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.
References
Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.
Bachem CWB, van der Hoeven RS, de Bruijn SM, Vreugdenhil D, Zabeau M and Visser RGF (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. The Plant J 9: 745–753.
Bauer D, Warthoe P, Rohde M, and Struss M (1994) PCR methods and applications manual supplement, pp S805-809. Cold Spring Harbor University Press, Cold Spring Harbor, NY.
Diachenko LB, Ledesma J, Chenchik AA and Siebert PD (1996) Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed mRNA. Biochem Biophys Res Com 219: 824–828.
Du-Toit R, Victor TC, and Van-Helden PD (1993) Empirical evaluation of conditions influencing the polymerase chain reaction: enterotoxigenic Escherichia coli as a test case. Euro J Clin Chem Clin Biochem 31: 225–231.
Habu Y, Fukadatanaka S, Hisatomi Y and Iida S (1997) Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence. Biochem Biophys Res Comm 234: 516–521.
Ivanova NB and Belyavsky AV (1995) Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting. Nucl Acids Res 23: 2954–2958.
Kato K (1996) RNA fingerprinting by molecular indexing. Nucl Acids Res 24: 394–395.
Liang P, Bauer D, Averboukh L, Warthoe P, Rohrwild M, Muller H, Strauss M and Pardee AB (1995) Analysis of altered gene expression by differential display. Meth Enzymol 254: 304–321.
Liang P and Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967–971.
Mathieu-Daudé F, Cheng C, Welsh J and McClelland M (1996) DNA rehybridization during PCR: the ‘C-ot effect’ and its consequences. Nucl Acids Res 24: 1504–1507.
Money T, Reader S, Qu IJ, Dunford RP and Moore G (1996) AFLP-based mRNA fingerprinting. Nucl Acids Res 24: 2616–2617.
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor University Press, Cold Spring Harbor, NY.
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Pelman J, Kuiper M and Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucl Acids Res 23: 4407–4414.
Welsh J, Chada K, Dalal SS, Ralph D, Cheng R and McClelland M (1992) Arbitrarily primed PCR fingerprinting of RNA. Nucl Acids Res 20: 4965–4970.
Zhao S, Ooi SL and Pardee AB (1995) New primer strategy improves precision of differential display. BioTechniques 18: 842–850.
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Bachem, C.W., Oomen, R.J. & Visser, R.G. Transcript Imaging with cDNA-AFLP: A Step-by-Step Protocol. Plant Molecular Biology Reporter 16, 157 (1998). https://doi.org/10.1023/A:1007468801806
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DOI: https://doi.org/10.1023/A:1007468801806