1. Digest the nucleotide sequence given using all pairwise combinations
of the following restriction enzymes:
2. Check if the secondary enzyme would cut the fragment at the primary
enzyme site. If it does, reject the fragment.
3. Reject fragments that are < maximum fragment length and >
minimum fragment length.
4. Verify whether the fragment overlaps the center of the nucleotide
sequence given and calculate the distance between the center of the
fragment and the center of the nucleotide sequence given. Sort by
proximity to the center. In the case
of RefSeqs, the center is the transcription start site.
5. Run primer3 (Untergasser A, Cutcutache I, Koressaar T, Ye J,
Faircloth BC, Remm M and Rozen SG. Primer3--new capabilities and
interfaces. Nucleic Acids Res. 2012 Aug 1;40(15):e
115) on each fragment.
6. Align all primers to the genome using BLAST (Altschul, S.F., Gish,
W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local
alignment search tool." J. Mol. Biol. 215:403-410) and accept those
that pass the following criteria.
The minimum % match varies slightly with primer length:
number of matches
Blast is being run with this command: blastn
-ungapped -db [blast_index] -query primers.fa -word_size 12 -evalue 10
-outfmt 7 -out blast.out
the schematic below for a graphical depiction of the 4C primer design
4C: Circular Chromatin Conformation Capture
1. 1st digestion with enzyme 1 results in 2 crosslinked DNA molecules, one being the viewpoint and another a distal unknown region.
2. 3C step: in 3C one of the primers has to be designed on the interacting region.
3. Ligation after 1st digestion: ligates distal ends of the 2 crosslinked DNA molecules.
4. 2nd digestion: enzyme 2 cuts the 2 crosslinked DNA molecules internally at a distance between 300-1500 bp from the dangling end of the viewpoint.
5. "4C step": circularization allows designing both primers on the viewpoint, allowing one to query arbitray interacting regions.
6. Ligation after 2nd digestion results in a circularized molecule containing sequences for the reading and non-reading primers.
Designer program operation
The program digitally digests the target region of
interest with the enzymes specified by the user and scans the accepted
fragments for primers with increasing lengths (18-23 bp or as specified
by the user).
All fragments with a primer pair identified by
primer3, given the parameters, are shown, even if they have too many
When a primer pair fulfills the BLAST criteria, the
program stops scanning for primers with increasing lengths. This means
that when 1 primer pair is found for a given primer length, good or
bad, all primer pairs identified by primer3 for all accepted fragments
will be shown.
If no results are shown at all, it means that either
no acceptable fragments for the specified enzymes were found or that
primer3 could not find suitable primers, given the parameters. The
number of acceptable fragments out of the total digitally digested is
shown for each primer length tested.