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.