Transcript profiles and genotyping of cancer tissue

Excerpt

Gene expression profiling has become one of the most attractive approaches to elucidate gene function. For these purposes, hybridization methods and SAGE have been the most commonly used techniques. We have developed an alternative strategy for cDNA tag analysis that results in a quantitative estimate of gene expression. The strategy relies on generation of 3'-tagged cDNA libraries and a new non-gel-based high throughput DNA-sequencing principle, pyrosequencing. Pyrosequencing is based on a sequencing by synthesis strategy in which single specific nucleotides are added to an extension substrate in the presence of a DNA polymerase. Incorporation is detected in real-time through an enzymatic cascade that produces a quantitative light signal, measured by a CCD-camera. A microtiter format is used, allowing sequencing of 96 samples within 40 min. In total, 2000 clones from a human tissue model system have been analysed by both conventional DNA sequencing and pyrosequencing. For the analysis of only a few cells, a cDNA amplification step, keeping the relative transcript levels, is used in the generation of the libraries. Furthermore, an SNP analysis technique based on pyrosequencing and the p53 tumor suppressor gene has been developed, that will allow correlation between expression and genotype of cancer tissue. The quantitative gene expression profiles from compared libraries are visualized by virtual chip technology. …