The cDNA-AFLP technology permits the display and quantification of transcripts based on AFLP® fingerprinting of double-stranded cDNA. The transcript profiles obtained using this technique are a reliable and efficient tool for the identification of differentially expressed mRNAs.
How it works
The fingerprinting procedure consists of the following steps:
isolation of mRNA from the samples of interest;
reverse transcription of mRNA using an oligo-dT primer to produce cDNA;
digestion of double stranded cDNA with a pair of restriction enzymes;
ligation of adapters specific for the two restriction sites;
pre-amplification of fragments with primers specific to the two adapter sequences;
selective amplification with adapter specific primer combinations with nucleotide extensions at their 3’ ends (usually varying between 1 and 3 selective nucleotides);
visualization of individual fragments on a polyacrylamide gel (using radioactive gels: one of the two primers is end labeled with 33P);
analysis by image-processing software. This software identifies the cDNA-AFLP fragments and quantifies the intensities that correspond to the original expression level.
Figure1. An example and a detail of a cDNA-AFLP fingerprint that shows up and down regulation as well as constant expression in two samples.
The cDNA-AFLP technology has a number of unique advantages:
it can visualize over 90% of all expressed genes;
it does not require prior knowledge of the genetic sequence of the analysed organism / genes;
since it is PCR based, it is very sensitive and can detect down to 1 mRNA molecule per cell;
bands corresponding to genes / expression profiles of interest can be excised and sequenced;
in case the sequence of relevant genes is known, the fragment size and gel position of the resulting cDNA-AFLP fragments can be predicted. As such the gene expression of known genes can be specifically followed.
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