The goal of this module was to familiarize the participants with a variety of bioinformatic approaches to predict gene content and structural characteristics encoded within the DNA of every organism.  In order to reach this goal the participents were involved with the shotgun sequencing a BAC clone from Brassica rapa.  This exercise also allowed the participants to contribute to the ongoing Brassica rapa sequencing project. Prior to the beginning of the workshop, the sequencing library was generated and approximately 700 sequences were obtained.  The objective was to have the workshop participants isolate and sequence DNA from approximately 300 additional clones in order to analyze a data set of 1000 total sequences.

The participants successfully utilized the robotic equipment located at the PMGF to isolate and sequence DNA in a high-throughput manner.  The returned sequences were then analyzed by a variety of software packages such as DNAStar SeqMan II and web based servers.  During the initial analysis, it was discovered that over 90% of the returned sequences were rRNA genes.  These genes have been shown to be arranged in a highly repetitive fashion.  Due do this fact, the alignment these sequences will prove to be extremely difficult.

The goal of this module was to introduce the participants to the methods and data analysis of gene expression using Affymetrix microarrays.  In this module, the participants were to isolate RNA from mutant and wild type Arabidopsis thaliana plants.  After successful isolation, the RNA was then to be converted into cDNA and finally into biotin labeled cRNA.  The samples were then to be taken to the micorarry facility at Children’s Hospital in Columbus for hybridization. 

The participants were able to successfully isolate RNA and generate labeled cRNA for hybridization to the Affymetrix GeneChip.  In order for the successful analysis of the GeneChip data, the participants spent time in the computer lab learning about developments in algorithms for the analysis of large-scale expression data, including statistical analysis, clustering tools, and methods for correlating data with other biological information After a successful hybridization, the data was then analyzed by the participants. 

The goal of this module was to introduce the participants to proteomic approaches using both wet-bench and in silico analysis.  For this module, the participants were to extract total proteins from ten-day-old Arabidopsis seedlings.  After extraction the protein, samples were then to be analyzed by 1-D and 2-D SDS-PAGE at the PMGF.  Next, interesting bands were to be removed and sent to the CCIC for mass spectrometry analysis.  For the in silico analysis, the participants were then to analyze the returned mass spectrometry data along with an introduction to database searching for predicted protein functions, subcellular targeting and topology predictions.

The participants were able to successfully isolate and analyze total protein extracts from Arabidopsis seedlings.  During the 1-D and 2-D analysis, the participants were able to see the inherent variation of performing these types of proteomic methods.  Each group of participants then cut out the same band from both the 1-D and 2-D SDS-PAGE and sent these for analysis by mass spectrometry.  After a successful analysis of the mass spectrometry, each group was then able to determine and compare the quality of their data with the other groups.