4.3. Gene Expression Profiling by Arrays
p. 41 in
Bioethics and the Impact of Human Genome Research in the 21st Century
Author: Bunsei Kawakami (Institute Biotechnology, TOYOBO Tsuruga)Editors: Norio Fujiki, Masakatu Sudo, and Darryl R. J. Macer
Eubios Ethics Institute
Copyright 2001, Eubios Ethics Institute
All commercial rights reserved. This publication may be reproduced for limited educational or academic use, however please enquire with the author.
The DNA array, a product of recent technical breakthroughs, is very useful for analyzing the expression of the total genes of a sample, and/or discovering novel genes. In this technique DNA densely arranged and immobilized on a solid-phase base is hybridized with labeled cDNA, and the image obtained is analyzed. As the expression of many genes can be investigated in a single experiment there are great expectations that the development of this technique will contribute to a major issue of the post-sequencing era, the functional analysis of genes and the clarification of their mutual interactions and networking. Many methods using for example slide-glasses, silicon or nylon as the solid-phase base have already been developed, but various drawbacks, false positives, sensitivity (false negatives) etc. still remain. In this lecture, a new DNA array system with substantial improvements with regard to these drawbacks will be introduced.
The greatest problems with present methods of DNA expression analysis using DNA arrays are false positives, sensitivity, reproducibility and cost. The occurrence of false positives, i.e. non-selective binding, has been reduced to a minimal level in our DNA array system by carefully selecting the DNA fragments to be immobilized on the solid phase using a computer-assisted, BLAST search for sequences without repeats and/or sequences homologous to other genes. To achieve the most suitable signal, special attention is also paid to the lengths and the GC contents of the DNA fragments. As to the sensitivity, the PCR-based cDNA labeling used in our DNA array system has increased this significantly, allowing the accurate analysis of the gene expression profiles from a small amount of a sample (as little as 0.1ug of mRNA). The reproducibility and quantitativeness of our system have been improved greatly by the adoption as the solid phase of a nylon membrane that permits the immobilization of DNA in adequate amounts with certainty. A quantifiable result can be obtained with between a few and several thousand copies per cell. The affordable price of our DNA array system has been achieved by the adoption of a CCD camera system instead of a more expensive laser scanner for the analysis of the hybridization images. Some of the actual data obtained by our DNA array system on cancerous tissue etc. will also be presented in the lecture.
(See slides in Japanese version)
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