In silico Double Digestion Fingerprinting (DDF)

Simulation of Subtracted Restriction Fingerprinting (SRF) experiment

SRF experiment will be simulate:
  • Both endonucleases will be palindromic
  • Recognition sequence has no degenerated nucleotides
  • One endonuclease cleaves genome very frequently and the other fewer times.
  • One restriction enzyme (RE1) allows a filling reaction to be performed with digoxigenin labelled Uridine (Dig-dUTP) and the other restriction enzyme allows a filling reaction with biotinylated Cytosine (Bio-dCTP).
  • From the four types of fragments yielded after digestion, RE1-RE2, RE2-RE1 and RE2-RE2 fragments (labelled in one or both ends with biotin) are captured by streptavidin-coated magnetic beads (subtracted).
  • The remaining RE1-RE1 fragments (which are digoxigenin labelled) will be separated by electrophoresis, transferred by Southern blot  and  band patterns analysed.
When filling the form (see image bellow)...
  • we will check the checkbox for "SRF", and the correct fragment type will be selected automatically ,
  • in this example we will select restriction enzymes MunI and TaqI from the lists of commercially available palindromic restriction enzymes,
  • although in the example the checkbox "For non-palindromic endonuclease discern complementary" is checked, unchecking it makes no difference in this case (due to palindromic nature of the selected endonucleases),
  • selective nucleotides are not included in the experiment (this method does not require them).

Image for example 1

After clicking the "Amplify" buttom we will get 83 bands (see result here).

In the example bellow the same experiment is performed, but in this case recognition sequence is introduced in the form.


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