American Journal of Molecular Biology, 2013, 3, 215-228 AJMB
http://dx.doi.org/10.4236/ajmb.2013.34028 Published Online October 2013 (http://www.scirp.org/journal/ajmb/)
DNA extraction method selection for agricultural soil using
TOPSIS multiple criteria decision-making model
Sepideh Pakpour1, Snizhana V. Olishevska2,3, Shiv O. Prasher2, Abbas S. Milani4,
Martin R. Chénier3,5*
1Department of Biology, University of British Columbia, Kelowna, Canada
2Department of Bioresource Engineering, McGill University, Montreal, Canada
3Department of Animal Science, McGill University, Montreal, Canada
4School of Engineering, University of British Columbia, Kelowna, Canada
5Department of Food Science and Agricultural Chemistry, McGill University, Montreal, Canada
Email: *martin.chenier@mcgill.ca
Received 20 June 2013; revised 22 July 2013; accepted 15 August 2013
Copyright © 2013 Sepideh Pakpour et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
There is an increased interest in the extraction of nu-
cleic acids from various environmental samples since
culture-independent molecular techniques contribute
to deepen and broaden the understanding of a greater
portion of uncultivable microorganisms. Due to diffi-
culties to select the optimum DNA extraction method
in view of downstream molecular analyses, this article
presents a straightforward mathematical framework
for comparing some of the most commonly used
methods. Four commercial DNA extraction kits and
two physical-chemical methods (bead-beating and
freeze-thaw) were compared for the extraction of
DNA under several quantitative DNA analysis crite-
ria: yield of extraction, purity o f extracted DNA (A260/280
and A260/230 ratios), degradation degree of DNA, easi-
ness of PCR amplification, duration of extraction,
and cost per extraction. From a practical point of view,
it is unlikely that a single DNA extraction strategy
can be optimum for all selected criteria. Hence, a sys-
tematic Technique for Order Preference by Simi-
larity to Ideal Solution (TOPSIS) was employed to
compare the methods. The PowerSoil® DNA Isolation
Kit was systematically defined as the best performing
method for extracting DNA from soil samples. More
specifically, for soil:manure and soil:manure:biochar
mixtures, the PowerSoil® DNA Isolation Kit method
performed best, while for neat soil samples its alter-
native version gained the first rank.
Keywords: DNA Extraction; Agricultural Soil; Biochar;
Poultry Manure; Multiple Criteria Decision-Making;
Technique for Order Preference by Similarity to Ideal
Solution
1. INTRODUCTION
Soil is a unique ecosystem containing many different
niches and creating favourable conditions for the devel-
opment of different groups of microorganisms [1,2].
Since less than 1% of soil microorganisms can be grown
in laboratory conditions using culture media [3,4] and the
vast majority are not cultivable [5,6], a significant num-
ber of studies dealing with microbial diversity utilize
molecular tools such as competitive PCR, real-time PCR,
denaturing gradient gel electrophoresis (DGGE) and lar-
ge-scale parallel-pyrosequencing based on the extraction
of environmental nucleic acids [7-11].
Numerous procedures exist for the isolation and puri-
fication of DNA from soil [1,9,10,12-14]. Studies sug-
gest that the selection of an appropriate extraction and
purification procedure depends on the physical and
chemical characteristics of the soil matrix, such as or-
ganic matter, clay content and pH value, as well as on
different amendments like biochar and poultry manure
used in agriculture for improvement of soil fertility [15-
19]. For example, environmental samples such as soil
and sediments often contain high levels of organic matter,
especially humic acids and phenolic compounds, as well
as heavy metals which can inhibit the activity of the Taq
DNA polymerase in PCR [20-22] and reduce the speci-
ficity of DNA hybridization analysis [10,23,24].
Indirect and direct approaches have been developed
for extracting nucleic acids from soil samples. Indirect
extraction of DNA from soil samples is based on the fol-
lowing steps: dispersion of soil particles, separation of
*Corresponding author.
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S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228
216
the cells from soil particles by centrifugation according
to sedimentation velocities, buoyant density or both, lysis
of extracted cells and finally DNA purification [1,25,26].
The most commonly used methods for direct extraction
of DNA are based on physical-chemical membrane dis-
ruption techniques (bead-beating and freeze-thaw) that
may allow greater yields of microbial DNA recovery
[9,12,14,15,20,27,28]. In spite of many advantages of
direct methods, they generally show lower DNA purity
with a high degree of DNA shearing that can negatively
affect PCR efficiency or specificity [1,20,27]. Due to the
fact that some of the steps of DNA extraction using these
direct methods might make DNA isolation expensive or
impractical for processing the large number of samples
usually required in ecological studies, commercial kits
have been increasingly utilized for DNA extraction and
purification from soil and sediments [9,10,14,29-34].
The main goal of this study was to choose a method
that would be inexpensive, able to process several sam-
ples quickly, and capable of obtaining high quality DNA
for PCR studies. Four commercial DNA extraction kits
and two physical-chemical methods (bead-beating and
freeze-thaw) were compared for the extraction of DNA
from a ferro-humic podzol soil amended or not with
poultry manure and biochar. No earlier work suggested a
mathematical decision aid tool to choose the optimum
method for DNA extraction from soil under several con-
flicting criteria. The present work introduces a system-
atic/mathematical approach for comparing the perform-
ance of different DNA extraction methods under a set of
simultaneous “multiple criteria”: yield of extraction, pu-
rity of extracted DNA (A260/280 and A260/230 ratios), deg-
radation degree of DNA, easiness of PCR amplification,
duration of extraction, and cost per extraction. The pro-
posed approach is formally called “multiple criteria deci-
sion-making” or MCDM [35].
In problems dealing with MCDM, which is a branch
of Operations Research (or OR) models, the main goal is
to consider a set of decision criteria and choose the best
performing option from a list of available alternatives
(i.e., options to choose from), which generally show no
obvious dominance one over another with respect to the
criteria (this is formally referred to as Pareto optimality
or Pareto efficiency condition). More precisely, it is as-
sumed that all given alternatives (e.g., here different
DNA extraction techniques) are feasible and there is al-
ways a trade-off in choosing one over another. In other
words, option A may be better than B under some criteria,
but worse under some other criteria. In such complex
decision-making scenarios, MCDM can aid the analyst
(the decision maker) to make a final decision considering
his/her experience, expectations, constraints, etc., into a
systematic mathematical model [35,36]. It is worth add-
ing that next to selection problems, there are also
MCDM models that are applicable to the sorting and
classification problems [37]. The application of MCDM
in decision-making processes in molecular biological
systems, and more especially in comparing DNA extrac-
tion kits, is rather new. Most recently, a basic MCDM
model, called “Weighted Sum Method” (WSM) was used
for comparing a set of sample preservation and DNA
extraction methods from swine feces [38]. Some other
example applications of MCDM in a diverse range of
practical problems include the use of decision analysis in
integrated manufacturing [39], in the evaluation of tech-
nology investment decisions [40], in sustainable energy
planning [41], and in prioritizing urban cultural heritage
values [42].
