7. Add 100 μL of sodium acetate and 400 μL of PEG to the supernatant. Allow the mixture to

precipitate at -20°C for 20 minutes in a deep freezer. Slowly thaw the tubes and then centrifuge at

12,000g, 5 min at 4°C.

8. Discard the supernatant and re-suspend the pellet with 500 μL of autoclaved TE buffer (10 mM Tris,

1 mM EDTA pH 8).

9. Prepare chloroform: isoamyl alcohol mixture in the ratio 24:1 and store it in a brown bottle at room

temperature.

10. Add equal volume (500 μL) of chloroform: isoamyl alcohol mixture and centrifuge at 12,000g for 5

min at 4°C.

11. Transfer the aqueous phase to a fresh eppendorf and add 500 μL of ice cold isopropanol.

12. Allow precipitation for 5 min at 4°C and centrifuge at 12,000g for 10 min at 4°C. Discard the

supernatant and wash the precipitate with 70% ethanol. Centrifuge at 12,000g for 2 min at 4°C.

13. Discard the supernatant, air dry the pellet and dissolve in 100 μL of TE buffer (pH 8).

doi:10.1371/journal.pone.0132441.t002

Metagenomic DNA Extraction

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Fig 1. Gel electrophoresis of metagenomic DNA extracted by methods M1 to M6 for four different soils. Samples were electrophoresed on 0.8%

agarose gel in 0.5X TBE buffer. 1A. Garden soil; 1B. Sewage sludge; 1C. Lake soil; 1D. Compost; Lane M represents 1Kb DNA ladder (Merck, India). Lanes

1 to 6 represents the methods M1 to M6 respectively. a,b,c represents the triplicates of corresponding method.

doi:10.1371/journal.pone.0132441.g001

Metagenomic DNA Extraction

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35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 1

min and a final extension of 72°C for 5 min. Amplification products were confirmed by loading

5 μL of samples along with 1Kb DNA ladder on 1% agarose gel (Fig 2 , S3 Fig). PCR efficiency

of the DNA isolated using the powdered glass method M6 was analyzed for a single soil sample

(sewage sludge) by qPCR (Lightcycler 480 system, Roche Life Sciences, US) using the primers

for 16S rDNA: forward (5

-AAGCAACGCGAAGAACCTTA-3

) and reverse (5

-ACCACCT

GTCACCTCTGTCC-3

). The reaction volume (10 μL) consisted of DNA (10 ng to 100 pg),

primers (5 pmoles) and 5 μL of 2x SYBR Green I master mix and the reaction was performed

in triplicate. The reaction without the template served as a non-template control (NTC). The

amplification conditions were 95°C for 7 min as initial denaturation followed by 40 cycles of

95°C for 20 s, 55°C for 20 s and 72°C for 20 s (S6 Fig).

Fig 2. (A) Gel electrophoresis of PCR amplified 16S rDNA for the DNA extracted using the powdered glass method M6. Samples were analyzed on

1% agarose gel in 0.5X TBE buffer. Lane M: 1 Kb DNA ladder (Merck, India); Lane 1: Garden soil; Lane 2: Sewage sludge; Lane 3: Lake soil; Lane 4:

Compost; Lane 5: E.coli MTCC 40; Lane 6: Bacillus subtilis NRRL-B-201. Lane 7: Negative control. (B). Gel electrophoresis of PCR amplified ITS-2 for

the DNA extracted using the powdered glass method M6. Lane M: 100bp DNA ladder (Merck, India); Lane 1: Negative control; Lane 2: PCR amplification

of ITS -2 region of Chlorella sorokiniana UTEX# 1666.

doi:10.1371/journal.pone.0132441.g002

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Partial restriction digestion and metagenomic library construction

Partial restriction digestion was performed using the enzyme BamHI (Fermentas, Germany)

for DNA extra cted by all the methods (one sample per triplicate) except M3 to examine the

suitability of the methods for downstream DNA manipulation. 1 μg of the DNA template was

digested with 1 U of the enzyme in a 50 μL reaction containing 5 μL of 10X assay buffer [1X

buffer composition: 10 mM Tris-HCl pH 8.0; 5 mM MgCl

; 100 mM KCl; 0.02% TritonX-100;

