Quicktest Primer and restriction sites

With the introduction of the Quicktest Primer tool in MacVector 12.6 primer design became even easier. Being able to slide your primer along a template sequence to see undesirable secondary structure and other attributes in realtime really does make it quicker and straightforward to design good primers. All user feedback we have had say it really does make a difference to primer design!

Now with the release of MacVector 13 it’s been enhanced to display “one out” and existing restriction enzyme sites around the primary binding site of the primer.

Sites that are created or destroyed by mismatches in the primer or due to the addition of a tail are now shown above the sequence. ‘One out’ sites can also be displayed below the sequence, color coded to indicate their potential effect on overlapping coding regions. The display is interactive so that when you “mouse over” a site, additional information is displayed. Clicking and holding on a “one out” site temporarily changes the primer and updates the entire dialog to reflect that change. Finally, double-clicking on a “one out” site makes a permanent change to the primer sequence.

Existing restriction sites are shown below the template sequence in black. However, sites that are unique to just the primer (including any tail that’s been added) will also be shown.

• Existing sites in the template sequence are shown below the sequence in black.
• New sites that will be introduced by mismatches in the primer are shown above the sequence in black.
• Existing sites that will be destroyed by mismatches in the primer are shown in grey.
• Putative “one out” sites are labelled with an asterisk as already used by the main restriction enzyme analysis tools.
• Putative “one out” sites that are translationally silent are shown in green.
• Putative “one out” sites that will change the amino acid sequence of the coding region are shown in red.

As well as displaying these sites the new tool makes it very easy to introduce the nucleotide change needed for the new restriction site into your primer.

1. If you mouse over one of the putative sites then the mismatched residue is shown in lower case and the recognition sequence of the site is displayed as a tooltip, aligned to the primer (as in the animation above).
2. If you click on a one-out site where the mismatched residue lies within the primer and hold the button down, the primer sequence temporarily changes, replacing the mismatched residue and showing any amino acid and restriction site changes above the primer.
3. Finally if you double-click the site it will make this change permanent in your primer.

Adding restriction sites to your PCR products has never been easier. With the usual MacVector way it’s just a few mouse clicks!