Despite the fact that WSM is known to be the earliest,
the simplest and probably the most widely used MCDM
method, it has some shortcomings in particular deci-
sion-making cases. Namely, it allows a direct trade-off
(compensation) among the criteria values in evaluating
the performance of each alternative. As a result, it may
choose an alternative that is excellent under some criteria
but at the same time poor or close to unacceptablely un-
der some other criteria, which in turn can induce a risk in
practice, especially under uncertain data or ambiguous
conditions. For instance, a biologist may choose a DNA
extraction kit using WSM that gives a very high yield but
at the same time the purity of extracted DNA may be at a
marginal level, which in turn can pose a primary risk/
concern for that particular decision maker in terms of the
quality of DNA. In turn, more advanced MCDM meth-
ods have been developed over years, among which is the
TOPSIS (the Technique for Order Preference by Similar-
ity to Ideal Solution) method developed by Hwang and
Yoon [43]. It carries several advantages such as “simplic-
ity, rationality, comprehensibility, good computational
efficiency and ability to measure the relative perform-
ance for each alternative in a simple mathematical form”
[44]. Most importantly, compared to other simple
MCDM methods such as WSM, TOPSIS respects the
fact that the decision maker sometimes likes to make as
much profit as possible, but also to avoid as much risk as
possible. The latter desire is satisfied with TOPSIS by in-
troducing the concept of the ideal and negative-ideal
(nadir) solutions. A selected alternative by this method
should have the shortest distance from the ideal solution
and the farthest distance from the negative-ideal solution
in a geometrical sense [43]. The mathematical frame-
work and application of this method in the context of the
optimum DNA extraction selection method for agricul-
tural soils are presented in a later section of the present
article, which is the main motivation of the work. It
should be added that the method is general enough to be
applied to other decision-making processes in biological
systems.
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S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228
Copyright © 2013 SciRes.
217
OPEN ACCESS
2. MATERIALS AND METHODS
2.1. Sample Collection and Preparation
Soil samples were collected in September 2011 at the
Emile A. Lods Agronomy Research Centre of the Mac-
Donald Campus Farm at McGill University, Quebec,
Canada, in the range of 5 - 10 cm depth of A1 horizon
(plowed soil). Collected samples were St-Amable sandy
(Ferro-Humic Podzol) soil with the physical-chemical
characteristics described in Tabl e 1. Soil was air dried at
room temperature to 10% - 20% water-holding capacity.
After sieving (stainless soil sieve with 2-mm mesh
size), soil samples were stored at 4˚C in the dark until
analysis. Poultry manure was collected from the Poultry
Complex of the Macdonald Campus Farm and subse-
quently air dried at room temperature, homogenized and
stored at 4˚C until analysis. Biochar used in this study
was obtained from wood lumbers and wastes by slow
pyrolysis at 450˚C (BlueLeaf Inc., Drummondville, QC,
Canada). Mixtures were formulated with soil:manure
[SM, 99:1(w/w)] and soil:manure:biochar [SMB, 98:1:1
(w/w)] and stored at 4˚C in the dark until analysis.
2.2. DNA Extraction
Total bacterial DNA was extracted from soil, soil:manure
[SM, 99:1(w/w)] and soil:manure:biochar [SMB, 98:1:1
(w/w)] mixtures using six different extraction methods
(Table 2). The latter included two previously described
home-made methods [38], as summarized below (a
bead-beating technique and a freeze-thaw technique), as
well as four commercial kits, as described by the manu-
facturers: PowerSoil® DNA Isolation Kit (MoBio Labo-
ratories, Inc., Carlsbad, CA, USA); UltraClean™ Soil
Table 1. Physical and chemical characteristics of soil.
Soil type Sand (%) Silt (%) pH Bulk density (Mg·m3) Organic matter (%)Cation exchange capacity (cmol·kg1) Hydraulic conductivity (cm·d1)
Sandy 92.2 4.3 5.5 1.350 2.97 4.9 1.67 ± 0.45
Table 2. Comparative analysis of DNA extraction techniques used in this study.
Commercial kits Physical-Chemical
techniques
PowerSoil UltraClean FastSPIN E.Z.N.A.
Bead-Beating Freeze-Thaw
Parameters
conventional alternative conventional alternativeconventionalalternativeconventionalalternative
Sample weight
(mg) 250 250 500 500 100 100
Beads Unknown Unknown Unknown Glass Glass None
Cell lysis solution Buffer contains sodium dodecyl sulphate (SDS)SDS Buffer, MT BufferBuffer of SLX Mlus SDS and proteinase K
FastPrep® Instrument for 40 sec. at speed setting of 6.0 Thermal shock
Cell lysis
technique
Vortex at
maximum
speed for 10
min.
Vortex 3 -
4 sec.
then
heating at
70˚ for 5
min.
Vortex at
maximum
speed for
10 min.
Vortex 3 -
4 sec. then
heating at
70˚ for 5
min.
No buffer,
no incubation
Buffer DS
and
incubation at
70˚ for 10
min.
Buffer DS
and
incubation at
70˚ for 10
min. then at
95˚ for 2 min.
No buffer,
no incubation
Protein removal Patented Inhibitor
Removal Technology Solution S2 Protein Precipitation
Solution Buffer XP1 PCI
Humic acid
removal
Patented Inhibitor
Removal Technology
Inhibitor Removal
Solution None HTR Reagent None
DNA precipitation High concentration salt solution Binding Matrix Isopropanol Polyethyleneglycol
and isopropanol
DNA purification Spin filter with silica membrane SPIN Filter HiBind DNA column CI§
Elution buffer 10 mM Tris-HCl, pH 8.0 10 mM Tris-HCl, pH
8.0 DNase/Pyrogen-Free WaterUnknown
T1E0.1 buffer:
1 mM Tris-HCl,
0.1 mM EDTA, pH 8.0
None
Incubation with
Binding Matrix for
5 min at 55˚C
Final volume (µl) 100 50 50 50 80
PowerSoil: PowerSoil® DNA Isolation Kit; UltraClean: UltraClean™ Soil DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.: E.Z.N.A.®
oil DNA Isolation Kit. PCI: phenol:chloroform:isoamyl alcohol, 25:24:1 (v/v); CI§: chloroform:isoamyl alcohol, 24:1 (v/v).
S
S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228
218
DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad,
CA, USA); FastDNA® SPIN Kit for Soil (MP Biomedi-
cals, LLC, Solon, OH, USA); E.Z.N.A.® Soil DNA Iso-
lation Kit (Omega Bio-Tek, Inc., Carlsbad, CA, USA).
Next to the conventional procedure for each of the four
DNA extraction commercial kits, to reduce DNA shear-
ing and/or increase DNA yields, alternative lysis meth-
ods were assessed as described by the manufacturers
(Tabl e 2), hence resulting in a total of 10 methods inclu-
ding the home-made methods.
Bead-Beating and Freeze-Thaw M eth o d s
Soil, soil:manure [SM, 99:1(w/w)] and soil:manure:bio-
char [SMB, 98:1:1(w/w)] mixtures (0.1 g) were resus-
pended in 1 ml of extraction buffer (500 mМ Tris-НСl
рН 8.0, 100 mM sodium EDTA рН 8.0, 1.5 М NaCl) and
homogenized by vortexing. For disrupting cells by bead-
beating, 0.1 g of 0.1 mm-diameter glass beads (BioSpec
Products, Bartlesville, OK, USA) were added and cells
were disrupted by shaking the tubes for 40 sec (speed =
6.0) in a Fast-Prep (Bio101 Fast-Prep model FP120,
Thermo Savant, Qiagen, Inc., Carlsbad, CA, USA), lea-
ving on ice for 5 min (to counteract heating of the tubes
in the Fast-Prep), and shaking a second time for 40 sec
(speed = 6.0). For disrupting cells by freeze-thaw, three
cycles of freezing in liquid nitrogen (196˚C) for 5 min
and thawing at 65˚C in a water bath for 10 min were
used.