0.1 mg/mL BSA] for 20 min at 37°C followed by heat inactivation of the enzyme at 70°C for 10

min. 5 μL of the digested products were analyzed on 0.8% agarose gel along with 1Kb DNA lad-

der (Fig 3, S4 Fig). A soil metagenomic library was constructed for the DNA isolated by pow-

dered glass method (M6) using the pUC19 vector in the following manner: 2 to 10 kb

fragments from the partially digested DNA were gel purified using EZ-Spin PCR purification

column (Biobasic Inc., Canada). pUC19 plasmid was linearized with BamHI and deph osphory-

lated using 10 U of calf intestinal alkaline phosphatase. Linearized vector was ligated with the

insert [vector: insert Molar ratio (1:3)] using 200 U of T4 DNA ligase (NEB, USA) in a 10 μL

reaction containing 1 μL of ligase buffer [1X buffer composition: 50 mM Tris-HCl, pH 7.5; 10

mM MgCl

; 1 mM ATP; 10 mM DTT] at 16°C overnight. 5 μL of the ligation mixture was then

Fig 3. Gel electrophoresis of partial restriction digestion of the metagenomic DNA extracted by powdered glass method (M6) using BamHI.

Samples were analyzed on 0.8% agarose gel in 0.5X TBE buffer. Lane M: 1Kb DNA ladder (Merck, India); Lane 1: Garden soil; Lane 2: Sewage sludge; Lane

3: Lake soil; Lane 4: Compost. Lane 5: Undigested DNA.

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added to 100 μL of chemically competent E.coli TOP 10 cells and incubated on ice for 20 min.

Heat shock was applied at 42°C for 90 s, then transferred to ice for 5 min followed by addition

of 1 mL of Luria Bertani (LB) broth and cells were allowed to grow at 37°C for 1 h. 100 μLof

cells were plated in LB media containing ampicillin (50 μg/mL), IPTG (0.1 mM) and X-gal

(40 μ g/mL). The recombinant colonies were re-plated on LB plate with ampicillin (50 μg/mL).

Statistical analysis

The experiments were performed in triplicate and the mean and standard deviations were esti-

mated for each experiment. Analysis of variance (One-way ANOVA) was performed with the

software GraphPad Prism 5.0 to determine the significant effects of extraction methods and

soil types on DNA yield. Paired T-test analysis was performed for the pairwise comparison of

DNA yields obtained by other methods (M1 to M5) against the powdered glass method M6 for

different soil samples. The comparisons were considered significant if p<0.05.

Results

Extraction of metagenomic DNA from different samples

Metagenomic DNA was extracted from different soil samples using earlier methods (M1, M2,

M3, M4) and commercially available Fast DNA spin kit for soil (MP Biomedicals, CA) (M5)

and compared with the currently developed powdered glass method (M6). Upon analysis of

the extracted soil samples on 0.8% agarose gels, method M6 is shown to produce intact and

bright bands for all samples (Fig 1A, 1B, 1C and 1D). Method M5 showed bright bands for

DNA extracted by lake and compost soils (Fig 1C and 1D), whereas M4 produced bright bands

for garden and compost soils (Fig 1A and 1D). Further illustrating the variation of results with

different soils, M2 showed bright bands for garden soil and sewage sludge (Fig 1A and 1B).

Methods M1 and M3 did not produce bright bands for any of the samples.

Comparison of DNA yield and purity using various methods

The amount of DNA (μg/g) for the four different soils is depicted in Table 3 and S3 Table.

Our improved powdered glass method M6 produced high average yield of DNA/g of soil

(5.87 ± 0.04 μg) followed by methods M5, M2 and M4 (3.51 ± 0.03, 3.42 ± 0.04, 3.38 ± 0.05 μg/g

respectively). Methods M3 and M1 produced the lowest DNA yield (1.47 ± 0.04, 1.29 ± 0.02 μg/g

respectively) per gram of soil. Analysis of variance (One-way ANOVA) revealed that the different

DNA extraction methods as well as various soil types had significant effects on the DNA yield

(P<0.0001). Statistically significant differences in DNA yields were obtained based on T-test

analysis for pairwise comparison of each method against the powdered glass method M6

(Table 3)).

