Optimization of cDNA-AFLP experiments using genomic sequence data.
BACKGROUND
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.
Kivioja
Teemu
T
Department of Computer Science, University of Helsinki, Helsinki, PO Box 68, FIN-00014, Finland. Teemu.Kivioja@vtt.fi
Arvas
Mikko
M
Saloheimo
Markku
M
Penttilä
Merja
M
Ukkonen
Esko
E
D000465:Algorithms; D002874:Chromosome Mapping / Q000379:methods*; D016172:DNA Fingerprinting / Q000379:methods*; D015342:DNA Probes / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D016415:Sequence Alignment / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
D015342:DNA Probes