Subsequent procedures for DNA purification after
both bead-beating and freeze-thaw were the same. After
physical disruption of the cells either by bead-beating or
freeze-thaw, 20 μl of proteinase K were added and tubes
were incubated at 37˚C with shaking at 180 rpm for 30
min to digest contaminating proteins. One hundred mi-
croliters of 20% (w/v) SDS (sodium dodecyl sulfate)
were added, tubes were mixed by inverting several times,
incubated at 65˚C for 1 h and centrifuged at 12,000 × g
for 5 min. The supernatants were transferred to new
tubes containing 0.5 volume of polyethylene glycol
(PEG) solution [30% (w/v) PEG, 1.5 M NaCl] and incu-
bated at room temperature for 2 h. Tubes were centri-
fuged at 16,100 × g for 20 min. The pellets were dis-
solved in 90 µl of T1E0.1 buffer (1 mM Tris-HCl, 0.1 mM
EDTA, pH 8.0). Thirty microliters of ammonium acetate
10 M were added, tubes were mixed by inverting several
times and left on ice for 5 min to counteract heating.
Tubes were centrifuged at 16,100 × g for 30 min at 4˚C
to precipitate proteins and polysaccharides. The DNA
was purified from the aqueous phase by phenolchloro-
form-isoamyl alcohol extraction [25:24:1 (v/v)] followed
by chloroform-isoamyl alcohol extraction [24:1 (v/v)].
The DNA was precipitated by adding 0.6 volume of iso-
propanol and incubating at 20˚C for 1 h. DNA was col-
lected by centrifuging at 16,100 × g for 10 min at 4˚C.
The pellets were washed twice with 70% ethanol. After
air drying for about 30 min, the pellets were resus-
pended in 80 μl of T1E0.1 buffer (1 mM Tris-HCl, 0.1
mM EDTA, pH 8.0). The resulting extracts were treated
with 10 μg of RNAse (Invitrogen, Burlington, ON, Can-
ada) for 10 min at 37˚C, and stored at 20˚C.
2.3. Polymerase Chain Reaction Amplification
PCR amplifications were carried out in a VeritiTM Ther-
mal Cycler (Applied Biosystems, Foster City, CA, USA)
using 10 ηg of DNA extracted from each sample by each
DNA extraction technique as template. The V3 region of
the bacteria 16S rDNA was targeted using the Bacteria
universal primers 341F (forward primer:
CCTACGGGAGGCAGCAG) and 534R (reverse primer:
ATTACCGCGGCTGCTGG), which yield amplicons of
about 193 bp [45]. The PCR reaction mixture contained
0.75 μM of each primer, 200 μM of each dNTP (Amer-
sham Biosciences Corp., Piscataway, NJ, USA), 1.25 U
Taq DNA polymerase (Invitrogen, Burlington, ON,
Canada), and the PCR buffer supplied with the enzyme
(10 mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2)
[46]. For each DNA extract, the following series of PCR
tubes were analyzed for the presence of the V3 region of
the bacteria 16S rDNA: 1) triplicate PCR tubes with 10
ηg of extracted DNA; 2) a positive control tube with 10
ηg of DNA extracted from a pure culture of Escherichia
coli ATCC 25922; 3) an inhibition control tube with 5 ηg
of DNA extracted from a pure culture of E. coli ATCC
25922 and 5 ηg of DNA extracted from each sample, in
order to assess the presence of PCR inhibitors in the ex-
tracts; 4) a negative control tube consisting of the reac-
tion mixture without DNA, in order to assess the pres-
ence of external or cross-contamination of the PCR reac-
tion mixtures by DNA.
The PCR conditions were 5 min at 99˚C (initial dena-
turation), then 2 cycles of 5 min at 94˚C (denaturation), 5
min at 55˚C (annealing) and 2 min at 72˚C (extension),
then 28 cycles of 1 min at 94˚C (denaturation), 1 min at
55˚C (annealing), 2 min at 72˚C (extension), and finally
an extension period of 10 min at 72˚C. The size (about
193 bp), specificity (unique band), and abundance of
PCR products were determined by comparison with
DNA standards (GeneRuler 100 bp DNA Ladder, MBI
Fermentas, Burlington, ON, Canada) after agarose gel
electrophoresis [46].
2.4. Identifying a Set of Criteria for Comparing
DNA Extraction Methods
All DNA extractions were performed in 5 replicates. For
each of the following criteria, errors are indicated in Ta-
ble 3 as the standard deviation of 3 replicate measure-
ents on 5 replicate extractions (n = 15). m
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S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228 219
Table 3. Performance of each DNA extraction method under seven decision criteria for soil, SM [soil:manure, 99:1(w/w)] and SMB
[soil:manure:biochar, 98:1:1(w/w)].
Performance/Decision Criteria
C1 C2 C3 C4 C5 C6 C7
Methods Sample
Yield (µg DNA/g
sample) A260/280 ratio A260/230 ratio Degree of DNA
degradation§
Easiness of
amplification#
Duration of
extraction (hrs.)
Cost per extraction
(CAD)††
Soil 8.21 ± 1.32 1.75 ± 0.02 1.30 ± 0.05 2 2 0.83 4.64
SM 14.21 ± 1.24 1.71 ± 0.06 1.43 ± 0.20 2 2 0.83 4.64
UltraClean
Conventional
SMB 13.03 ± 1.23 1.77 ± 0.04 1.21 ± 0.30 2 2 0.83 4.64
Soil 5.59 ± 0.58 1.63 ± 0.23 1.22 ± 0.06 1 2 0.92 4.64
SM 10.77 ± 1.39 1.50 ± 0.04 0.81 ± 0.04 1 2 0.92 4.64
UltraClean
Alternative
SMB 9.40 ± 1.36 1.53 ± 0.06 0.88 ± 0.03 1 2 0.92 4.64
Soil 6.76 ± 0.84 1.67 ± 0.08 1.28 ± 0.18 1 2 1.00 5.54
SM 10.52 ± 1.69 1.96 ± 0.05 1.67 ± 0.13 1 2 1.00 5.54
PowerSoil
Conventional
SMB 8.75 ± 1.43 1.90 ± 0.07 1.57 ± 0.15 1 2 1.00 5.54
Soil 3.78 ± 0.32 1.74 ± 0.34 2.07 ± 0.10 1 2 1.08 5.54
SM 10.48 ± 3.58 1.55 ± 0.06 0.86 ± 0.11 1 2 1.08 5.54
PowerSoil
Alternative
SMB 11.97 ± 2.17 1.55 ± 0.05 0.96 ± 0.11 1 2 1.08 5.54
Soil 17.70 ± 1.72 1.73 ± 0.02 0.25 ± 0.02 3 1 1.16 6.58
SM 20.00 ± 1.64 1.77 ± 0.03 0.39 ± 0.04 3 1 1.16 6.58
FastSpin
Conventional
SMB 23.87 ± 2.60 1.84 ± 0.02 0.60 ± 0.07 3 1 1.16 6.58
Soil 23.29 ± 1.54 1.70 ± 0.02 0.34 ± 0.12 3 1 1.25 6.58
SM 20.36 ± 0.88 1.77 ± 0.04 0.50 ± 0.09 3 1 1.25 6.58
FastSpin
Alternative
SMB 21.45 ± 1.95 1.79 ± 0.01 0.47 ± 0.01 3 1 1.25 6.58
Soil 4.33 ± 0.18 1.67 ± 0.08 0.43 ± 0.42 3 1 3.17 4.38
SM 7.26 ± 0.1.30 1.85 ± 0.06 1.57 ± 0.28 3 1 3.17 4.38
E.Z.N.A.
