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Table 5 Pre-restriction of immobilized cDNA

From: Improved coverage of cDNA-AFLP by sequential digestion 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 %
  1. 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).