In silico Double Digestion Fingerprinting (DDF)

Simulation of Double Digestion Fingerprinting experiment and detection of bands with labelled adaptors

This approach has not yet been tried in the lab.

This procedure may be considered as a modification of Subtracted Restriction Fingerprinting (SRF), but it does not require the subtraction step.

 The basic procedure for this experiment may be sorted as follows:
  • Two endonucleases are used to cleave the genome
    • One endonuclease (RE1) cleaves the genome a few times
    • The other endonuclease (RE2) cleaves the genome very often
Consequently, we will get a lot of RE2-RE2 fragments, fewer  RE1-RE2, RE2-RE1, and probably no RE1-RE1 fragments.
  • Labelled adaptors (for example with biotin) will be ligated to fragments with one or two RE1 ends.
  • Fragments will be separated by electrophoresis, transferred to a membrane, and biotinylated bands detected by using conventional methods.
When filling the form (see image bellow)...
  • we will select three fragment types: RE1-RE1, RE1-RE2 and RE2-RE1
  • in this example we will select restriction enzymes XbaI and MseI 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),
  • no selective nucleotides are used
After clicking the "Amplify" buttom we will get 78 bands for E. coli K12.

Example 4

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