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PIRA-PCR

Source sequence

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.

Position of SNP from start of sequence

The position of the SNP allele in the source sequence.

Allele1

The SNP allele as indicated in the source sequence.

Allele2

The other SNP allele (the one not in the source sequence).

Optimum primer size

The value specifies the optimum length of the primers (in bases). The program will pick primers close to this length.

Maximum primer size

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

Minimum primer size

The value specifies the minimum acceptable length of the primers (in bases).

Optimum product size

The value specifies the optimum length of the PCR products.

Maximum product size

The value specifies the maximum length of the PCR products.

Minimum product size

The value specifies the minimum length of the PCR products.

Optimum primer Tm

Optimum melting temperature(Celsius) for a primer oligo. The program will try to pick primers with melting temperatures are close to this temperature.

Maximum primer Tm

Maximum acceptable melting temperature(Celsius) for a primer oligo.

Minimum primer Tm

Minimum acceptable melting temperature(Celsius) for a primer oligo.

Maximum primer GC%

Maximum allowable percentage of Gs and Cs in any primer.

Minimum primer GC%

Minimum allowable percentage of Gs and Cs in any primer.

Maximum complementarity

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.

Maximum 3' complementarity

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.

Salt concentration (mM)

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.

Annealing primer concentration (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.

Number of positions for mismatches on either side of SNP

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.

Number of outputs

The maximum number of primer pair combinations to return. Setting this parameter to a large value (upper limit is 1000) will increase running time.

Others

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  • 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 xke@soton.ac.uk.

    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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