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

� 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 (3-

methyl-1-butanol) followed by precipitation with

2-propanol. The extracted DNA is purified by mod-

ified 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 CTAB-

based 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,

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

Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-014-1591-5) contains supple-mentary 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: anyingfeng666@163.com

S. Jiang

State Key Laboratory of Forest and Soil Ecology, Institute

of Applied Ecology, Chinese Academy of Sciences,

Shenyang, People’s Republic of China

123

Biotechnol Lett

DOI 10.1007/s10529-014-1591-5

2001; Verma and Satyanarayana 2011). For example,

PEG, powdered activated charcoal (PAC), polyvinyl-

polypyrrolidone (PVPP), and silica-based columns

have been used for soil DNA purification (Martin-

Laurent 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 coef-

ficient of drag alteration (SCODA) instrument (Boreal

Genomics) was used to selectively concentrate high-

molecular 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 Univer-

sity 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 contain-

ing 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

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 Biosci-

ences, Inc., California, USA), respectively. The slur-

ries 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 super-

natants 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 extrac-

tion 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 mea-

sured 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

123

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 break-

ing 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

Fig. 1 Outline of the two-

kit 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)

Biotechnol Lett

123

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 puri-

fication 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 micro-

organisms 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

purification method was efficient to remove contam-

inations 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. Addi-

tionally, 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 purifi-

cation method were successful, while neither restric-

tion 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

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kits (e.g., SoilGen, UltraClean, Powersoil, and Soil-

Master), 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., QIA-

quick, GeneJET, and StrataPrep) would affect the

efficiency of the two-kit purification method. There-

fore, further studies would be necessary to investigate

the efficiency of the method and its scope of applica-

tion. 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.

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

Biotechnol Lett

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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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