Abstract
Motivation: cDNA amplified fragment length polymorphism (cDNA-AFLP) is one of the few genome-wide level expression profiling methods capable of finding genes that have not yet been cloned or even predicted from sequence but have interesting expression patterns under the studied conditions. In cDNA-AFLP, a complex cDNA mixture is divided into small subsets using restriction enzymes and selective PCR. A large cDNA-AFLP experiment can require a substantial amount of resources, such as hundreds of PCR amplifications and gel electrophoresis runs, followed by manual cutting of a large number of bands from the gels. Our aim was to test whether this workload can be reduced by rational design of the experiment. Results: We used the available genomic sequence information to optimize cDNA-AFLP experiments beforehand so that as many transcripts as possible could be profiled with a given amount of resources. Optimization of the selection of both restriction enzymes and selective primers for cDNA-AFLP experiments has not been performed previously. The in silico tests performed suggest that substantial amounts of resources can be saved by the optimization of cDNA-AFLP experiments.
Original language | English |
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Pages (from-to) | 2573-2579 |
Number of pages | 7 |
Journal | Bioinformatics |
Volume | 21 |
Issue number | 11 |
DOIs | |
Publication status | Published - 1 Jun 2005 |
MoE publication type | A1 Journal article-refereed |
Funding
The authors would like to thank Mr T. Mielikäinen for helpful discussions and Dr T. Pasanen for his comments on the manuscript. This work was supported by the Academy of Finland (grants 201560 and 77304), the European Union Biotechnology Program BIO4CT96-0535, the European Union V Framework Program Eurofung QLK3-CT-1999-00729 and, the research program Life 2000 (Academy of Finland and National Technology Agency of Finland, 2000-2003, Project no. 50023).
Keywords
- cDNA microarrays
- amplified fragment length polymorphism
- AFLP
- gene expression
- genome-wide expression analysis