- PersatuanGenetik Malaysia

ORAL PRESENTATIONS: MICROB|AL GENETTCS
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DNA Extractions and PCR Amplifications of Agriculture and Virgin
Forest Soils from Malaysia for Construction of Metagenomic Libraries
Seri lntan Mokhtar, Rafidah Saadun, lsnazunita lsmail, Rohani Hashim
Environmentatandrilrl#"""1o.:n,ji":,Elt"1?:sHMBerhad,Nol
Persiaran Dato' Menteri, section 2, P.o. Box 7035, Shah Alam 40911,
Selangor, Malaysia
E-mail: intan
@
sirim.mv
lntroduction
Most microbes that are being identified and used today come from soil-dwelling
microbes cultured in laboratories (Rondon et al., 1999) and represent only 0.1 to 1 %
of their total number (Handelsman, 2005). The inability to culture most microbes from
environmental samples is a fundamental obstacle in understanding microbial ecology
and diversity. Metagenomics is a culture-independent genomic analysis of microbial
communities that can be used to overcome this obstacle. lt opens a window to assess
the remaining gg "/o of the total number organisms in the soil (Schloss and
Handelsman, 2003). However, some techniques used to extract DNA from soil may
result in contamination of the DNA with humic acid that interJeres the subsequent
molecular biological manipulations (Yeates et. a1.,1998). This work describes the use
of several methods for DNA extraction and the assessment of the DNA quality and
diversity by PCR amplification prior to the construction of metagenomic libraries.
Materials and Methods
Soil samples were obtained from three locations: Cameron Highlands, Pahang,
Gunung Mandi Angin, Terengganu and Pulau Perak, Kedah. Soil samples from Cameron
Highlands were obtained from vegetable and flower farms while those from Gunung
Mandi Angin and Pulau Perak were virgin forests.
Soil samples collected from Cameron Highland were kept on ice during sampling
and kept frozen at -20'C prior to DNA extraction except for Epicentre extraction method
where the DNA was extracted fresh from soil. Soil samples collected from Gunung Mandi
Angin were left at room temperature for 5 days during sampling expedition before being
trozen al -70 oC for four months prior to DNA extraction. Soil samples received from
Pulau Perak were kept at room temperature for two months prior to DNA extraction.
Physico-chemical analyses of soil samples collected from Cameron Highland were
carried out to determine soil properties. Genomic DNA were extracted from soil samples
using SoilMaster DNA Extraction Kit (Epicentre), QlAamp DNA Stool Mini Kit and QlAamp
DNA Blood Midi Kit (Qiagen), three modified methods from Yeates et. al. (1998) and one
from Vazquez-Marruf o et. al. (2002). Genomic DNA was assessed using polymerase
chain reaction (PCR) using three pairs of primers namely, prokaryotic 16 S rDNA, Fungal
28 S rDNA and Eukaryotic rDNA (lTS) region.
Results and Discussions
Soil handling and storage prior to DNA extraction were important factors to be
considered in order to prevent the degradation of DNA in the soil samples. Soil samples
obtained from Cameron Highlands were categorized as silt, loam or both. The majority of
the samples were acidic to near neutral in pH with organic matter contents of below 10%.
Results show that all the methods used allow the successful extraction of DNA except for
one method, namely, QlAamp DNA Stool Mini Kit and QlAamp DNA Blood Midi Kit
(Qiagen) However, when PCRs were carried out, only products from the DNA extracted
by Soilmaster DNA Extraction Kit (Epicentre) were obtained. Approximately 1.4 kb PCR
Proceedings of the dh National Congress on Genetics,
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2-14 May 2005, Kuala Lumpur
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ORAL PRESENTATIONS: MICROBIAL GENETICS
product was amplified using 16 S rDNA primers, 550-650 kb PCR product was amplified
using Fungal 28 S rDNA primers and 1 kb PCR product was amplified using Eukaryotic
rDNA (lTS) region primers.
Acknowledgement
Authors are grateful to the Malaysian Government for providing funds through
National Biotechnology Directorate (NBD) Project no. IRPA 09-02-02-008-BTi(ER-36 to
carry out this research.
References
Handelsman, J. (2005). "Sorting out metagenomes". Nature Biotechnology, Vol. 23, No. 1,
p.38-39.
Rondon, M. R., Goodman, R. M. and Handelsman, J. (1999). "The eafth's bounty:
Assessing and accessing soil mirobial diversity". TibTech, Vol. 17, p. 403-409.
Schloss, P. and Handelsman, J. (2003). "Biotechnological prospects from metagenomic".
Current opinion in Biotechnology, Vol. 14, p. 303-310.
Vazquez-Marrufo, G., Soledad, M.A., Yazquez-garciduenas, Gomez-Luna, B.E. and
Olalde-Portugal, V. (2002). "DNA lsolation from Forest Soil Suitable for PCR Assays of
Fungal and Plants rRNA Genes. Plant Molecular Biology Reporte/'. 20:379-390.
Yeates, C., Gillings, M.R., Davison, A.D., Altavilla, N and Veal, D.A. (1998). "Methods for
Microbial DNA Extraction from Soil for PCR Amplification". Biological Procedure Online
Vol.1, No. 1 . http://www.biologicalprocedures.com.
Proceedings of the dh National Congress on Genetics, 12-14 May 2005, Kuata
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