|FORTUNA, ANN-MARIE - WASHINGTON STATE UNIV.
|GUNNING, KERRY - INTEGRATED DNA TECHNOLOG
Submitted to: American Society of Agronomy Meetings
Publication Type: Abstract Only
Publication Acceptance Date: 9/15/2008
Publication Date: 10/5/2008
Citation: Fortuna, A., Gunning, K., Honeycutt, C.W. 2008. Comparison of DGGE and Microarray Technologies for the Detection of 16S rRNA Obtained from Beta-Ammonia Oxidizer Cultures and Soil. American Society of Agronomy Meetings. Published on CD-Rom.
Technical Abstract: Methods linking environmental function and microbial community structure are limited by phylogenetic resolution and throughput time. Many keystone species are uncultured and account for less than 1% of the microbial population in soil. Rapid genomic screening tools select for organisms present in large numbers and provide poor or limited resolution of species. Denaturing gradient gel electrophoresis allows detection of strains separated by a one base pair in rRNA sequence but sample preparation time is lengthy. Microarray technologies are high throughput and can provide resolution of microbial strains. We have designed a microarray to identify environmental strains of beta-ammonia oxidizers using 16S rRNA sequences. Probe design is based on 74 reference organisms isolated from cultured and all uncultured organisms using DGGE primers, CTO189f-GC and CTO654r. A set of PCR amplicons was obtained from pure culture reference organisms representing 6 known beta-ammonia oxidizer cluster groups. Amplicons obtained with Cy3 labeled CTO primers were hybridized to the microarray. Control organisms were added to the microarray as a single template and amplicon mixtures of varying concentration. Mixtures were used to simulate environmental sample conditions using known sequences and concentrations. An internal control organism absent from soil was added to all environmental samples prior to hybridization on the microarray. A second set of amplicons generated with CTO primers containing a GC clamp were resolved using a DGGE system. Preliminary data indicate that microarray technology can provide information on the eveness of community members and resolution of beta-ammonia community structure comparable to that of DGGE.