Alignment based design of PCR primers

In this mode, a single or a pair of PCR primers are designed. CLC Genomics Workbench allows the user to design primers which will specifically amplify a group of included sequences but not amplify the remainder of the sequences, the excluded sequences. 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. To design primers that are general for all primers in an alignment, simply add them all to the set of included sequences by checking all selection boxes. Specificity of priming is determined by criteria set by the user in the dialog box which is shown when the Calculate button is pressed (see below).

Different options can be chosen concerning the match of the primer to the template sequences in the included group:

When in Standard PCR mode, clicking the Calculate button will prompt the dialog shown in figure 16.14.

The top part of this dialog shows the single-primer parameter settings chosen in the Primer parameters preference group which will be used by the design algorithm.

The central part of the dialog contains parameters pertaining to primer specificity (this is omitted if all sequences belong to the included group). Here, three parameters can be set:

The lower part of the dialog contains parameters pertaining to primer pairs (this is omitted when only designing a single primer). Here, three parameters can be set:

The output of the design process is a table of single primers or primer pairs as described for primer design based on single sequences. These primers are specific to the included sequences in the alignment according to the criteria defined for specificity. The only novelty in the table, is that melting temperatures are displayed with both a maximum, a minimum and an average value to reflect that degenerate primers or primers with mismatches may have heterogeneous behavior on the different templates in the group of included sequences.

Image alignmentpcrcalculationdialog
Figure 16.14: Calculation dialog shown when designing alignment based PCR primers.