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:
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.
Authors
Primer designer code written by Alex Gileta under
guidance by Scott Smemo and Noboru Sakabe. Modified and extended by Noboru Sakabe. Web
interface created by Noboru Sakabe.
Nobrega lab - Human Genetics Department - University of Chicago, USA
