Supporting Genome and Post-Genome Research by High-Throughput Technologies
The information flow in a living cell proceeds from DNA (genes) to mRNA (transcripts) to proteins to metabolites. With recent techniques we are able to determine the entirety of these molecules in living systems, called genome, transcriptome, proteome, and metabolome, respectively.
Technologies and Research
The Technology Platform Genomics (TPG) comprises four sections: genomics, transcriptomics, proteomics, and metabolomics, the most important technologies in genome and post-genome research.
Genomics at the TPG mainly comprises genome sequencing. Two of the most recent high-throughput sequencing systems, the Roche Genome Sequencer flx and the Illumina Genome Analyzer GA IIx, are currently installed and running. These sequencing machines have been used to sequence the entire genomes of bacteria and yeasts and are currently applied to even larger genomes as that of plants or the Chinese Hamster and to complex DNA samples like meta-genomes.
Transcriptomics at the TPG cover all techniques used to analyze transcripts, with a focus on mRNA. This includes determination of gene expression by microarrays as well as gene-specific, quantitative approaches by real-time reverse-transcriptase (RT)-PCR. Currently, transcriptomics also applies sequencing of cDNA generated from reverse-transcribed transcriptomes. Although computationally highly demanding, this method provides unprecedented sensitivity and resolution. The transcriptomics techniques are mostly applied to bacteria, many of them of industrial relevance.
Proteomics at the TPG involve gel-based and non-gel-based separation of proteomes as well as identification and quantification of proteins by tryptic fingerprints and MALDI-TOF mass spectrometry (Bruker ultrafleXtreme) or analysis of protein modification by MALDI-TOF MS/MS or by LC-ESI mass spectrometry. Generally, a first step in proteomics is to fractionate the cellular proteomes. In the bacteria analysed, the extracellular proteome can be separated from the outer or the inner membrane proteome, and the cytosolic proteome fraction. In higher organisms as in Chinese Hamster Ovary cells, more subcellular compartments are present and even more proteomic fractions can be generated.
Metabolomics at the TPG embrace metabolic profiling and flux analysis by gas-chromatography (GC) or liquid-chromatography (LC) coupled to mass spectrometry (MS). At the TPG, routinely GC-MS analysis is applied to samples obtained from bacteria, plants and even from humans. GC-MS is very sensitive and able to separate hydrophobic molecules. The coupling to mass spectrometry again allows the determination of masses of specific ions and comparison with mass spectral databases helps in identification of a specific substance. The TPG is running three GC or GCxGC mass spectrometers. However, a number of very hydrophilic molecules can not be separated by GC-MS. Therefore, LC-MS is an ideal complement to GC-MS, since it enables the separation of water-soluble molecules. Two LC-MS instruments are available at the TPG, a quadrupole TOF and an ESI ion-trap mass spectrometer.