Conventional
SMB 7.69 ± 0.1.32 1.87 ± 0.06 1.70 ± 0.16 3 1 3.17 4.38
Soil 25.98 ± 4.86 1.56 ± 0.02 0.30 ± 0.20 3 1 3.25 4.38
SM 25.90 ± 2.92 1.57 ± 0.04 0.55 ± 0.20 3 1 3.25 4.38
E.Z.N.A.
Alternative
SMB 15.73 ± 3.53 1.65 ± 0.01 0.47 ± 0.33 3 1 3.25 4.38
Soil 5.24 ± 1.03 0.77 ± 0.05 0.12 ± 0.01 ND 3 7.5 1
SM 6.53 ± 0.36 0.87 ± 0.06 0.11 ± 0.01 ND 3 7.5 1
Bead-Beating
SMB 4.87 ± 0.44 0.78 ± 0.09 0.09 ± 0.01 ND 3 7.5 1
Soil 5.49 ± 1.10 1.13 ± 0.18 0.19 ± 0.07 ND 3 8.5 1
SM 6.21 ± 0.64 0.85 ± 0.07 0.11 ± 0.01 ND 3 8.5 1 Freeze-Thaw
SMB 8.90 ± 2.73 0.86 ± 0.06 0.13 ± 0.02 ND 3 8.5 1
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil® DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.: E.Z.N.A.®
Soil DNA Isolation Kit. PCI; : Each test was repeated five times and the ± values refer to their standard deviations. §Degree of DNA degradation (Lemarchand
et al., 2005): 1 = low (mean fragment size between 23 and 2 kb); 2 = medium (mean fragment size between 23 and 0.5 kb); 3 = high (mean fragment size be-
tween 23 and < 0.5 kb). #1 = low (mean fragment size between 23 and 2 kb); 2 = medium (mean fragment size between 23 and 0.5 kb); 3 = high (mean frag-
ent size between 23 and <0.5 kb); ††Canadian dollars; Not determined since extracted DNA was not visible on the agarose gel stained with ethidium bromide. m
Copyright © 2013 SciRes. OPEN ACCESS
S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228
220
DNA concentration (ng DNA·µl–1), A260/280 ratio (ab-
sorbance at 260 ηm/absorbance at 280 ηm) and A260/230
ratio (absorbance at 260 ηm/absorbance at 230 ηm) of
each extract were determined using a NanoDrop 2000
Spectrophotometer (Thermo Fisher Scientific, Marietta,
OH, USA). The yield for each DNA extraction method
was calculated as follows: Yield of extraction (µg of
DNA/g of sample) = concentration of DNA in the extract
(ηg/µl) × (1 µg/1000 ηg) × final volume of extract (µl)/
dry weight of sample (g).
The A260/280 ratio and the A260/230 ratio were used to
evaluate the purity of DNA extracts. An A260/280 ratio
higher than 1.8 indicates the absence of proteins in DNA
extracts. When the A260/280 ratio is lower than 1.8, pro-
teins or other contaminants (co-extracted with DNA) that
absorb strongly at or near 280 ηm may be present. An
A260/280 ratio over 2.0 indicates RNA contamination of
the sample.
An A260/230 ratio between 2.0 and 2.2 is indicative of
the high purity of extracted DNA. When the A260/230 ratio
is lower than 2, humic acids, carbohydrates, phenol, gua-
nidine HCl or other contaminants that absorb at or near
230 ηm, may be presented [47].
The DNA in each extract was checked for integrity
(degradation degree) by agarose gel electrophoresis by
comparing with Lambda DNA HindIII Digest standards
(New England BioLabs, Ipswich, MA, USA) using Al-
phaEaseFC software version 3.1.2 (AlphaInnotech Cor-
poration, San Leandro, CA, USA). The degradation de-
gree of the DNA in each extract was evaluated using the
scale proposed by Lemarchand et al. (2005): 1 = low
(mean fragment size between 23 and 2 kb); 2 = medium
(mean fragment size between 23 and 0.5 kb); 3 = high
(mean fragment size between 23 and <0.5 kb).
In addition to the above, in this study we used a new
criterion “Easiness of amplification” for comparing the
ten DNA extraction methods (Tab le 2). The number of
PCR bands as well as the presence or absence of Taq
DNA polymerase inhibitors dictated the “Easiness of
amplification”, which was expressed by a quantitative
scheme as follows: [1 = easy to perform (one band de-
tected, absence of inhibitors), 2 = moderately difficult to
perform (1 band detected and presence of inhibitors, or 2
bands detected and absence of inhibitors), 3 = very dif-
ficult to perform (2 bands detected and presence of in-
hibitors, or no band detected)].
Two other criteria, namely duration of extraction and
cost per extraction, were included for a total of seven
decision criteria to compare the ten different DNA ex-
traction methods and choose the best one via a system-
atic mathematical method as follows.
2.5. Multiple Criteria Decision-Making (MCDM):
The Entropy and TOPSIS Techniques
A decision-making process often involves making pref-
erence decisions over multitude alternatives (given op-
tions) that are characterized by multiple, usually con-
flicting criteria (Ahn, 2011). A typical decision matrix, X,
used in MCDM is shown in Figure 1, where Cj represent
the decision criteria (j = 1,···, n); Ai represent the alter-
natives (i = 1,···, m), and xij represent the value of the
i-th alternative under the j-th criterion. Wj (j = 1,···, n)
are the criteria weights, indicating the relative impor-
tance among them.
Among different criteria weight assignment techniques
used in the MCDM field, the “Entropy” method is
among the very few techniques that are independent of
the decision maker’s subjective priorities/judgments. In
the entropy method, the criteria weights are calculated
based on the actual measured data in the decision matrix;
i.e., by means of an objective/statistical process.
Following Section 2.4 in the present study, the ten dif-
ferent DNA extraction methods (alternatives) are to be
compared under seven performance criteria (Cj, j = 1,
2,···, 7) including: (C1) yield of extraction (the higher the
better); (C2) A260/280 ratio (the higher the better); (C3)
A260/230 ratio (the higher the better); (C4) degree of DNA
degradation (the lower the better); (C5) easiness of am-
plification ranking index (the lower the better); (C6) du-
ration of extraction (the lower the better); and (C7) cost
per extraction (the lower the better). Decision weights
using the entropy technique were calculated for all crite-
ria via the following steps [48].
Step 1. Transferring the decision matrix to the nor-
malized mode.
In order to adjust the entropy measure for the j-th cri-
terion, the related values in the decision matrix are first
normalized as Pij;
ij
ij m
ij
i
x
P
x
(1)
Step 2. Calculating the entropy of dataset for each
criterion.