An assessment of the extracted DNA purity demonstrates that the method M6 provides

more pure DNA with an average absorbance ratio (A

260/280

) of 1.76 ± 0.05, as compared to

absorbance ratios lower than 1.5 for the other methods. The A

340

absorbance value was taken

as an indication of humic acid contamination rather than A

230

because it has less overlap with

the absorbance of DNA which is measured at A

260

and the associated use of A

260

230

as a

typical ratio indicator of DNA purity [22]. The average A

340

value for method M6 is low

(0.047 ± 0.03) as compared to the other methods (Table 4, S4 and S5 Tables) which is consis-

tent with the recent report of Sharma et al. (2014) [22] that demonstrated effective use of PAC

for the reduction in humic substances.

An assessment of the efficiency of DNA extraction from soil for glass powder relative to

glass beads, and the impact of combining the PAC as a single step is presented in Table 5,

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S5 Fig and S6 Table. The average yield of DNA using glass beads was 2.1 ± 0.08 μg/g of soil;

much lower than glass powder (5 ± 0.11 μ g/g of soil). The addition of PAC to the glass powder

did not adversely affect the DNA yield with an average yield of 5.4 ± 0.04 μg/g for the four soil

types analyzed. In addition, the purity obtained by the use of glass powder combined with PAC

was higher when compared to the use of glass beads or glass powder alone.

Further, as an indicator of the superiority of our improved methodology, method M6 also

yielded a higher concentration and higher purity DNA when tested for its applicability on culti-

vatable organisms. The concentration (ng of DNA/μL) and purity A

260/280

were respectively

found to be 71.1 ± 0.25 ng/μL and 1.80 ± 0.04 for Gram negative E. coli MTCC40, 42.2 ± 0.42

ng/μL and 1.73 ± 0.02 for Gram positive B. subtilis NRRL-B-201 and 39.0 ± 0.68 ng/μL and

1.62 ± 0.07 for C. sorokiniana UTEX# 1666 (S2 Fig). The presence of peptidoglycan in the cell

wall of Gram positive bacteria and glycoprotein rich cell wall of microalga e likely contributes

to the lower yield of DNA as compared to Gram negative bacteria.

Table 3. Assessment of yield of metagenomic DNA obtained by methods M1 to M6 for different soils.

Yield (μg/g of soil)

Methods Garden soil Sewage sludge Lake soil Compost

M1 1.34 ± 0.01**** 1.49 ± 0.02**** 1.42 ± 0.02*** 0.93 ± 0.03****

M2 4.92 ± 0.03** 5.01 ± 0.04*** 1.52 ± 0.04*** 2.24 ± 0.07****

M3 3.63 ± 0.06*** 0.56 ± 0.05**** 1.08 ± 0.02*** 0.62 ± 0.03****

M4 5.06 ± 0.05* 2.06 ± 0.06**** 2.25 ± 0.04**** 4.15 ± 0.06***

M5 2.01 ± 0.03*** 2.56 ± 0.05**** 4.58 ± 0.02*** 4.89 ± 0.03

M6 5.48 ± 0.03 7.81 ± 0.04 5.19 ± 0.06 4.98 ± 0.04

DNA concentration (μg/mL) was quantiﬁed in a spectrophotometer (A

260

nm).

Yield in μg per gram of soil = concentration of DNA (μg/μL). Volume used to suspend DNA (μL) /weight of soil (g)

P values were generated by Graph Pad Prism 5.0 software using paired T-test analysis.

P value < 0.05 is considered to be statistically signiﬁcant. DNA extraction methods performing worse at a statistically signiﬁcant level as compared with

the best performing method M6 are indicated by asterisks

**** indicates P < 0.0001

*** indicates P < 0.001

** indicates P < 0.01

* indicates P < 0.05

indicates P > 0.05 which is statistically non-signiﬁcant.

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Table 4. Assessment of purity of metagenomic DNA by methods M1 to M6 for different soils.

