Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples.Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) [558272/2009-6]Conselho Nacional de Pesquisa e Desenvolvimento (CNPq)Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB)Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB

Ewing

G.V. Lacerda-Junior

J.C.M. Cascardo

J.C.T. Dias

J.H. Amorim

M. Brendel

R.O. Vidal

R.P. Rezende

Sambrook

Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples

Amorim, J. H.

Vidal, R. O.

Lacerda-Junior, G. V.

Dias, J. C. T.

Brendel, M.

Rezende, R. P.

Cascardo, J. C. M.

RCAAP - Repositório Científico de Acesso Aberto de Portugal

A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content

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Genetics and Molecular Research

Universidade de São Paulo

Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP)

  Universidade de São Paulo
 
2012
 
A simple boiling-based DNA extraction for
RAPD profiling of landfarm soil to provide
representative metagenomic content
 
 
GENETICS AND MOLECULAR RESEARCH, RIBEIRAO PRETO, v. 11, n. 1, supl. 1, Part 3, pp. 182-
189, DEC, 2012
http://www.producao.usp.br/handle/BDPI/40902
 
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©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (1): 182-189 (2012)
A simple boiling-based DNA extraction for 
RAPD profiling of landfarm soil to provide 
representative metagenomic content 
J.H. Amorim1, R.O. Vidal2, G.V. Lacerda-Junior3, J.C.T. Dias3,
M. Brendel3, R.P. Rezende3 and J.C.M. Cascardo3
1Departamento de Microbiologia, Universidade de São Paulo, São Paulo, SP, Brasil
2Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, 
Ilhéus, BA, Brasil
3Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brasil 
Corresponding author: R.P. Rezende
E-mail: rezende.rachel@gmail.com
Genet. Mol. Res. 11 (1): 182-189 (2012)
Received September 27, 2011
Accepted December 8, 2011
Published January 27, 2012
DOI http://dx.doi.org/10.4238/2012.January.27.5
ABSTRACT. Landfarm soils are employed in industrial and 
petrochemical residue bioremediation. This process induces selective 
pressure directed towards microorganisms capable of degrading 
toxic compounds. Detailed description of taxa in these environments 
is difficult due to a lack of knowledge of culture conditions required 
for unknown microorganisms. A metagenomic approach permits 
identification of organisms without the need for culture. However, 
a DNA extraction step is first required, which can bias taxonomic 
representativeness and interfere with cloning steps by extracting 
interference substances. We developed a simplified DNA extraction 
procedure coupled with metagenomic DNA amplification in an effort 
to overcome these limitations. The amplified sequences were used to 
generate a metagenomic data set and the taxonomic and functional 
representativeness were evaluated in comparison with a data set 
183
©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (1): 182-189 (2012)
DNA extraction for RAPD profiling of landfarm soil
built with DNA extracted by conventional methods. The simplified 
and optimized method of RAPD to access metagenomic information 
provides better representativeness of the taxonomical and metabolic 
aspects of the environmental samples.
Key words: Landfarm area; Metagenomic; RAPD;
Microbiota representativeness
INTRODUCTION
Landfarm soils are employed in industrial and petrochemical residue treatment by bio-
remediation (Maciel et al., 2007). A range of microorganisms and genes with potential biotech-
nological application may be present in environments such as these, where a selective pressure 
on metabolic pathways involved in the degradation of toxic compounds is intense (Schloss and 
Handelsman, 2003; Maciel et al., 2007). Detailed descriptions of taxa, metabolic pathways, pro-
tein/peptide interactions, and molecular relationships of all microorganisms from environmental 
microbiota present in landfarm soils are not possible using culture-dependent techniques, due to 
the lack of knowledge of culturing conditions for unknown microorganisms (Yun et al., 2004; Ri-
esenfeld et al., 2004). Thus, a metagenomic approach has been indicated as a way to further access 
data contained in these ecosystems (Johnson and Slatkin, 2006; McHardy and Rigoutsos, 2007).
The term metagenomic derives from the concepts of meta-analysis and genomic 
(Schloss and Handelsman, 2003). This technology allows access to data at the phylogenetic 
level and the isolation and cloning of novel genes, with several biological functions, such as 
enzymatic, antimicrobial and antitumor (Streit and Schmitz, 2004; Roh et al., 2006; McHardy 
and Rigoutsos, 2007). Accessing metagenomic data is based on conventional DNA extraction 
methods that usually bias the representativeness of microbial taxa (Robe et al., 2003; Daniel, 
2004; Roh et al., 2006). Besides, conventional cloning steps based on the cloning of polymerase 