In this step, the entropy of the j-th criterion, Ej, is cal-
culated as follows:

1In
1, 2,,,1, 2,,
m
jijij
i
Eapp
imj



n
mn
(2)
12
12
11 121
1
21 2222
12
()
n
n
n
n
mmm
criteria C CC
weightswww
alternatives
xx x
A
xx xA
A
x
xx









Figure 1. A typical decision matrix
in MCDM.
Copyright © 2013 SciRes. OPEN ACCESS
S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228 221
where, α = 1/ln(m); “m” is the total number of alterna-
tives (in this study, the DNA extraction methods over
different samples);
Next, the operation of subtraction is used to measure
the degree of diversity relative to the corresponding an-
chor value (unity), Dj, using the following formula:
1
j
j
DE (3)
Step 3. Defining criteria weights.
The entropy weight of each criterion is calculated us-
ing:
1
m
j
j
i
WD D
j
(4)
These weights are then incorporated into the so-called
TOPSIS MCDM technique to calculate an overall score
for each DNA extraction method. The TOPSIS technique
was chosen because of its high speed, accuracy, and
compatibility [49]. The algorithm of this technique is
summarized as follows:
1) Transfer the decision matrix to the normalized
mode;

2
1
1,2, ,,1,2, ,
ij
ij m
ij
i
x
rimj
x
 
n
(5)
2) Weigh the normalized decision matrix;

1, 2,,,1, 2,,
ijjij
vWri mjn  (6)
3) Define the “ideal positive”
V and “ideal negative
(nadir)”
V solutions;





12
12
,,,max
min1, 2,,
,,, max
max1,2,,
niij
iij
niij
iij
VVVVj J
Vj Jim
VVVVj J
Vj Jim
 





,
,
(7)
4) Measure the distances, i
d
and i, i = 1, 2,···, n,
from the ideal and negative ideal solutions;
d


12
2
1
12
2
1
,1,2,,
,1,2,,
n
iijj
i
n
iijj
i
dVVi
dVVi



 

 

 


m
m
(8)
5) Determine the relative closeness of alternatives to
the ideal solution;
,1,2,,
i
i
ii
d
Ci
dd


m
(9)
where 0 1. Alternatives with higher magnitudes
of closeness are more preferred.
i
C
i
C
3. RESULTS
3.1. Revealing Conflicts among the Selection
Criteria: A One-Criterion-at-a-Time
Analysis
The performance of the ten DNA extraction methods
under the seven decision criteria for soil, SM [soil:ma-
nure, 99:1(w/w)] and SMB [soil:manure:biochar, 98:1:1
(w/w)] are displayed in Table 3 (which in fact can be
considered as the given decision matrix in the MCDM
terminology). Altogether, 30 different DNA extracts were
obtained (10 extraction methods for each of the three
sample types). Subsequently, using the values presented
in Tabl e 3, the 10 extraction methods were evaluated by
seven decision criteria for each of the three sample types
and were ranked in Table 4. The ranking results in Table
4 show that the E.Z.N.A alternative extraction method
provided the highest yield of extraction for soil and SM
samples, whereas for SMB samples, the FastSPIN con-
ventional method had the best performance in terms of
yield of extraction compared to the other commercial kits
and home-made physical-chemical techniques.
Under the purity (ratio A260/280 and A260/230) criteria, the
PowerSoil conventional and E.Z.N.A. conventional
methods ranked 1st and 2nd, respectively, for SM and
SMB samples (Table 4). In contrast, under the A260/280
criterion for soil samples, the UltraClean conventional
method gained the first rank, followed by the PowerSoil
alternative method. However, under the A260/230 criterion
for soil samples, the latter method gained the first rank
and the UltraClean conventional method received the
second rank.
The degree of degradation of extracted DNA also var-
ied depending on the extraction technique applied (Table
3). It was the highest when the conventional and alterna-
tive lysis methods of the FastSPIN and E.Z.N.A. kits
were used (Ta bl e 4), and was the lowest using the Pow-
erSoil conventional and alternative methods as well as
the UltraClean alternative method. Among all commer-
cial kits, it is also worth noticing from Tab l e 3 that the
UltraClean kit was the only one for which the alternative
method reduced the degree of DNA degradation.
Under the “Easiness of amplification” criterion, con-
sistent specific PCR amplification (unique band) of ~193
bp amplicons corresponding to the V3 region of the Bac-
teria 16S rDNA was successfully obtained for DNA ex-
tracted from soil, SM and SMB mixtures using both
conventional and alternative methods of FastSPIN and
E.Z.N.A. (results not shown): hence they resulted in the
top rank under this criterion (Tabl e 4 ). In contrast, two
bands were detected after amplification of DNA ex-
tracted from soil, SM and SMB mixtures using both
conventional and alternative methods of UltraClean and
owerSoil commercial kits (results not shown). In addi- P
Copyright © 2013 SciRes. OPEN ACCESS
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Copyright © 2013 SciRes.
222
OPEN ACCESS
Table 4. Ranks of DNA extraction methods for seven decision criteria; each group of three numbers separated with commas indicate:
soil, SM [soil:manure, 99:1(w/w)] and SMB [soil:manure:biochar, 98:1:1(w/w)].
Ranks in decision criteria
C1 C2 C3 C4 C5 C6 C7
Methods
Yield of extraction A260/280 ratio A260/230 ratioDegree of DNA
degradation
Easiness of
amplification
Duration of
extraction
Cost per
extraction
UltraClean Conventional 4, 4, 4 1, 5, 5 2,3, 3 2, 2, 2 2, 2, 2 1, 1, 1 3, 3, 3
UltraClean Alternative 6, 5, 6 7, 8, 8 4, 5, 5 1, 1, 1 2, 2, 2 2, 2, 2 3, 3, 3
PowerSoil Conventional 5, 6, 8 5, 1, 1 3, 1, 1 1, 1, 1 2, 2, 2 3, 3, 3 4, 4, 4
PowerSoil Alternative 10, 7, 5 2, 7, 7 1, 4, 4 1, 1, 1 2, 2, 2 4, 4, 4 4, 4, 4
FastSpin Conventional 3, 3, 1 3, 3, 3 8, 8, 6 3, 3, 3 1, 1, 1 5, 5, 5 5, 5, 5
FastSpin Alternative 2, 2, 2 4, 4, 4 7, 7, 8 3, 3, 3 1, 1, 1 6, 6, 6 5, 5, 5
E.Z.N.A. Conventional 9, 8, 9 6, 2, 2 6, 2, 2 3, 3, 3 1, 1, 1 7, 7, 7 2, 2, 2
E.Z.N.A. Alternative 1, 1, 3 8, 6, 6 5, 6, 7 3, 3, 3 1, 1, 1 8, 8, 8 2, 2, 2
Bead-Beating 8, 9, 10 10, 9, 10 10, 9, 10 ND* 3, 3, 3 9, 9, 9 1, 1, 1
Freeze-Thaw 7, 10, 7 9, 10, 9 9, 10, 9 ND* 3, 3, 3 10, 10, 10 1, 1, 1
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil® DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.: E.Z.N.A.®
Soil DNA Isolation Kit. Rank of each DNA extraction method for soil, soil:manure, and soil:manure:biochar, respectively, under each decision criterion. *: Not
determined.
tion, no band was detected after amplification of DNA
extracted by the Bead-Beating and Freeze-Thaw tech-
niques. These results imply a requirement for optimiza-
tion of the PCR reaction mixture and/or program, as well
as the necessity for assessing the presence of Taq DNA
polymerase inhibitor(s).