260/280

340

Methods Garden soil Sewage sludge Lake soil Compost Garden soil Sewage sludge Lake soil Compost

M1 1.14 ± 0.02 1.22 ± 0.03 1.22 ± 0.09 1.29 ± 0.04 0.09 ± 0.03 0.08 ± 0.02 0.09 ± 0.03 0.08 ± 0.01

M2 1.53 ± 0.06 1.48 ± 0.03 1.56 ± 0.01 1.40 ± 0.07 0.06 ± 0.02 0.07 ± 0.03 0.08 ± 0.02 0.06 ± 0.04

M3 1.32 ± 0.03 1.25 ± 0.05 1.34 ± 0.01 1.37 ± 0.06 0.07 ± 0.01 0.08 ± 0.03 0.06 ± 0.04 0.06 ± 0.05

M4 1.33 ± 0.05 1.34 ± 0.06 1.46 ± 0.09 1.48 ± 0.03 0.09 ± 0.04 0.08 ± 0.02 0.08 ± 0.03 0.09 ± 0.02

M5 1.60 ± 0.04 1.62 ± 0.08 1.56 ± 0.03 1.50 ± 0.07 0.06 ± 0.04 0.06 ± 0.03 0.07 ± 0.08 0.08 ± 0.01

M6 1.82 ± 0.08 1.72 ± 0.04 1.78 ± 0.02 1.73 ± 0.05 0.04

± 0.03 0.06 ± 0.04 0.05 ± 0.01 0.04 ± 0.06

260/280

represents protein contamination. A

340

is an indication for humic acid presence.

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PCR Amplification and quantitative PCR analysis of ribosomal RNA

genes

DNA samples were tested for their suitability for PCR amplification of 16S rDNA. Powdered

glass method (M6) produced an amplificatio n product of 1.5 kb for all the samples including

Gram positive and Gram negative bacteria (Fig 2A). Genomic DNA of C. sorokiniana UTEX#

1666 obtained by method M6 was also amplified using algal specific ITS-2 primers producing a

PCR product of 800 bp (Fig 2B). Methods M1, M3 and M4 did not provide amplification for

any of the soil samples indicating that those methods would require further purification of

DNA to remove humic substances. M2 and M5 provided amplification for a few samples (S3

Fig); however, these methods would require additional purification to be suitable for all the soil

types. The qPCR efficiency (indicative of the exponential increase in DNA during successive

PCR cycles) for the DNA isolated using powdered glass method (M6) was 1.986 (99.3%) with a

slope of -3.356 (S6 Fig).

Partial restriction digestion and metagenomic library construction

All the soil DNA samples extracted by powdered glass method (M6) were partially digested

with the enzyme BamHI for metagenomic library construction (Fig 3). Method M5 was also

suitable for digestion of all DNA extracts, where the other methods failed to achieve digestion

for at least one of the samples (S4 Fig). Method M3 was not subjected to restriction digestion

because of the shear-degraded, sonicated DNA. The suitability of the other methods for restric-

tion digestion and 16S rDNA PCR is presented in S2 Table. The partially digested DNA by

powdered glass method M6 was subjected to metagenomic library construction using pUC19

vector and colo nies were obtained with the transformation efficiency of 4 X 10

CFU mL

-1

This confirms the efficacy of the method for the required downstream metagenomic DNA pro-

cessing including PCR, restriction digestion and library construction.

Table 5. Assessment of yield and purity of the DNA extracted by method M2 and its modification (glass beads, glass powder and glass powder

+ powdered activated charcoal).

Method/Soil Glass Beads Glass Powder Glass Powder + PAC

DNA yield

(μg/g)

260/280

340

DNA yield

(μg/g)

260/280

340

DNA yield

(μg/g)

260/280

340

Garden soil 3.3 ± 0.05 *** 1.50 ± 0.03 0.06 ± 0.04 5.5 ± 0.07 *** 1.50 ± 0.01 0.03 ± 0.02 6.2 ± 0.04 1.80 ± 0.03 0.05 ± 0.02

