Plant Molecular Biology Reporter

, Volume 16, Issue 2, pp 157–157

Transcript Imaging with cDNA-AFLP: A Step-by-Step Protocol

Authors

  • Christian W.B. Bachem
    • Laboratory of Plant Breeding, Graduate School of Experimental Plant SciencesWageningen Agricultural University
  • Ronald J.F.J. Oomen
    • Laboratory of Plant Breeding, Graduate School of Experimental Plant SciencesWageningen Agricultural University
  • Richard G.F. Visser
    • Laboratory of Plant Breeding, Graduate School of Experimental Plant SciencesWageningen Agricultural University
Article

DOI: 10.1023/A:1007468801806

Cite this article as:
Bachem, C.W., Oomen, R.J. & Visser, R.G. Plant Molecular Biology Reporter (1998) 16: 157. doi:10.1023/A:1007468801806

Abstract

The method of cDNA-AFLP allows detection of differentially expressed transcripts using PCR. This report provides a detailed and updated protocol for the cDNA-AFLP procedure and an analysis of interactions between its various parameters. We studied the effects of PCR cycle number and template dilution level on the number of transcript derived fragments (TDFs). We also examined the use of magnetic beads to synthesise cDNA and the effect of MgCl2 concentration during amplification. Finally, we determined the detection level of the cDNA-AFLP method using TDFs of various sizes and composition. We could detect TDFs corresponding to a single copy per cell of a specific transcript in a cDNA-AFLP pattern, indicating high sensitivity of the method. Also, there was no correlation between concentration of detectable TDF and the fragment size, stressing the high stringency of the amplification reactions. Theoretical considerations and specific applications of the method are discussed.

cDNA-AFLPplant developmentRNA-fingerprinting

Copyright information

© Kluwer Academic Publishers 1998