Tool Usage

Fill out the initial form, noting that the accessions are from the “Nordborg 96” list of accessions, and you must pick different ones. You must enter a beginning and end position, which can be no more than 10Mbp apart. Whole chromosome analysis is possible but must be completed in several sessions.

At this point the primer design options can be adapted; their default values correspond to standard recommendations for high-throughput genotyping.

All the fragments found in the search interval are displayed. Those carrying one or more SNPs are highlighted. This gives you an indication of polymorphism between your selected accessions. Not all SNPs can be used for making a marker. Select one candidate to take forward.

Usable SNPs from the selected fragment are displayed. A short sequence of ~25 bp containing the SNP at position ~20 appears. It serves as input for CAPS/dCAPS design. If the SNP appears on the north half of the fragment, the sequence is presented in “forward” sense. If the SNP is on the south part of the fragment, the sequence appears in the “reverse” sense. If the program finds none that can be used, it display “0” and you need to select another candidate.

When you have found a successful candidate the selected SNP is input in a dCAPS finder. All enzymes tested are commercially available, and isoschizomers appear separately for practical reasons.

The dCAPS finder results are filtered. Only enzymes that cut once and at the target SNP on the PCR product are shown. The primers are also filtered out according to primer design specifications. They are presented with their length, melting temperature and GC content.

Following this the whole target PCR product is displayed. You can therefore verify the PCR product and where cleavage occurs. Selecting a forward primer will allow you to proceed.

In some cases, despite a SNP, no enzyme is found that can help make a polymorphic marker of the SNP. Or all found enzymes cleave at multiple points. In such case, you need to look at other SNPs on the same fragment or at fragments nearby.

A series of reverse primers is generated upon selection of a CAPS/dCAPS forward primer. The primers are ordered by their capacity to form primer dimers. The reverse primers also meet the specifications of primer design as selected in the first window.

In some instances the program cannot generate a reverse primer due to sequence complexity (multiple or large indels). It is however possible to generate a consistent primer manually from the previous window.

Select a reverse primer to complete the process and save the results in the “cart” window.

The selected primer couple with the corresponding enzyme and relevant primer information are passed to a table. If several markers are being designed, they all add to the list.