Sewage sludge 1.2 ± 0.03 **** 1.45 ± 0.04 0.06 ± 0.03 5.8 ± 0.07

1.52 ± 0.03 0.06 ± 0.05 5.9 ± 0.03 1.69 ± 0.04 0.03 ± 0.02

Lake soil 1.0 ± 0.07*** 1.30 ± 0.02 0.13 ± 0.07 4.2 ± 0.15

1.42 ± 0.04 0.07 ± 0.04 4.4 ± 0.03 1.73 ± 0.04 0.03 ± 0.02

Compost 3.0 ± 0.18 *** 1.44 ± 0.05 0.09 ± 0.06 4.1 ± 0.15 ** 1.50 ± 0.04 0.08 ± 0.04 5.0 ± 0.09 1.74 ± 0.06 0.04 ± 0.05

DNA concentration (μg/mL) was quantiﬁed in a spectrophotometer (A

260

nm).

Yield in μg per gram of soil = concentration of DNA (μg/μL). Volume used to suspend DNA (μL) /weight of soil (g).

260/280

represents protein contamination. A

340

is an indication for humic acid presence.

PAC represents powdered activated charcoal.

P values were generated by Graph Pad Prism 5.0 software using paired T-test analysis.

P value < 0.05 is considered to be statistically signiﬁcant. DNA extraction methods performing worse at a statistically signiﬁcant level as compared with

the best performing method (Glass powder + PAC) are indicated by asterisks

**** indicates P < 0.0001

*** indicates P < 0.001

** indicates P < 0.01

indicates P > 0.05 which is statistically non-signiﬁcant.

doi:10.1371/journal.pone.0132441.t005

Metagenomic DNA Extraction

PLOS ONE | DOI:10.1371/journal.pone.0132441 July 13, 2015 10 / 16

Discussion

Various methods are available for metagenom ic DNA extraction based on chemical or

mechanical lysis of microbial cells present in the soil. Among these methods, glass bead beating

is considered to be an effective technique for metagenomic DNA extraction [6]. This method

has also been modified in previous reports to be suitable for different soil types [25]. Commer-

cial kits such as Fast DNA SPIN kit for soil, MP Biomedicals, Santa Ana, CA) and Ultra Clean

Mo Bio Soil DNA isolation kit are also based on the method of bead beating. Even though this

method was adopted for different soils, it requires ‘bead beater’ for cell lysis and the method’s

efficiency depends on the size of beads and duration of agitation. While ‘bead beating’ and our

improved method M6 both utilize glass, the mechanism of cell breakup is fundamentally differ-

ent. Bead mills and other suspension-based cell breakup approaches rely on the fluid-dynamic

shear that resu lts in flows between particles [26], or more accurately, the mechanisms of dissi-

pating the imparted energy at the turbulent length scales of the cells [27]. In contrast, the

improved method M6 utilizes direct mechanical grinding of the soil and glass shards (Table 2).

Therefore, in contra st to shear forces of bead beating, the mortar and pestle grinding represents

a direct physical maceration, which is apparently more effective means of disrupting the cells

in a metagenom ic soil sample based on DNA recovery yield (Table 3). A potential reason for

reduced performance of cell disruption in soil samples by bead beating could result from

increased viscosity due to the presence of a high concentration of insoluble materials during

the beating process [28].

Our improved method M6 involves the fine grinding of soil samples with the glass powder

which takes less than 5 min. In addition to providing an excellent mechanical force for cell

breakup, we suggest that enhanced DNA adsorption on the finely powdered silica may also be

playing a role [18]. Upon grinding, the cells are lysed and the DNA is adsorbed by the silica

particles which are subsequently extracted by addition of extraction buffer [21, 16]. The size of

glass powder varies from 223 to 261 nm. This extremely small size of powdered glass relative to

typical glass milling beads (~100 microns) provides greatly enhanced surface area as well as

freshly fractured silica surfaces to contribute to DNA adsorption. The adsorption of DNA to

silica has been intensely studied in relevance to sequencing analysis and microfluidics [29], and

reports the advantage of large silica surface area for DNA extraction [ 30]. This behavior of

DNA adsorption to glass has the potential to effect the application of these methods for PCR

amplification and sequencing of the environmental samples to study microbial diversity and

functionality in complex communities [16]. Li et al. 2015 [31] reported that the use of silica col-

umns improves viral sequence recovery when compared to other methods of DNA extra ction.

