Testing the Specificity of Primers to Environmental Ammonia Monooxygenase (amoA) Genes in Groundwater Treated with Urea to Promote Calcite Precipitation

Journal Article · Sun Jan 01 04:00:00 EST 2006 · Journal of Undergraduate Research

OSTI ID:1051817

Freeman, S; Reed, D W; Fujita, Y

Bacterial ammonia monooxygenase (amoA) genes in DNA isolated from microorganisms in groundwater were characterized by amplification of amoA DNA using polymerase chain reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis, and sequencing. The amoA gene is characteristic of ammonia oxidizing bacteria (AOB). The DNA extracts were acquired from an experiment where dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho to examine whether such amendments could stimulate enhanced ureolytic activity. The hydrolysis of urea into ammonium and carbonate serves as the basis for a potential remediation technique for trace metals and radionuclide contaminants that can co-precipitate in calcite. The ammonium ion resulting from ureolysis can promote the growth of AOB. The goal of this work was to investigate the effectiveness of primers designed for quantitative PCR of environmental amoA genes and to evaluate the effect of the molasses and urea amendments upon the population diversity of groundwater AOB. PCR primers designed to target a portion of the amoA gene were used to amplify amoA gene sequences in the groundwater DNA extracts. Following PCR, amplified gene products were cloned and the clones were characterized by RFLP, a DNA restriction technique that can distinguish different DNA sequences, to gauge the initial diversity. Clones exhibiting unique RFLP patterns were subjected to DNA sequencing. Initial sequencing results suggest that the primers were successful at specific detection of amoA sequences and the RFLP analyses indicated that the diversity of detected amoA sequences in the ESRPA decreased with the additions of molasses and urea.

• Research Organization: DOESC (USDOE Office of Science (SC) (United States))
• Sponsoring Organization: USDOE Office of Science (SC)
• OSTI ID: 1051817
• Journal Information: Journal of Undergraduate Research, Journal Name: Journal of Undergraduate Research Vol. 6
• Country of Publication: United States
• Language: English