Table 5.
Pre-restriction of immobilized cDNA
| No pre-restriction | Partial pre-restriction | Full pre-restriction | ||||
| Arabidopsis thaliana | ||||||
| Marking enzymes | FatI | MboI | HinPI | MboI | FatI | MboI |
| Releasing enzymes | MboI | FatI | MseI | MseI | MboI | FatI |
| MseI | MseI | TasI | TasI | MseI | MseI | |
| TasI | TasI | TaqI | TaqI | TasI | TasI | |
| Sequence coverage | 78 % | 70 % | 52 % | |||
| Sequence redundancy | 30 % | 30 % | 0 % | |||
| Average fragment length (nt) | 183 | 237 | 142 | |||
| PCR reactions | 100 % | 125 % | 36 % | |||
| Mouse | ||||||
| Marking enzymes | MboI | Csp6I | HinPI | MboI | MboI | Csp6I |
| Releasing enzymes | Csp6I | MseI | TasI | TasI | Csp6I | MseI |
| MseI | MboI | MseI | MseI | MseI | MboI | |
| FatI | FatI | TaqI | TaqI | FatI | FatI | |
| Sequence coverage | 87 % | 81 % | 50 % | |||
| Sequence redundancy | 41 % | 35 % | 0 % | |||
| Average fragment length (nt) | 270 | 271 | 159 | |||
| PCR reactions | 100 % | 39 % | 25 % | |||
| Human | ||||||
| Marking enzymes | MboI | Csp6I | HinPI | MboI | MboI | Csp6I |
| Releasing enzymes | CspI | MboI | MseI | MseI | CspI | MboI |
| HinPI | FatI | TasI | TasI | HinPI | FatI | |
| FatI | HinPI | TaqI | TaqI | FatI | HinPI | |
| Sequence coverage | 85 % | 84 % | 63 % | |||
| Sequence redundancy | 35 % | 40 % | 0 % | |||
| Average fragment length (nt) | 274 | 292 | 182 | |||
| PCR reactions | 100 % | 86 % | 61 % | |||
Simulation of cDNA-AFLP with sequential digestion protocol on Arabidopsis, human and mouse RefSeq ESTs was performed. Optimal combination of restriction enzymes (2 marking in combination with 3 releasing enzymes) with the highest sequence coverage was used to compare the effect of partial pre-restriction and full pre-restriction options on maximal transcript coverage, sequence redundancy, average fragment length and reduction of PCR effort (relative to the maximal number of PCR by one of the options).