Silica nanoparticles have been fabricated for the effective immobilization and sensitive

sequence-specific detection of DNA [32]. An observation to be validated using the above meth-

odology is its efficiency in the recovery of high molecular weight DNA (>20Kb) for use in

large sized libraries. For this behavior to not affect metagenomic analysis, one must assume

that the process of cell disruption and subsequent shearing of DNA is similar for different

microorganisms. We suggest that the mechanical grinding of the method (M6), as compared to

hydrodynamic shearing described above, may provide for a more representative sampling of

the microbial populations. Future work combining defined mixtures of microorganisms to

sterile soil could address this issue.

The efficiency of using powdered glass in mechanical cell lysis and enhanced surface area is

confirmed by the yield of DNA which is much higher in method M6 than the other methods.

Another advantage of this methodology is the inclusion of PAC in the extraction buffer to min-

imize additional purification steps. PAC is very porous with a vast surface area which allows

the absorption of humic substances , lignin sulphonate, tannic acid, heavy metals and non-

Metagenomic DNA Extraction

PLOS ONE | DOI:10.1371/journal.pone.0132441 July 13, 2015 11 / 16

degradable coloured substances [33]. PAC has previously been used as a purifying agent in

other reports of soil DNA extraction [11, 22]. In th e present study, 1% PAC was added directly

to the extraction buffer in order to absorb contaminating components of the metagenomic

DNA that would otherwise inhibit subsequent DNA manipulation. This simplifies the need for

subsequent column purification. Polyethylene glycol (30% PEG) has been used in other meta-

genomic DNA extraction metho ds because it does not co-precipitate humic acids with the

DNA as occurs with ethanol or isopropanol precipitation [34]. In the current procedure (M6) ,

the humic substances are precipitated in a simple intermediate step using chloroform and iso-

propanol as reported previously [35].

The powdered glass method (M6) is compared with four other previously reported methods

and a commercial kit. All the methods follow the same basic steps: cell lysis and precipitation

of DNA but they differ from each other on the homogenization approach and duration of the

total protocol (Table 1). Methods M2, M3, M4 and M5 require homogenizing equipment for

cell lysis whereas M1 is based on chemical lysis and M6 is based on glass powder grinding.

Among all the methods studied, powdered glass method (M6) shows higher efficiency of cell

lysis during DNA extraction which is evidenced in terms of DNA yield. The methods were also

tested for four different soil samples that that differed in texture, pH, percentage of organic

matter and the amount of metals (S1 Table). The presence or absence of clay and other particu-

late matter present in each soil is an important consideration because they tend to decrease the

DNA yield by adsorbing the free DNA [36, 37]. Grinding with high surface area powdered

glass is expected to out-compete such adsorption [38]. We feel that this characteristic helps

contribute to the robust performance of method M6 for different soil types.

As noted above, metho ds M1 to M4 did not achieve either high yield or purity. Though

method M1 [39] did not require any sophisticated equipment for cell lysis, it failed to meet

high purity and yield for some of the soil samples, while method M3 apparently suffered from

excessive shearing of the DNA due to sonication. Method M2 based on bead beating is often

recommended as the most effective method; however, in our study, this method did not yield

pure DNA for all the soil samples. Method M2 also requires the bead beater equipment for the

cell lysis. Method M4 that used a combination of lysozyme and freeze thaw did not reduce the

humic acid content. The Fast DNA kit for soil resulted in a better DNA yield and purity but

still contained humic acid residues. Other reports also demonstrate the failure of commercial

resins to provide DNA free from humic acids as they compete with the DNA for column bind-

ing [40 , 41].

Downstream applications such as PCR amplification and restriction digestion involve enzy-

matic reactions that can be significantly inhibited by humic acids. Humic substances chelate

the Mg

ions required for the activity of Taq polymerase, restriction enzymes and ligases [42].

