Alignment-based TaqMan probe design

CLC Genomics Workbench allows the user to design solutions for TaqMan quantitative PCR which consist of four oligos: a general primer pair which will amplify all sequences in the alignment, a specific TaqMan probe which will match the group of included sequences but not match the excluded sequences and a specific TaqMan probe which will match the group of excluded sequences but not match the included sequences. As above, the selection boxes are used to indicate the status of a sequence, if the box is checked the sequence belongs to the included sequences, if not, it belongs to the excluded sequences. We use the terms included and excluded here to be consistent with the section above although a probe solution is presented for both groups. In TaqMan mode, primers are not allowed degeneracy or mismatches to any template sequence in the alignment, variation is only allowed/required in the TaqMan probes.

Pushing the Calculate button will cause the dialog shown in figure 16.15 to appear.

The top part of this dialog is identical to the Standard PCR dialog for designing primer pairs described above.

The central part of the dialog contains parameters to define the specificity of TaqMan probes. Two parameters can be set:

The lower part of the dialog contains parameters pertaining to primer pairs and the comparison between the outer oligos(primers) and the inner oligos (TaqMan probes). Here, five options can be set:

The output of the design process is a table of solution sets. Each solution set contains the following: a set of primers which are general to all sequences in the alignment, a TaqMan probe which is specific to the set of included sequences (sequences where selection boxes are checked) and a TaqMan probe which is specific to the set of excluded sequences (marked by *). Otherwise, the table is similar to that described above for TaqMan probe prediction on single sequences.

Image alignmenttaqmancalculationdialog
Figure 16.15: Calculation dialog shown when designing alignment based TaqMan probes.