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