The DNA isolated using methods M1 to M4 did not provide sufficiently pure DNA for further

studies. Kit based method M5 provided DNA suitable for the restriction digestion assay; how-

ever, it was not found suitable for PCR amplification when teste d on different soil samples. The

results reveal that the methods M1 to M5 requires further purification of the DNA for molecu-

lar analysis. The DNA isolated using the powdered glass method M6 displays a high qPCR effi-

ciency; greater than 99%, where the accepted PCR efficiency for qPCR analysis ranges from 90

to 110 percent (Roche Life Sciences, US). The suitability of the DNA for PCR, qPCR, restriction

digestion assay and metagenomic library construction confir ms that sufficient purity was

achieved using method M6; a simplified method that integrates cell lysis and DNA purification

steps (Table 2).

Despite the diversity in the sources of the soil samples, our improve d powdered glass

method (M6) produced high yield of DNA, demonstrating broader applicability of the method.

The average DNA yield of the four soil samples was 5.87 μg/g which was considerably higher

Metagenomic DNA Extraction

PLOS ONE | DOI:10.1371/journal.pone.0132441 July 13, 2015 12 / 16

than the other methods (Table 1). In addition, protein and humic acid contamination was low

when compared to the other tested methods. The purity of the metagenomic DNA obtained by

this method M6 was further substantiated by facile construction of a metagenomic library. Fur-

ther, this method was also successfully used for DNA extraction from Gram positive, Gram

negative bacteria and also microalgae. The statistically significant improved efficiency of DNA

extraction invariably contributes to downstream manipulations by effectively reducing the

contaminants.

As a final added advantage, the overall processing time for powdered glass method (M6)

was shorter than the other tested methods. The earlier reported methods (M1 to M4) took

about 5 to 7 h for proc essing while the method M6 requires only 1.5 h which was considerably

shorter than the recently reported method of Sagar et al. (2014) [35]. The commercial kit

(method M5) took the least time for DNA extraction (Table 1); however, the cost of processing

1 g of soil for a single reaction is about $8 (US) which is quite high if large numbers of samples

are to be processed. The use of glass powder is an economical method for cell lysis that does

not require sophisticated equipment or enzymes for cell lysis.

Conclusion

A robust method of DNA extraction from soil for metagenomic analysis and cultivated organ-

isms is demonstrated which uses glass powder for cell lysis and powdered activated carbon for

purification. This method is rapid, efficient and economical, and found to be robustly applica-

ble for DNA extraction where intact and pure DNA is requir ed, and may include additional

advantages for providing a representative sampling of the metabiome population.

Supporting Information

S1 Fig. Size determination of the glass powder by FE-SEM analysis.

(DOC)

S2 Fig. Gel electrophoresis of genomic DNA isolated by method M6 for Gram positive,

Gram negative bacteria and microalgae .

(DOC)

S3 Fig. Gel electrophoresis of PCR amplified 16S rDNA for the metagenomic DNA

extracted by methods M1 to M5.

(DOC)

S4 Fig. Gel electrophoresis of partial restriction digestion of the metagenomic DNA

extracted by methods M1, M2, M4 and M5 usi ng BamHI.

(DOC)

S5 Fig. Gel electrophoresis of metagenomic DNA extraction by the glass beads, glass pow-

der and glass powder + powdered activated charcoal for four different soils.

(DOC)

S6 Fig. qPCR analysis for method M6.

(DOC)

S1 Table. Characteristics of the four different soil samples.

(DOC)

S2 Table. Suitabi lity of the DNA samples extracted by methods M1 to M6 for 16S rDNA

PCR amplification and partial restriction digestion by BamHI.

(DOC)

Metagenomic DNA Extraction

PLOS ONE | DOI:10.1371/journal.pone.0132441 July 13, 2015 13 / 16

S3 Table. Triplicate values for DNA DNA yield (μg/g of soil) for methods M1 to M6.

(DOC)

S4 Table. Triplicate values for A

260/280

(DOC)

S5 Table. Triplicate values for A

340

(DOC)

S6 Table. Triplicate values for DNA yield (μg/g of soil) for method M2 and its modifica-

tion.

(DOC)

Acknowledgments

The authors acknowledge SRM University for the infrastructure facilities.

Author Contributions

Conceived and designed the experiments: MR. Performed the experiments: SGD SR AAF RA.

Analyzed the data: SGD MR. Contributed reagents/materials/analysis tools: SR AAF. Wrote

the paper: WRC SGD.

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