GK primer tool help
The GK primer design tool is implemented within the visualisation part and displays the location of the designed primers. It only requires the absolute minimum of parameter definition.
There are three ways to enter the primer design tool:
- via the navigation menu following the link ‘GK primer design’
- via a link from an insertion prediction (‘go to primer design’) that leads directly to the respective insertion position
- via a button in the visualisation (‘go to primer design’) for the current position. The desired position for the primer can be adjusted in the user interface by using the chromosomal position according to the TAIRv10 genomic sequence or the AGI code of the gene of interest.
Primers design is triggered using the respective button, and primer sequences are listed (after a little while that can take quite some seconds) below the buttons. Primer positions on the pseudochromosome sequence are presented as well. After one primer pair has been designed, additional primer pairs can be made. Primer pairs are shown in the visualisation as dark-red arrowheads. When more than one primer pair has been computed, the one generated last is shown.
For using the tool the following parameters have to be defined by the user:
- the desired genomic position
- the distance of the primers to the position (default: 300 to 800bp); range can be changed between 100 to 1500bp
- the optimal annealing temperature (default: 60.5 °C); 50 to 72 °C allowed
Primers for genotyping
- If the primer pair that is designed by the tool spans a (predicted) GABI-Kat insertion site, primer and genotyping-related information is supplied via a mouse-over window (hover over the "HERE" link).
- The mouse-over window can be fixed to allow access by copy-and-paste to the data presented. A fixed window can be de-selected by pointing on the "HERE"-button with the mouse.
- The following data are presented:
(i) the size of the amplimer defined by the forward and reverse primer;
(ii) the border primers (including their sequence) which should be used in the PCRs for the detection of both borders in combination with the primers designed by the tool;
(iii) the approximate sizes of the resulting PCR products, which are valid if "ideal insertions" (with "textbook transitions" from border sequence to gDNA sequence) are present in the respective line.
- If primers designed by the tool do not span a GABI-Kat insertion site prediction, only the size of the amplimer defined by the primers is shown.
- Primers are designed between MIN and MAX distance (in bp) to the specified position on the pseudochromosome.
- The larger the region, the better the chances to obtain a unique primer. This means essentially that the selection of a larger area often allows the design of better primers.
- The primers can either be used in combination with T-DNA border primers (LB or RB, see here) to confirm a T-DNA insertion prediction of interest, or simply to amplify PCR products in the chosen genomic region (e.g. a "genotyping amplicon" that spans a confirmed T-DNA insertion position for selective detection of the wt allele).
- The tool is suitable for all regions of the genome and takes paralogous regions into account, if necessary.
- The tool attemps to offer the best primer with respect to genome-wide uniqueness and selective power for paralogs.
- The tool checks the 3'-ends of the primers for redundancy in the genome. A primer is regarded as “unique” if its 12bp-3’-end has only one hit in the genome.
- The number of occurrences of each 12bp sequence within the genome has been precomputed and stored in our db. Primers with the least 12bp-hits are returned by the tool.
- One of two different methods for primer design is chosen by our tool automatically. In "normal cases", a Primer3-based method is applied. In case of paralogous regions, the primer design is based upon multiple sequence alignments. Mismatches detected in these alignments are then used as a basis for primer design.