Similarity overlap
If two single primers overlap with at least this many nucleotides, they are considered similar and regarded, for all practical purposes, as the same primer. This parameter therefore has a profound impact on which primers are reported. Consider two primer sets P and Q consisting of P.fw/P.rv and Q.fw/Q.rv. If both P.fw and Q.fw are similar and P.rv and Q.rv are similar, and Q has a higher score than P, then P is not reported (since it is considered to be more or less the same set as Q). Furthermore, if some primer p is similar to two other primers already reported, p (i.e., any set that includes p) is not reported (note that such sets would have lower scores than the sets already reported). Thus, the primer sets eventually reported are the highest scoring sets under these restrictions. This way suggesting several almost identical pairs is avoided. If the Similarity Overlap parameter is increased, the reported primer sets most likely will be highly overlapping. If the parameter is decreased, the program is forced to suggest primers that don't overlap as much and hence are located in different regions. That leads to higher variation among the suggested primer sets but may end up including sets which have a much lower score compared to sets not reported. Care is needed when changing this parameter.

Tail length
The length of the 3'-end tail in which the number of A's and T's are counted.

Maximum primer length
Maximum primer length allowed (10-80 nt).

Minimum primer length
Minimum primer length allowed (10-80 nt).

Maximum melting temperature
Maximum primer melting temperature allowed.

Minimum melting temperature
Minimum primer melting temperature allowed.

Critical melting temperature
If both primer melting temperatures are below this value, penalize the pair.

Minimum melting temperature with ambiguity positions
If a primer melting temperature is below this value, the primer can have no ambiguity positions.

Minimum number of 3'-end matches
A primer has to have at least this many perfect matches in its 3'-end.

Maximum number of ambiguity positions
A primer may have at most this many ambiguity positions. A primer is based on a section of the alignment; each mismatch column in this section corresponds to an ambiguity position.

Minimum length with three or more ambiguity positions
A primer which is shorter than this value may have at most two ambiguity positions.

Minimum length with two ambiguity positions
A primer which is shorter than this value may have at most one ambiguity position.

Critical ambiguity position distance from 3'-end
Penalize ambiguity positions closer than this distance in nucleotides to the 3'-end.

Maximum diversity per nucleotide position
A primer cannot cover a nucleotide position in the alignment with more than this many different nucleotides (gaps are ignored).

Maximum number of ambiguity positions with maximum diversity
A primer may cover at most this many nucleotide positions of the alignment with the maximum diversity allowed.

Minimum number of nucleotides per nucleotide position
In all nucleotide positions in the alignment covered by a primer, at least this many sequences must have a nucleotide. Thus, with the value 2 no primer may cover a part of the alignment in which only 1 sequence is represented (has a nucleotide).

Primer concentration in nanomolar
The concentration of the primers in the PCR reaction.

Salt concentration in molar
The salt concentration in the 1 x PCR buffer.

Maximum melting temperature difference
Maximum difference allowed between the melting temperatures of the two primers in a pair.

Optimal primer length interval
A primer length between these two values (both inclusive) is considered optimal.

Optimal PCR product length interval
A PCR product length between the middle two of these four values (both inclusive) is considered optimal. A length between the first two values is considered acceptable but less than optimal, and likewise with a length between the last two values. A length outside the full range is penalized. The product length is the length of the two primers plus the distance between them, measured in nucleotides.

Minimum PCR product length
Minimum length of the PCR product allowed.

Maximum PCR product length
Maximum length of the PCR product allowed.

Optimal primer length dispensation with no ambiguity positions
If a primer has no ambiguity positions, its length can be this many nucleotides shorter than the otherwise smallest optimal length value and still be considered optimal.

Good conserved region length
Ambiguity positions in a primer preferably are surrounded by perfectly matching regions without ambiguity positions. A region of at least this length is considered an acceptable such region; if an ambiguity position is flanked by a perfectly conserved region of less than this length (e.g. of length 0), it is penalized.

Conservation window length
Primers (partly) based on only two sequences must lie in a highly conserved region. The window length used as a basis for the conservation calculation has this length. See Minimum conservation %.

Minimum conservation-% in conservation window
When calculating the overall conservation in the conservation window, there must be at least this percentage of perfect matches (gaps are allowed) in the window.

Introns in sequences
If this option is set to 'yes', X'es in the sequences are interpreted as special intron markers following this translation scheme:
XXX : intron, <= 200 bp
XXXX : 201 - 500 bp intron
XXXXX : 501 - 1000 bp
XXXXXX : > 1000 bp
Further, the primers in a primer set *must* have an intron between them to increase the probability of finding polymorphisms in the PCR product, and some other evaluation criteria apply when scoring the found primer sets. If the option is set to 'no', X's are regular wild card symbols, and primer sets are not required to have an intron between them.

At least one primer must be based on three sequences
If this option is set to 'yes' and one of the primers is based on only two sequences, the other must be based on at least three. If the option is set to 'no', both primers may be based on two sequences only (they may not score very high, but they won't be discarded).

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