chain reaction (PCR) products or large metagenomic DNA fragments are unfeasible in some as-
pects (Schloss and Handelsman, 2003). The interference of co-extracted substances in the PCR 
procedure or small number of vector copies and difficulties in connecting large DNA fragments 
to vectors are points to be considered (Robe et al., 2003; Roh et al., 2006; Amorim et al., 2008). 
Another aspect that limits the study of metagenomic content concerns the bioinfor-
matics methods used to analyze the genomic data generated (Rondon et al., 2000; Roh et al., 
2006; Huson et al., 2007). Notably, most information is computed by methods that depend on 
sequences of the same gene to determine taxonomic and metabolic profiles of the environment 
(Rondon et al., 2000; Roh et al., 2006). Thus, they do not allow the determination of taxo-
nomic and metabolic content at the same time (Rondon et al., 2000; Roh et al., 2006). 
Here, we present a low-cost and practical alternative to accessing metagenomic con-
tent by boiling the soil sample and directly applying the random amplified polymorphic DNA 
(RAPD) technique to cover the metagenome of a landfarm area as much as possible, with a 
subsequent cloning step of the RAPD products into pTZ57R/T vectors, to finally construct a 
metagenomic library. A robust bioinformatic analysis was carried out focusing on open read-
ing frames (ORFs) to compare the taxonomic and metabolic representativeness of metage-
nomic libraries constructed using traditional or boiling/RAPD-based approaches. The results 
presented in this study suggest new conditions to properly access metagenomic content.
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J.H. Amorim et al.
MATERIAL AND METHODS
Soil sampling 
A soil sample was collected at Landulpho Alves Refinery, in São Francisco do Conde, 
Bahia, Brazil. Sampling was performed as described elsewhere (Maciel et al., 2007).
DNA extraction and cloning of RAPD products 
In a 1.5-mL sterile tube, 1 g landfarm soil was suspended in 1 mL sterile ultrapure 
water, followed by a boiling step of 20 min. Afterwards, the tube was centrifuged for 10 min 
at 13,400 rpm. The supernatant was kept and aliquots were diluted in sterile ultrapure water 
to 1:10, 1:50, 1:100, and 1:200, to optimize the best condition to obtain the highest yield of 
metagenomic RAPD product. The best dilution was directly used to perform RAPD reactions 
with the primers OPC01, OPK08, OPI07, OPN18, OPN19, OPK20, OPB13, OPS07, OPS10, 
D13, T17, R08, Q08, OPJ04 E T20 (Operon kit, Gibco), with the following reagents and con-
ditions: 0.975 U Taq DNA polymerase; 3.5 mM MgCl2; 0.48 mM dNTPs; 2.5 ng RAPD primer; 
0.5 mM BSA and DNA (4 µL of best dilution) to a final volume of 12.5 µL. A conventional 
metagenomic DNA extraction procedure was performed as previously described (Amorim et 
al., 2008). The amount of DNA extracted by this method used as template in RAPD reactions 
was determined by screening dilutions, as done with DNA extracted by simple boiling. RAPD 
products were ligated into the T/A cloning site of the vector pTZ57R/T, according to manufac-
turer instructions (InsTAclone PCR cloning kit, Fermentas). Plasmids ligated to RAPD prod-
ucts were then transformed into chemically competent Escherichia coli TOP10 and recombi-
nant bacterial colonies were selected by blue/white colony screening (Sambrook et al., 1989).
Sequencing and construction of metagenomic libraries 
From 1600 selected colonies, 800 were used to clone DNA extracted by the conven-
tional method and 800 were used to clone DNA extracted by simple boiling. The clones were 
individually cultured in Luria-Bertani medium containing ampicillin (100 µg/mL) in 96-well 
culture plates and then submitted to plasmid DNA extraction (Sambrook et al., 1989). Extracted 
plasmids were then used as templates to perform sequencing reactions, which were read in a 
MegaBace 1000 (GE Healthcare). Empty plasmids or plasmids containing inserts up to 100 
bp were discarded. Plasmids containing inserts greater than 100 bp were used to compose 
metagenomic libraries. 
Bioinformatics and statistics
Sequences were treated and filtered by Phred, Crossmatch (Ewing et al., 1998), Lucy 
and RepeatMasker (Smit et al., 2004; http://www.repeatmasker.org) and after they were mined 
by the Getorf program version 5.0.0 (from EMBOSS program family). ORFs with a minimum 
of 50 amino acids were further submitted to similarity analysis using BLASTP against the 
NR database. Sequences with a hit with a minimal e-value of 1e-7 were accepted. Selected 
sequences were then blasted against NR (NCBI non-redundant database), GO (Ashburner 
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DNA extraction for RAPD profiling of landfarm soil
et al., 2000; Gene Ontology http://www.geneontology.org) and KEGG (Kanehisa and Goto, 
2000; Kyoto Encyclopedia of Genes and Genome http://www.genome.jp/kegg) to determine 
the taxonomic, functional and metabolic profiles, respectively. The results of these annotations 
