4C primer designer logo

Procedure implemented in this website

1. Digest the nucleotide sequence given using all pairwise combinations of the following restriction enzymes:
EcoRI: GAATTC
HindIII: AAGCTT
BglII: AGATCT
DpnII: GATC
Csp6I: GTAC
NlaIII: CATG

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:
primer length
minimum number of matches
18
16
19
17
20
17
21
18
22
19

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

See the schematic below for a graphical depiction of the 4C primer design procedure:

4C: Circular Chromatin Conformation Capture

Schematic showing the steps of the design of a 4C primer set. A textual description can be found below.

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.

For more information on designing primers for 4C viewpoints see:
Methods Enzymol. (2012) 513:89-112
Nat Methods. (2012) 9:969-72

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 BLAST hits.
    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.