Design PCR Primers - Inputs

1. 3’-end Model:

   Determines whether the 3’-end model is employed to make use of mismatches located near the primer's 3’-end. This model was developed using Taq DNA polymerase with 30 second annealing and elongation steps. If using a DNA polymerase with proofreading activity (e.g., Phusion), or substantially longer annealing or elongation times, then the use of the 3'-end model is not recommended.
   
   It is possible to have the algorithm induce a mismatch at the primer's 6^th position from the 3'-end to further increase specificity to the target group. It is recommended to use this option only when necessary, as the program will generate primers that are mismatched to the target group. In some circumstances inducing a mismatch may be the only way to achieve the desired level of specificity.

2. Primers:

   Set constraints on the designed primers by limiting their lengths and maximum number of different permutations in order to cover the target group.

3. PCR Product Amplicon:

   Specify the desired amplicon size, which determines the distance separation between the start of the forward and reverse primers on the target template. The amplicon length may be a range between 50 and 10,000 base pairs.

4. Reaction Conditions:

   These inputs are used to determine the optimal length for each primer that will maximize sensitivity to the target group, and minimize bias between different primer permutations at the specified annealing temperature. Concentrations should be provided as they are in the final PCR reaction. For example, if you add 1µL of a 10µM stock of each primer to a 25µL total reaction volume then the final primer concentration is 1µL*10µM/25µL = 400nM. The program will calculate a Sodium Equivalent concentration based on the formula [Na⁺] + 3.33*([Mg⁺⁺] - [dNTP])^0.5. To use an alternative sodium equivalent concentration simply enter it as the sodium concentration and enter zero for the magnesium and dNTP concentrations.

5. Target group:

   Set the name of sequences belonging to the target group and the minimum fraction of sequences that must be covered by the primers. For example, specifying a coverage of 90% will ensure that the designed set of forward/reverse primers perfectly matches at least 90% of sequences in the target group. The target group should match the FASTA identifier of one or more sequences in the FASTA file.

6. FASTA File:

   Choose a text file containing the sequence records to use for primer design. Here are some remarks on the input file:

   • Sequences must be in FASTA format where each new sequence record begins with a ">" symbol on a single line containing the name of the group where it belongs, and subsequent lines contain the actual sequence. There are no restrictions on the number of nucleotides per line, however a maximum of 10,000 nucleotides are permitted per sequence.
   • Sequences must be aligned (with gaps; "-") such that all input sequences are the same total length. Standard IUPAC ambiguity codes are permitted.
   • The size of the uploaded file is restricted to be less than 50 MB. This limit corresponds to roughly 35,000 sequences of length 1,000 nucleotides plus gaps. To analyze more than 50MB of sequences, please download DECIPHER for use on your computer.
   • Each FASTA record must have an identifier listing only the group to which it belongs. One or more sequences must be named the same as the target group. Up to 100 non-target groups are permitted. For example, a correctly formatted input file for the target group "XYZ" might look like:
     >XYZ
     ACTG-ACTG...
     >XYZ
     ACTGGACTG...
     >ABC
     AGGG-ACTG...
     >ABC
     -GGC-ACTG...
     >Etc.
     ...