FastPCR Software for PCR, In Silico PCR, and Oligonucleotide Assembly and Analysis - DNA Cloning and Assembly Methods - page 278

“{− ln20-23 -tm55-68 -3tm37-50 -cg41-70 -q70
-lc75 -npr400 -c5[NN] -c3[SWW SSW SWS WSS]}, ”
where “ -ln20-23 ” determines the range of primer length (20-
23 bases), “ -tm55-68 ” determines the range of primer T m (55-
68 °C), “ -3tm37-50 ” determines the range of primer T m at 3′
end (37-50 °C), “- cg41- 70 ” determines the range of primer
CG% contents (between 41 and 70 %), “ -npr400 ” shows the
maximum number of primers (400) designed to each target,
“ -c5[NN] ” denotes a primer having no specific sequence pattern
for 5′ ends, and “- c3[SWW SSW SWS WSS] ” specifies primers
that conform to particular patterns of ambiguity, such as that
shown here for example.

5.3 Examples for
Primer Selection
Regions

Users can specify, individually for each sequence, multiple locations
for both forward and reverse primer placement with the commands:
“ -FpdN1-N2 ” for forward primers and “ -RpdN1-N2 ” for reverse
primers, where from N1 to N2 are bases from the selected regions;
“ -pdN1-N2 ” (see more at: http://primerdigital.com/soft/pcr_
help.html ) . Alternatively, users can specify multiple locations for
both forward and reverse primers using [ “and” ] inside each
sequence: the software allows multiple and independent locations
for both forward and reverse primers inside each of the sequences.
Whilst PCR primer design will be performed independently for
different targets, multiplex PCR primer design can be performed
simultaneously with multiple amplicons within a single sequence as
well as for different sequences, i.e., all possible combinations of
[ “and” ] inside one or more sequences. By default, the software
designs primers within the entire sequence length. Optionally,
users can specify, individually for each sequence, multiple locations
for both forward and reverse primers with the commands:

1. The same location for both forward and reverse primers will be
designed in the central [nnnnnnnnnn] part (“ [] ” is used
only once):
.......[nnnnnn]....
2. Different locations for forward and reverse primers; forward
primers will be chosen inside the “ [1nnnnnn] ” location and
reverse primers inside “ [2nnnnnn] ” location (twice “ [] ”):
.......[1nnnnnn]....[2nnnnnn].....
3. Primers must flank the central “ ]nnnnnn[ ”; forward primers
will be chosen from 1 to “ A] ” bases and reverse primers will be
chosen from base “ [C ” to the end of sequence: ......A]
nnnnnn[C.....
4. Forward and reverse primers have an overlapping part
“ [nnnnnn] ”; forward primers will be chosen from “[A to n]”
bases and reverse primers will be chosen from “ [n base to C] ”:
......[A.....[nnnnnn]......C]....