Tuesday, November 14, 2006

Development of Microarray-Based PCR for Analysis of Nitrogen Cycling Functional Guilds.

Bruno P. Lima and John Kelly. Loyola University Chicago, 6525 N Sheridan Rd., Chicago, IL 60626

The nitrogen cycle is an important chemical process that controls the availability of nitrogen to plants and other organisms. This process is driven by microorganisms in several functional guilds specific for each step of the cycle. Although great progress has been made in understanding the microorganisms involved with these processes, much is still unknown due to difficulties in cultivating these microbes in the lab. A variety of molecular tools have contributed to the study of these microbes, but there are still limitations to many of these techniques. In an attempt to minimize these limitations we are applying a new strategy to the study of nitrogen cycling organisms. This strategy combines two powerful molecular techniques, microarray analysis and multiplex PCR, to allow parallel amplification and detection of a large number of different genes within a short period of time. Our objective is to optimize this technique to be used to assay microorganisms involved with the nitrogen cycle.  To test this technique, we have created a sequence database for functional genes coding for key enzymes in each of the stages of the nitrogen cycle and we have utilized this sequence database to design PCR primers for each of these functional genes. Reference organisms containing each of these targeted genes have been obtained, cultured in the lab, and their functional gene sequences verified by cloning and sequencing. The primer sets we designed have been tested against these reference organisms using solution phase PCR to determine the effectiveness and specificity of the primers. Several primer sets are now ready to be mounted onto the microarrays chips for further testing.