Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA.
BACKGROUND
cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively.


RESULTS
With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation) was written in Perl. cDNA-AFLP protocols described in the literature and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set.


CONCLUSIONS
Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.
Molecular Phytopathology and Mycotoxin Research Division, University of Goettingen, Goettingen, Germany. aweiber1@gwdg.de
Arne
A
Weiberg
Dirk
D
Pöhler
Burkhard
B
Morgenstern
Petr
P
Karlovsky
D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D003198:Computer Simulation; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012984:Software
D017931:DNA Primers; D018076:DNA, Complementary; D004262:DNA Restriction Enzymes