For DNA extracted by both conventional and alterna-
tive methods of the four commercial kits as well as
home-made methods (Bead-Beating and Freeze-Thaw
techniques), the specific amplification of ~193 bp am-
plicons was obtained for each extract in the inhibition
controls (mixture of DNA extracted from E. coli ATCC
25922 and DNA extracted from each sample), indicating
the absence of Taq DNA polymerase inhibitors in the
extracts from these DNA extraction techniques. The ab-
sence of PCR band is in line with the low yields and pu-
rities of DNA extracted with these two techniques (Table
3), making them hold the lowest (3rd) rank under the
easiness of amplification criterion.
The duration of extraction and the cost per extraction
are also very important in selecting an optimum DNA
extraction technique in view of processing a large num-
ber of environmental samples in ecological studies. Un-
der the duration of extraction criterion, UltraClean con-
ventional method obtained the first rank, followed by the
alternative type of UltraClean method (Table 4). The
home-made Bead-Beating and Freeze-Thaw techniques
performed the poorest under all the criteria except for the
cost where they were ranked as the preferred options
(least costly). Since the DNA extracted with the Bead-
Beating and Freeze-Thaw methods was not visible on the
agarose gel stained with ethidium bromide because of
very low concentrations (6.08 and 8.16 ng of DNA/µl of
extract, respectively, Ta bl e 3 ), the degree of DNA deg-
radation could not be determined.
The above comparison of the DNA extraction methods
under each criterion individually (Table 4) clearly de-
monstrates the presence of conflicts among criteria in the
given decision-making problem. An example of such
conflict is for the FastSpin conventional extraction me-
thod which resulted in a good performance in terms of
yield of DNA extraction from soil, SM, and SMB (ranks
of 3, 3, and 1 respectively), but performed poorly under
the degree of DNA degradation criterion. In contrast, the
DNA extracted by the E.Z.N.A. conventional method
offered a good performance under Easiness of amplifica-
tion (rank 1 for all three sample types), but its yield of
extraction was one of the lowest (ranks of 9, 8, and 9 for
soil, SM, and SMB, respectively). Because of such con-
flicts, the MCDM Entropy-TOPSIS approach was
deemed necessary and implemented in order to choose
overall the best DNA extraction method under simulta-
neous decision-making criteria for each specific soil
mixture.
3.2. Multiple Criteria Decision-Making (MCDM):
The Entropy Method
Following Section 2.5, as the first step to the MCDM
solution, criteria importance weights needed to be calcu-
lated for all seven criteria for soil, SM and SMB using
the entropy method according to Formulas 1-4. For ex-
ample, to calculate the weight of criterion C1 for DNA
extraction from soil, the normalization was first per-
formed to calculate pij values using formula 1 (Ta ble 5).
Then, pij × ln(pij) values were calculated (see the exam-
S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228 223
Table 5. Normalized decision matrix for DNA extraction methods for soil.
Normalized Decision Matrix Data (pij)
C1 C2 C3 C4 C5 C6 C7
Methods
Yield of
extraction
A260/280
ratio
A260/230
ratio
Degree of DNA
degradation
Easiness of
amplification
Duration of
extraction
Cost per
extraction
UltraClean Conventional 0.09‡ 0.13 0.18 0.12 0.10 0.07 0.11
UltraClean Alternative 0.06 0.12 0.16 0.06 0.10 0.07 0.11
PowerSoil Conventional 0.07 0.12 0.17 0.06 0.10 0.08 0.13
PowerSoil Alternative 0.04 0.13 0.28 0.06 0.10 0.09 0.13
FastSpin Conventional 0.19 0.13 0.03 0.18 0.15 0.09 0.16
FastSpin Alternative 0.24 0.13 0.05 0.18 0.15 0.10 0.16
E.Z.N.A. Conventional 0.05 0.12 0.06 0.18 0.15 0.25 0.10
E.Z.N.A. Alternative 0.27 0.12 0.07 0.18 0.15 0.26 0.10
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil® DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.: E.Z.N.A.®
Soil DNA Isolation Kit. Example of calculation: Yield of extraction criterion for soil (using Table 3): 0.09 = 8.21/(8.21 + 5.59 + 6.76 + 3.78 + 17.70 + 23.29 +
4.33 + 25.98).
ple of calculation in Table 6) followed by the calculation
of Ej and Dj using Formulas 2 and 3, respectively (Table
6). Finally the weight of each criterion was calculated
using Formula 4. All criteria weights for soil, SM and
SMB are summarized in Table 7. It should be added that
in some cases the decision maker is experienced enough
to have his/her own (subjective) weights, which can be
combined by the (objective) Entropy weights extracted
from Equation (4). The mathematical framework for the
latter combined weighting scheme can be found in [43].
Here it is assumed that the decision maker is inexperi-
enced and/or conservative where he/she has an equal
preference towards the performance criteria and hence
prefers to purely rely on the Entropy weights.
3.3. Selecting an Optimal DNA Extraction
Method for Each Sample Type: The
TOPSIS Method
The obtained weights of criteria were incorporated into
the TOPSIS technique (Formulas 5-9) to calculate an
overall score for each DNA extraction method for soil,
SM, and SMB (Tables 8-12). The final rakings of the
extraction methods for different sample types are shown
in Tables 10-12. Based on these results, the extraction
methods were ranked as follows:
For soil (Ta b le 10, descending order): PowerSoil Al-
ternative > FastSpin Alternative > E.Z.N.A. Alternative >
UltraClean Conventional > PowerSoil Conventional >
UltraClean Alternative > FastSpin Conventional >
E.Z.N.A. Conventional > Bead-Beating = Freeze-Thaw;
For SM (Table 1 1, descending order): PowerSoil Con-
ventional > UltraClean Conventional > UltraClean Al-
ternative > PowerSoil Alternative > FastSpin Alternative
> FastSpin Conventional > E.Z.N.A. Conventional >
E.Z.N.A. Alternative > Bead-Beating = Freeze-Thaw;
For SMB (Table 12, descending order): PowerSoil
Conventional > UltraClean Conventional > PowerSoil
Alternative > UltraClean Alternative > FastSpin Conven-
tional > FastSpin Alternative > E.Z.N.A. Conventional >
E.Z.N.A. Alternative > Bead-Beating = Freeze-Thaw.
4. DISCUSSION
The selection of an appropriate method for extracting
DNA from complex ecosystems such as soil has a critical
impact on the composition and richness of detected mi-
crobial communities using culture-independent molecu-
lar microbiological methods, such as competitive PCR,
real-time PCR, denaturing gradient gel electrophoresis
(DGGE) and Next-Generation DNA Sequencing (NGS)
technologies [9,10,11,29]. In addition, it has been dem-
onstrated that the quality of extracted DNA can interfere
with microarray hybridizations, yielding high back-
ground noise and false positives [9,10,24,50]. These ar-
guments show the necessity for carefully selecting a
suitable DNA extraction method for each given sample
type and proposed downstream DNA-based analyses.
