Plant & Animal Genome IX Conference Plant & Animal Genome IX Conference
Poster: Sequencing & EST
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The Center for Animal Functional Genomics (CAFG) at Michigan State University is dedicated to production, dissemination, and application of cDNA microarrays to address complex biological problems in livestock and companion animals. As part of its mission, the CAFG has prepared normalized cDNA libraries to facilitate studies on complex production diseases in cattle (e.g., mastitis and Johne's disease) and muscle development and meat quality in swine. Bovine leukocyte and swine skeletal muscle libraries were constructed using a commercial system (Life Technologies, Inc. Gaithersburg, MD) and amplified in semi-solid media to preserve relative clone abundance. Amplified libraries were normalized using single-stranded DNA circles and controlled hybridization to reduce the prevalence of high-abundance transcript clones. Effectiveness of normalization was assayed by DNA sequencing of >200 random clones picked pre- and post-normalization, and by Southern blot hybridization using probes representing different mRNA abundance classes. Normalization increased the relative abundance of small clones and empty vectors, highlighting the need for size selection. Final libraries were plated and colonies robotically picked and subjected to high-throughput DNA sequence analysis. Clone data, including BLAST results and keywords are stored in a web-accessible database that includes an array building tool to direct re-racking and arraying efforts. Our system utilizes genes or gene fragments identified by DDRT-PCR and verified to be differentially expressed in specific systems as landmarks, genetic elements that change expression level in known ways that will serve as quality controls during future array hybridizations