ArticleBMC genomics2008
Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA.
Article in BMC genomics, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- Isolation and characterization of a novel PDR-type ABC transporter gene PgPDR3 from Panax ginseng C.A. Meyer induced by methyl jasmonate.Molecular biology reports · 2013Article
- Identification of candidate genes associated with senescence in durum wheat (Triticum turgidum subsp. durum) using cDNA-AFLP.Molecular biology reports · 2011Article
- Typing of genetic markers involved in stress response by fluorescent cDNA-amplified fragment length polymorphism technique.Molecular biotechnology · 2010Article
- Eukaryotic transcriptomics in silico: optimizing cDNA-AFLP efficiency.BMC genomics · 2009Article
- Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP).Electrophoresis · 2009Article
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Authors and funding
4 authors.
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Abstract
backgroundcDNA-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.
resultsWith 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.
conclusionSimulation 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.
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