were inserted into the MEGAN software (Huson et al., 2007). Statistical analyses were per-
formed using two-way ANOVA, with statistical significance set at P < 0.05. MEGAN analysis 
corresponding to taxonomic, functional or metabolic profiles of clones from DNA extracted 
by the two different methods was performed. 
RESULTS AND DISCUSSION
The aim of this study was to establish a simple and low-cost process to access the 
metagenomic content of impacted soils. The best conditions to allow cloning of RAPD prod-
ucts were determined. Hence, cloning RAPD products allowed not only the representation of 
different taxa, but also different biological functions and metabolic pathways.
The best conditions to obtain the highest metagenomic amplified DNA yield by 
RAPD were established using template DNA extracted by simple boiling at a 1:200 dilu-
tion (Figure 1A). The same conditions were shown to be more suitable for amplifying DNA 
extracted by the conventional method (Figure 1B). Using this dilution, co-extracted interfer-
ence substances (Amorim et al., 2008) were also diluted, probably allowing better annealing 
of primers and efficient action of DNA polymerase. After cloning, sequencing and sequence 
treatment, metagenomic sequence sets were determined; 500 reads regarding DNA extracted 
by the conventional method, labeled as Land 1, and 570 reads regarding DNA extracted by 
simple boiling, labeled as Land 2.
Figure 1. Optimization of RAPD conditions using A. DNA extracted by simple boiling method or B. DNA extracted 
by conventional method, as template. Reactions were performed with the following DNA dilutions: lane 1 = 1:10; 
lane 2 = 1:50; lane 3 = 1:100; lane 4 = 1:200. Lane M is the molecular weight marker, with molecular weights 
shown on the left side of the figure. Lane 5 corresponds to the negative control of RAPD reactions.
Both, taxonomic and functional representativeness were affected by the DNA extrac-
tion method. As shown in Figure 2, the Land 1 dataset failed to represent several taxa pres-
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J.H. Amorim et al.
ent in the landfarm soil studied. On the other hand, Land 1 was shown to represent more 
efficiently biological activities present in the environment studied (Figure 3). However, the 
representativeness regarding metabolic pathways was shown to be the same between the Land 
1 and Land 2 datasets (Figure 4). Taken together, these results indicate that the extraction 
method affects metagenomic representativeness in the taxonomic, functional and metabolic 
fields. Moreover, extracting DNA by simple boiling seems to improve the determination of 
taxonomic representativeness of the environment studied.
Figure 2. Representativeness of taxon content by Land 1 and Land 2 datasets. The number of sequences 
representative of each taxon is recorded. Land 2 dataset is shown to be more representative (P < 0.05).
The simple boiling of soil, applied in the construction of Land 2, allows lysis in situ, 
which can permit a more efficient access to DNA (Robe et al., 2003; Roh et al., 2006). As we 
have shown in Figure 2, it was demonstrated that Land 1 represents extra biological activities, 
which can be due to the generation of RAPD products using DNA from a lower number of ge-
nomes of representative taxa as template. This may allow primers to anneal in a larger number 
of gene families, subsequently amplifying them. The better representativeness of metabolic 
content observed in the Land 2 dataset confirms that disruption of bacterial cells in situ pro-
motes more availability of DNA than using the conventional DNA extraction method. A pos-
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DNA extraction for RAPD profiling of landfarm soil
sible cause of this may be the fact that in ex situ extraction methods a considerable number of 
bacterial cell DNA donors may be discarded in the process of separating cells and soil particles 
(Robe et al., 2003; Roh et al., 2006).
Figure 3. Representativeness of biological activity content by Land 1 and Land 2 datasets. The lighter the area 
is, the more sequences related to the corresponding biological activity are scored. Dark areas mean lack of 
representativeness.
Land 2 showed a higher diversity of microorganisms in comparison to Land 1 and 
was shown to be more representative (Figure 2). However, it is important to note that neither 
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J.H. Amorim et al.
represents an entire sample, as there are missing taxa in both samples. Reflecting the saturation 
problem, both samples show some exclusive biological activities and metabolic pathways, 
although Land 2 appears to be more representative (Figures 3 and 4). Regarding the biological 
activities, we noted the presence of some related to biological transport, such as ATP-binding 
cassette transporter, gluconate transporter and two-component sensor. The biosynthetic activ-