In the present study, we compared both the quantity
and quality of DNA extracted from soil, SM and SMB
mixtures using 10 different DNA extraction methods to
be used subsequently for PCR analysis. The results
showed that both the quantity (yield of extraction) and
the quality (purity, degree of degradation, easiness of
amplification) of the extracted DNA depended on the
extraction method and the type of environmental sample
(Ta bl e 3), which was in agreement with earlier observa-
tions reported in the literature [9,10,14,32,33]. Moreover,
Copyright © 2013 SciRes. OPEN ACCESS
S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228
224
Table 6. Calculating the entropy of data (column-wise) for each decision criterion for soil.
pij × lnpij
C1 C2 C3 C4 C5 C6 C7
Methods
Yield of
extraction
A260/280
ratio
A260/230
ratio
Degree of DNA
degradation
Easiness of
amplification
Duration of
extraction
Cost per
extraction
UltraClean Conventional 0.21 0.27 0.31 0.25 0.23 0.18 0.24
UltraClean Alternative 0.17 0.26 0.30 0.17 0.23 0.19 0.24
PowerSoil Conventional 0.19 0.26 0.30 0.17 0.23 0.20 0.27
PowerSoil Alternative 0.13 0.26 0.36 0.17 0.23 0.21 0.27
Fast Spin Conventional 0.31 0.26 0.11 0.31 0.28 0.22 0.29
Fast Spin Alternative 0.34 0.26 0.14 0.31 0.28 0.23 0.29
E.Z.N.A. Conventional 0.14 0.26 0.16 0.31 0.28 0.35 0.23
E.Z.N.A. Alternative 0.35 0.25 0.19 0.31 0.28 0.35 0.23
Sum 1.84 2.08 1.87 1.98 2.06 1.92 2.07
Ej§ 0.89 1.00 0.90 0.95 0.99 0.92 0.99
Dj# 0.11†† 0.00 0.10 0.05 0.01 0.08 0.01
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil® DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.: E.Z.N.A.®
Soil DNA Isolation Kit. Example of calculation: p11 × lnp11 (using Table 5, Formula 1) = 0.09 × ln(0.09). §Ej = entropy of the set of normalized data. Example
of calculation of E1 where α = 0.48 (Formula 2): 0.65 = 0.48 × [(0.21) + (0.17) + (0.19)) + (0.13) + (0.31) + (0.34)]. #Dj = degree of diversity.
††Example of calculation of D1: 0.35 = 1 0.65 (Formula 3).
Table 7. Criteria weights (Wj, j = 1, 2,···, 7) for soil, SM [soil:manure, 99:1(w/w)] and SMB [soil:manure:biochar, 98:1:1(w/w)].
C1 C2 C3 C4 C5 C6 C7
Soil 0.32 0.00 0.28 0.14 0.03 0.21 0.02
SM 0.16 0.01 0.25 0.21 0.04 0.31 0.03
SMB 0.16 0.01 0.22 0.22 0.04 0.33 0.03
Example of calculation of W1 (weight of yield of extraction for soil, formula 4), using D1 in Table 6: 0.32 = 0.11/(0.11 + 0.00 + 0.10 + 0.05 + 0.01 + 0.08
+0.01).
Table 8. Summary of normalized decision matrix data for TOPSIS method using seven decision criteria for each DNA extraction
method for soil.
Normalized Decision Matrix Data
C1 C2 C3 C4 C5 C6 C7
Methods
Yield of
extraction A260/280 ratio A260/230 ratio Degree of DNA
degradation
Easiness of
amplification
Duration of
extraction
Cost per
extraction
UltraClean Conventional 0.20 0.37 0.42 0.30 0.28 0.16 0.31
UltraClean Alternative 0.14 0.34 0.39 0.15 0.28 0.18 0.31
PowerSoil Conventional 0.16 0.35 0.41 0.15 0.28 0.19 0.37
PowerSoil Alternative 0.09 0.37 0.66 0.15 0.28 0.21 0.37
FastSpin Conventional 0.43 0.36 0.08 0.46 0.42 0.22 0.43
FastSpin Alternative 0.56 0.36 0.11 0.46 0.42 0.24 0.43
E.Z.N.A. Conventional 0.10 0.35 0.14 0.46 0.42 0.61 0.29
E.Z.N.A. Alternative 0.63 0.33 0.17 0.46 0.42 0.62 0.29
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil® DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.: E.Z.N.A.®
Soil DNA Isolation Kit. Example of calculation: Yield of extraction criterion for soil (using Table 3): 0.20 = 8.21/[(8.21)2 + (5.59)2 + (6.76)2 + (3.78)2 +
(17.70)2 + (23.29)2 + (4.33)2 + (25.98)2 + (5.24)2 + (5.49)2]0.5.
Copyright © 2013 SciRes. OPEN ACCESS
S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228 225
Table 9. Summary of the weighted normalized decision matrix data for the TOPSIS method using seven decision criteria for each
DNA extraction method for soil.
Vij
C1 C2 C3 C4 C5 C6 C7
Methods
Yield of
extraction A260/280 ratio A260/230 ratio Degree of DNA
degradation
Easiness of
amplification
Duration of
extraction
Cost per
extraction
UltraClean Conventional 0.06 0.00 0.12 0.04 0.01 0.03 0.01
UltraClean Alternative 0.04 0.00 0.11 0.02 0.01 0.04 0.01
PowerSoil Conventional 0.05 0.00 0.11 0.02 0.01 0.04 0.01
PowerSoil Alternative 0.03 0.00 0.19 0.02 0.01 0.04 0.01
FastSpin Conventional 0.14 0.00 0.02 0.06 0.01 0.05 0.01
FastSpin Alternative 0.18 0.00 0.03 0.06 0.01 0.05 0.01
E.Z.N.A. Conventional 0.03 0.00 0.04 0.06 0.01 0.13 0.01
E.Z.N.A. Alternative 0.20 0.00 0.05 0.06 0.01 0.13 0.01
Sum 0.74 0.00 0.67 0.36 0.08 0.51 0.06
V+ § 0.20 0.00 0.19 0.06 0.01 0.13 0.01
V- 0.03 0.00 0.02 0.02 0.01 0.03 0.01
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil® DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.: E.Z.N.A.®
Soil DNA Isolation Kit. Example of calculation: 0.06 = 0.20 (normalized data for UltraClean Conventional under the yield of extraction criterion, Table 8) ×
0.32 (weight of yield of extraction criterion for soil, Table 7). §Ideal positive solution. Ideal negative solution.
