QuickTest Primer

Quicktest Primer simplifies the design of primers with mismatches and/or tails. Quicktest Primer allows you to quickly evaluate the suitability of any short (~20 residues) DNA sequence for use in PCR or sequencing experiments.This can be used in conjunction with Design Primers (Primer3) to design pairs of primers with mismatches and/or tails and to easily generate the predicted product of the reaction, complete with tails and/or mismatches.

QuickTest Primer displays a variety of information of any primer entered in the main edit box

• Tm
• Presence of "bad" residues like non-G/C 3' end and runs of >3 homopolymers (esp G)
• "Score" of hairpin loops and primer dimers
• Graphical display of hairpin loops and primer dimers.
• Location of any other matches on the target sequence

The main editor pane also shows the primary binding site of the primer on the currently active sequence window (if one is open). Any secondary binding sites are shown in a scrolling list and can be clicked on and directly viewed in a separate alignment panel. No binding information is displayed if the primer does not bind to the active nucleic acid sequence or if no nucleic acid sequence is active. If you bring another sequence window to the front, it will instantly update to display the potential binding sites of the primer (if any) on that sequence. You can use the arrow buttons to "nudge" the primer along the sequence to try to reduce potential hairpin or primer dimer/duplex formation. The translations of any CDS features in the sequence are displayed and you can edit the primer sequence to introduce mismatch mutations and any amino acid changes that would result are instantly displayed

Information panes above the primer editing box show graphical representations of the highest scoring primer dimer and/or primer duplex that can be formed by the primer with itself (if any) and any hairpin loop structures that may be present in the sequence. Scrollable text boxes also display primer characteristics including molecular weight, Tm, thermodynamic properties, etc.

Restriction Sites

As well as existing restriction sites in the template sequence Quicktest primer also shows restriction sites that will be introduced into the product with changes in the primer. It will also show putative "one out" sites that can be introduced with a single base change and whether these are translationally silent.

• Existing sites in the template sequence are shown below the sequence in black.
• New sites that will be introduced by the primer are shown above the sequence in black.
• Existing sites that will be destroyed by the primer are shown in grey.
• Putative "one out" sites are labelled with an asterix as in 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 coding region are shown in red.

If you mouse over one of the putative sites then the mismatched residue will be shown in lower case. 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 the amino acid change. When you release, it will return to the previous sequence. However, if you double-click the site it will make this change permanent in your primer.

Primer Database

The Primer Database section allows you to save designed primers direct to your own primer database. The Choose and Open buttons allow you to open and select which primer database file you are using. The Add to.. button allows you to save your designed primer to the database. The Primer Selector allows you to edit an existing primer that is stored in your primer database.

Quickstart

1. Select a short region of a sequence that approximately corresponds to the area you want your primer to be
2. Go to ANALYZE > PRIMERS > QUICKTEST PRIMER

To assess the suitability of a sequence as a primer

1. Choose Analyze | Primers | Quicktest Primer... from the menu. The Quicktest Primer dialog box is displayed
2. Type or paste the primer sequence you want to evaluate into the central primer editing box, with the white background.
3. Click in the Optional tail region of the primer editing box and type in a sequence to add a tail to the primer.When you make these changes, the Quicktest Primer display updates dynamically.
4. Click Copy Primer & Tail to copy the full primer to the clipboard for pasting into other applications.
5. Click Show Report to generate a report summarizing all the information displayed in the Quicktest Primer dialog.
6. Click Primer3 to open the Primer Design (Primer3) dialog box, prepopulated with the primer you have been assessing.

To assess the suitability of a portion of an existing sequence as a primer

1. Open the nucleic acid sequence file you want to analyze.
2. Select a primer-sized portion (less than 80 residues) of the sequence in the Editor, Map or Features tabs.
3. Choose Analyze | Primers | Quicktest Primer... from the menu.
4. Click the left and right arrow icons to nudge the primer selection left and right along the sequence.
5. Click in the Optional tail region of the primer editing box and type in a sequence to add a tail to the primer.
6. Click Copy Primer & Tail to copy the full primer to the clipboard for pasting into other applications.
7. Click Show Report to generate a report summarizing all the information displayed in the Quicktest Primer dialog.
8. Click Primer3 to open the Primer Design (Primer3) dialog box, prepopulated with the primer you have been assessing.

Related Topics.

Primers

How to design primers with tails and mismatches.

Primer Database

How QuickTest Primer scores hairpin loops and primer dimers.

Design Primers (Primer3): Advanced Options

Sequencing primers/probes

Test sequencing primer/probe