ity was also assigned in a significant number together with unknown biological processes. 
Together, these results indicate the potential of this environment as a possible reservoir of 
unclassified transport mechanisms and source of new molecules with biotechnological ap-
plications. For example, new substances involved in biosynthetic or catabolic pathways could 
contribute to improving some important industrial processes (Rondon et al., 2000; Roh et al., 
2006; McHardy and Rigoutsos, 2007).
Figure 4. Representativeness of metabolic pathways contained in the environment studied. The number of 
sequences representative of each taxon is recorded. Land 2 dataset is shown to be more representative (P < 0.05).
Regarding the pathways in both samples, we noted the presence of some related to nitro-
gen metabolism, a substance regularly added as a nutrient to landfarms to favor microorganism 
metabolism and growth (Maciel et al., 2007). Although the presence of other pathways such 
as butanoate, propanoate and sulfur pathways, related to the degradation of toxic compounds, 
deserves attention, a predominant amount of no hits in this analysis corroborates the possibility 
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DNA extraction for RAPD profiling of landfarm soil
of this environment being a reservoir of new metabolic pathways and compounds, with the most 
diverse biotechnological applications, a result also found in the analysis of biological activities. 
CONCLUSIONS
The simplified and optimized method presented here to access metagenomic data was 
shown to better determine the representativeness of the environment studied, in taxonomic and 
metabolic fields, allowing a more efficient profiling of the landfarm area. Data presented in 
this study suggest new conditions to properly access metagenomic content.
ACKNOWLEDGMENTS
We should always thank Professor Julio Cesar de Mattos Cascardo, a great scientist and 
best friend, who devoted his life to science and teaching. Julio passed away in 2010. Research sup-
ported by Conselho Nacional de Pesquisa e Desenvolvimento (CNPq, #558272/2009-6) and Fun-
dação de Amparo à Pesquisa do Estado da Bahia (FAPESB, scholarship granted to J.H. Amorin). 
REFERENCES
Amorim JH, Macena TN, Lacerda GV Jr, Rezende RP, et al. (2008). An improved extraction protocol for metagenomic 
DNA from a soil of the Brazilian Atlantic Rainforest. Genet. Mol. Res. 7: 1226-1232.
Ashburner M, Ball CA, Blake JA, Botstein D, et al. (2000). Gene Ontology: tool for the unification of biology. The Gene 
Ontology Consortium. Nat. Genet. 25: 25-29. 
Daniel R (2004). The soil metagenome - a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol. 
15: 199-204.
Ewing B, Hillier L, Wendl MC and Green P (1998). Base-calling of automated sequencer traces using phred. I. Accuracy 
assessment. Genome Res. 8: 175-185.
Huson DH, Auch AF, Qi J and Schuster SC (2007). MEGAN analysis of metagenomic data. Genome Res. 17: 377-386.
Johnson PL and Slatkin M (2006). Inference of population genetic parameters in metagenomics: a clean look at messy 
data. Genome Res. 16: 1320-1327.
Kanehisa M and Goto S (2000). KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28: 27-30.
Maciel BM, Dias JC, Dos Santos AC, Argolo Filho RC, et al. (2007). Microbial surfactant activities from a petrochemical 
landfarm in a humid tropical region of Brazil. Can. J. Microbiol. 53: 937-943.
McHardy AC and Rigoutsos I (2007). What’s in the mix: phylogenetic classification of metagenome sequence samples. 
Curr. Opin. Microbiol. 10: 499-503.
Riesenfeld CS, Schloss PD and Handelsman J (2004). Metagenomics: genomic analysis of microbial communities. Annu. 
Rev. Genet. 38: 525-552.
Robe P, Nalin R, Capellano C, Vogel TM, et al. (2003). Extraction of DNA from soil. Eur. J. Soil. Biol. 39: 183-190.
Roh C, Villatte F, Kim BG and Schmid RD (2006). Comparative study of methods for extraction and purification of 
enviromental DNA from soil and sludge samples. Appl. Biochem. Biotech. 134: 97-112.
Rondon MR, August PR, Bettermann AD, Brady SF, et al. (2000). Cloning the soil metagenome: a strategy for accessing 
the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66: 2541-2547.
Sambrook J, Fritsch EF and Maniatis T (1989). Molecular Cloning: A Laboratory Manual. 3rd edn. Cold Spring Harbor 
Laboratory Press, New York.
Schloss PD and Handelsman J (2003). Biotechnological prospects from metagenomics. Curr. Opin. Biotechnol. 14: 303-310.
Smit AFA, Hubley R and Green P (2004). RepeatMasker Open-3.0. 1996-2007. 
Streit WR and Schmitz RA (2004). Metagenomics - the key to the uncultured microbes. Curr. Opin. Microbiol. 7: 492-498.
Yun J, Kang S, Park S, Yoon H, et al. (2004). Characterization of a novel amylolytic enzyme encoded by a gene from a 
soil-derived metagenomic library. Appl. Environ. Microbiol. 70: 7229-7235.


A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content

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