The sequence from which to choose primers. The sequence must be presented 5'-3'. The bases may be upper or lower
case. Whitespace characters and digits are ignored, and any other unrecognized character, other than A, T, C, G,
R, K, H, D, Y, S, B, M, W, V, N, is treated as N.
The position of the SNP allele in the source sequence.
The SNP allele as indicated in the source sequence.
The other SNP allele (the one not in the source sequence).
The value specifies the optimum length of the primers (in bases). The program will pick primers close
to this length.
The value specifies the maximum acceptable length of the primers (in bases). This parameter should not
be larger than 35. This limit is governed by maximum oligo size for which the program's melting-
temperature is valid
The value specifies the minimum acceptable length of the primers (in bases).
The value specifies the optimum length of the PCR products.
The value specifies the maximum length of the PCR products.
The value specifies the minimum length of the PCR products.
Optimum melting temperature(Celsius) for a primer oligo. The program will try to pick primers with
melting temperatures are close to this temperature.
Maximum acceptable melting temperature(Celsius) for a primer oligo.
Minimum acceptable melting temperature(Celsius) for a primer oligo.
Maximum allowable percentage of Gs and Cs in any primer.
Minimum allowable percentage of Gs and Cs in any primer.
The maximum allowable local alignment score when testing a single primer for (local) self-complementarity
and the maximum allowable local alignment score when testing for complementarity between left and right
primers. Local self-complementarity is taken to predict the tendency of primers to anneal to each other
without necessarily causing self-priming in the PCR. Scores are non-negative, and a score of 0.00
indicates that there is no reasonable local alignment between two oligos.
The maximum allowable 3'-anchored global alignment score when testing a single primer for self-
complementarity, and the maximum allowable 3'-anchored global alignment score when testing for
complementarity between left and right primers. The 3'-anchored global alignment score is taken to
predict the likelihood of PCR-priming primer-dimers. The scoring system is as for the Maximum
Complementarity argument. Scores are non-negative, and a score of 0.00 indicates that there is no
reasonable 3'-anchored global alignment between two oligos. It is nonsensical to provide a larger value
for this parameter than for the Maximum (local) Complementarity parameter because the score of a local
alignment will always be at least as great as the score of a global alignment.
The millimolar concentration of salt (usually KCl) in the PCR. The program uses this argument to
calculate oligo melting temperatures. Default is 50.0 mM.
The nanomolar concentration of annealing oligos in the PCR. This argument is used to calculate oligo
melting temperatures. The default value is set to 50 (nM). The value of the parameter is less than
the actual concentration of oligos in the reaction because it is the concentration of annealing oligos,
which in turn depends on the amount of template (including PCR product). This concentration increases
a great deal during a PCR. However, PCR seems quite robust for a variety of oligo melting temperatures.
The number of bases allowed for mismatches on either side of SNP, excluding SNP itself and the base
immediately close to the SNP. Because most of the Type II enzymes have recogition sequences not longer
than six base pairs, setting this value to the default of four would be sufficient in most cases. The
largest value allowed is 12.
The maximum number of primer pair combinations to return. Setting this parameter to a large value (upper
limit is 1000) will increase running time.
To update the pages with Netscape: do reload while pressing the "shift" key.
To update a frame: hold right button of mouse, then reload frame while pressing the "shift" key.
To update a page in Internet Explorer: delete the corresponding page from your computer (usually in C:\Windows\temporary internet files\ directory), then load. You could delete the particular html file manually or delete all temporary internet files as follows: go to settings -> control panel -> Internet options -> General -> Click "Delete Files..." button in the "Temporary Internet files" box.
If you encounter errors, please have quick check on your inputs first. For example, make sure your source sequence is less than the recommended limit of 1kb. This should sufficient in most cases. If you really need sequence longer than that, please stay below 2kb limit.
For further questions: contact firstname.lastname@example.org.
For reference, please cite the following paper:
Xiayi Ke, Andrew Collins and Shu Ye
PIRA PCR designer for restriction analysis of single nucleotide polymorphisms.
Bioinformatics, 2001, Vol.17, No.9, Pages 838-839
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