This website was planned to be shut off on or after 1st June 2024. However a number of people have contacted us to say it is vital to their research so we are working on moving it to new hosting instead. For more information, or feedback, contact Christopher Gutteridge at totl@soton.ac.uk.

PCR Designer for Restriction Analysis of Polymorphisms

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.

Starting position of mutation in sequence

The starting position of the polymorphic alleles in the source sequence. In the rare case of a combination of mutations where they are close to each other, you can treat them as a "single" combined mutation. Therefore, the comined mutation would range from the begining of the first mutation to the end of the last mutation, and use the starting position of the first mutation as the current input.

Allele1

The normal allele as indicated in the source sequence.

Allele2

The other mutated allele (the one not in the source sequence). It could be a deletion, an insertion, a substitution such as a SNP, or a combination of them. In the rare case of a combination of mutations where they are close to each other, you can treat them as a "single" combined mutation. Therefore, the comined mutation would range from the begining of the first mutation to the end of the last mutation.

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 mutation

The number of bases allowed for mismatches on either side of mutation, excluding mutation itself and the base immediately close to the mutation. 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 restriction sites

The maximum number of restriction sites choosen by the program. Setting this parameter to a large value will increase running time.

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

  • 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.


  • If you are running the program for a combination of mutations, please refer to Starting position of mutation in sequence and Allele2.

    • For computing questions: contact xiayi@well.ox.ac.uk.

    For questions relating to laboratory applications: contact Shu.Ye@soton.ac.uk.

    Please cite the following paper in your resulting publications of using this program:

    Xiayi Ke, Andrew R. Collins, Shu Ye (2002) PCR Designer for Restriction Analysis of Various Types of Sequence Mutation. Bioinformatics 18: 1688-1689.

    Back to top