As the first application of the Seegene's ACP™ Technology, GeneFishing™ DEG Premix Kits for identifying differentially expressed genes (DEGs) in two or more nucleic acid samples is specially designed to overcome the disadvantages and limitations of the current Differentially Display related methodologies. GeneFishing™ DEG Premix Kits requires the following 3 steps including a two-stage PCR (GeneFishing™ PCR)

Step 1:
RT is conducted using oligo dT-ACP to synthesise first-strand cDNAs from samples, wherein the 3'-end core portion of the oligo dT-ACP comprises a hybridising sequence complementary to a poly A region of mRNA transcripts.

Step 2:
1st stage PCR for only one cycle is conducted using an arbitrary ACP to synthesise second-strand cDNAs under conditions that the 3'-end core portion of the oligo-dT ACP is prevented from annealing to the first-strand cDNAs and only the 3'-end core portion (10-mer) of the arbitrary ACP comprising a hybridising sequence sufficiently complementary to a region of the first-strand cDNAs is involved in annealing to the first-strand cDNAs. This step makes it possible to initially and fundamentally exclude the results from the problems of the arbitrary/arbitrary products or the oligo-dT/oligo-dT products.

Step 3:
2nd stage PCR follows under high stringency conditions to amplify only the arbitrary-primed second cDNA strands generated from Step 2. Both the oligo-dT and arbitrary ACP set are involved in annealing only to the sites or complementary sites of 3'- and 5'-ends of the second cDNA strands, which results in the amplification of only real PCR products with NO false products.

Fig. 2. Detailed flow chart of cDNA synthesis and GeneFishing™ PCR.