Table 10. Summary of the positive and negative distances and
the final TOPSIS scores of DNA extractions methods for soil.
i
d i
d TOPSIS Score : Ci + Rank
Methods
Value Value Value
UltraClean Conventional 0.156 0.140§ 0.474 4
UltraClean Alternative 0.175 0.134 0.433 6
PowerSoil Conventional 0.165 0.136 0.452 5
PowerSoil Alternative 0.172 0.189 0.522 1
FastSpin Conventional 0.181 0.136 0.429 7
FastSpin Alternative 0.164 0.171 0.511 2
E.Z.N.A. Conventional 0.247 0.018 0.068 8
E.Z.N.A. Alternative 0.176 0.174 0.496 3
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil®
DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.:
E.Z.N.A.® Soil DNA Isolation Kit. Example of calculation using Table 9:
0.156 = (0.06 0.20)2 + (0.00 0.00)2 + (0.12 0.19)2 + (0.04 0.06)2 +
(0.01 0.01)2 + (0.03 0.013)2 + (0.01 0.01)2; §0.140 = (0.06 0.03)2 +
(0.00 0.00)2 + (0.12 0.02) 2 + (0.04 0.02)2 + (0.01 0.01)2 + (0.03
0.03)2 + (0.01 0.01)0.5; 0.474 = 0.140/(0.140 + 0.156).
the application of an alternative lysis step for most of the
extraction methods did not improve their performance
significantly, except for the E.Z.N.A. method (Ta b le 3 ).
This method, both its conventional and alternative ver-
sions, resulted in high values of DNA purity. However,
the E.Z.N.A. conventional method provided much lower
yields of extraction in comparison to its alternative method,
as was also observed in other investigations [32,33].
Because of the demonstrated conflicts among different
Table 11. Summary of the positive and negative distances and
the final TOPSIS scores of DNA extractions methods for SM
[soil:manure, 99:1(w/w)].
i
d
i
d TOPSIS Score : Ci + Rank
Methods
ValueValue Value
UltraClean Conventional0.0560.176 0.757 2
UltraClean Alternative0.0870.160 0.647 3
PowerSoil Conventional0.0550.184 0.770 1
PowerSoil Alternative0.0860.153 0.640 4
FastSpin Conventional0.1250.138 0.524 6
FastSpin Alternative 0.1190.133 0.529 5
E.Z.N.A. Conventional0.1680.095 0.362 7
E.Z.N.A. Alternative 0.1830.067 0.268 8
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil®
DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.:
E.Z.N.A.® Soil DNA Isolation Kit.
extraction methods and criteria, the selection of the op-
timum method for different types of soil was not strai-
ghtforward (Table 4). For this reason, for the first time in
the field, the application of a systematic MCDM ap-
proach was proposed and implemented to select overall
the optimum DNA extraction method for each type of
soil. The PowerSoil method was systematically defined
as the best performing method for extracting DNA from
soil samples; more specifically, for soil, the alternative
version of the PowerSoil method gained the first rank
(Table 10), while for SM and SMB its conventional ver-
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S. Pakpour et al. / American Journal of Molecular Biology 3 (2013) 215-228
226
Table 12. Summary of the positive and negative distances and
the final TOPSIS scores of DNA extraction methods for SMB
[soil:manure:biochar, 98:1:1(w/w)].
i
d i
d
TOPSIS Score : Ci + Rank
Methods
Value Value Value
UltraClean Conventional 0.065 0.162 0.714 2
UltraClean Alternative 0.080 0.160 0.666 4
PowerSoil Conventional 0.059 0.172 0.744 1
PowerSoil Alternative 0.072 0.153 0.681 3
FastSpin Conventional 0.108 0.141 0.567 5
FastSpin Alternative 0.116 0.132 0.531 6
E.Z.N.A. Conventional 0.176 0.088 0.333 7
E.Z.N.A. Alternative 0.193 0.030 0.134 8
UltraClean: UltraClean™ Soil DNA Isolation Kit; PowerSoil: PowerSoil®
DNA Isolation Kit; FastSPIN: FastDNA® SPIN Kit for Soil; E.Z.N.A.:
E.Z.N.A.® Soil DNA Isolation Kit.
sion performed best (Tables 11 and 12, respectively).
The performances of the DNA extraction kits assessed
in the present study (Table 3) were similar or better than
those reported in the literature. For the PowerSoil kit,
which ranked first in the present work, the yields of ex-
traction ranged between 3.78 and 11.97 µg of DNA/g of
soil, while the A260/280 and A260/230 ratios varied from 1.55
to 1.96 and 0.86 to 2.07, respectively. Previous studies
reported yields of extraction of 0.12 to 23.0 µg of DNA/g
of soil, as well as A260/280 and A260/230 ratios of 1.34 - 1.65
and 0.55 - 0.61, respectively [10,14,32,33].
Using the UltraClean kit, we obtained yields of extrac-
tion ranging from 5.59 to 14.21 µg of DNA/g of soil,
A260/280 ratios between 1.50 and 1.77, and A260/230 ratios
between 0.81 and 1.43. Other authors presented yields of
extraction between 0.31 and 2.81 µg of DNA/g of soil,
an average A260/280 ratio of 1.33, and A260/230 ratios be-
tween 0.67 and 2.20 [10,29,31,34].
In the present study, the yields of extraction with the
FastSpin kit ranged between 17.70 and 23.87 µg of
DNA/g of soil, whereas the A260/280 and A260/230 ratios
were 1.70 - 1.84 and 0.25 - 0.60, respectively. The lit-
erature indicates lower yields of extraction (0.80 to 9.12
µg of DNA/g of soil), A260/280 ratios (1.53 to 1.64) and
A260/230 ratios (0.24 to 0.28) [10,29-31,33].
Regarding the E.Z.N.A. kit, Table 3 indicates yields of
extraction of 4.33 to 25.98 µg of DNA/g of soil (with
significant improvements using the alternative method),
A260/280 ratios of 1.56 to 1.87, and A260/230 ratios of 0.30 to
1.70. Previous publications included similar or lower
yields of extraction (between 0.60 to 12.5 µg of DNA/g
of soil), similar A260/280 ratios ranging from 1.75 to 1.87,
and generally higher A260/230 ratios varying from 1.59 to
1.87 [32,33].
5. CONCLUDING REMARK
In summary, the choice of a DNA extraction method for
microbial ecology studies is critical to obtain reliable
results since each method can affect the composition and
the richness of microbial communities of tested samples.
Hence, in selecting a suitable extraction method, it is
necessary to take into account the type of the environ-
mental sample (in our case, soil, SM, and SMB), quanti-
tative and qualitative characteristics of extracted DNA
(e.g., yield of extraction, purity, degradation degree,
quality of PCR products), and downstream molecular
analyses such as competitive PCR, real-time PCR, dena-
turing gradient gel electrophoresis (DGGE) and large-scale
parallel-pyrosequencing. Based on the results of the per-
formed case study, overall we recommend the Power-
Soil® DNA Isolation Kit as an optimum method for ob-
taining total bacterial DNA from soil and soil-containing
mixtures such as soil:manure and soil:manure:biochar.
The standardization/selection of DNA extraction techni-
ques in the field is a current problem, and hence the po-
werful MCDM approaches such as entropy/TOPSIS
which were used in this study are recommended as a first
step towards comparing similar methods in other studies.
6. ACKNOWLEDGEMENTS
This work was supported by the National Sciences and Engineering
Research Council of Canada (NSERC—Discovery Grant) and the
Fonds de recherche du Quebec sur la Nature et les Technologies
(FRQNT—Team Research Grant Project) for operating funds, and by
the Canada Foundation for Innovation (CFI—Leaders Opportunity
Fund) for infrastructure funds to Martin R. Chénier. Snizhana V. Oli-
shevska benefited from the CFI—Infrastructure Operating Fund for
personal support. The financial support by UBC’s Work-Study Pro-
gram to Sepideh Pakpour is also greatly acknowledged.
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