Isolation of PCR quality microbial community DNA from heavily contaminated environments.
Asphalts, biochemically degraded oil, contain persistent, water-soluble compounds that pose a significant challenge to the isolation of PCR quality DNA. The adaptation of existing DNA purification protocols and commercial kits proved unsuccessful at overcoming this hurdle. Treatment of aqueous asphalt extracts with a polyamide resin afforded genomic microbial DNA templates that could readily be amplified by PCR. Physicochemically distinct asphalt samples from five natural oil seeps successfully generated the expected 291 bp amplicons targeting a region of the 16S rRNA gene, illustrating the robustness of the method. DNA recovery yields were in the 50-80% range depending on how the asphalt sample was seeded with exogenous DNA. The scope of the new method was expanded to include soil with high humic acid content. DNA from soil samples spiked with a range of humic acid concentrations was extracted with a commercial kit followed by treatment with the polyamide resin. The additional step significantly improved the purity of the DNA templates, especially at high humic acid concentrations, based on qPCR analysis of the bacterial 16S rRNA genes. The new method has the advantages of being inexpensive, simple, and rapid and should provide a valuable addition to protocols in the field of petroleum and soil microbiology.
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Manjula
M
Gunawardana
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Simon
S
Chang
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Abraham
A
Jimenez
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Daniel
D
Holland-Moritz
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Hannah
H
Holland-Moritz
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Taylor P
TP
La Val
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Craig
C
Lund
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Madeline
M
Mullen
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
John
J
Olsen
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Terra A
TA
Sztain
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
Jennifer
J
Yoo
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA.
John A
JA
Moss
Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd., Pasadena, CA 91107, USA. Electronic address: m.baum@oak-crest.org.
Marc M
MM
Baum
D003365:Costs and Cost Analysis; D004247:DNA / Q000302:isolation & purification*; D004787:Environmental Pollution; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D023281:Genomics / Q000379:methods*; D009757:Nylons / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000302:isolation & purification; D013997:Time Factors
D004791:Enzyme Inhibitors; D009757:Nylons; D012336:RNA, Ribosomal, 16S; D012989:Soil Pollutants; D004247:DNA
Asphalt; DNA extraction; PCR; Polyamide resin; Polyphenols; Solid phase extraction