Figure 1

Schematic presentation for the protocol of cDNA-RDA (supplied by D. Schatz) and its modification. A. Classical strategy [1]. Double stranded cDNA is derived from the original mRNAs by RT. After digestion with DpnII, the resulted 4-nucleotide protruding ends are ligated to suitable linkers (12 and 24-mers) and subjected to PCR to generate amplifiable populations named "Representations". Following the hybridization step, the tester homoduplexes are enriched in the mix by 18 cycles of PCR (pre-PCR). The mixture is treated with Mung Bean nuclease to enrich the mix in tester sequences and is subjected to a second round of PCR (final PCR) to generate a final Difference Product. B. Modified protocol. After DpnII-restriction, the representations are filled-in with dNTPs. Following the subtractive hybridization, the 5' recessed ends of the target tester homoduplexes and the driver strand of the heteroduplexes are filled-in with α-thio-dNTPs, to render them resistant to Exonuclease III cleavage. The other components in the mix are degraded by Exonuclease III and by Mung Bean nuclease. The mixture is subjected to 28 cycles of PCR to generate a Difference Product.