In the first article in this series we took an overview of all the primer design tools in MacVector. In this second look at primer design in MacVector I’m going to look in more detail at our latest tool – Design Primers (Primer3).
Primer3 is a powerful, highly configurable command line tool for designing primers. However, using it from the command line is fairly complicated, and it has a hard learning curve. For example you need to supply your template sequence and parameters using a pre-XML data exchange format called BoulderIO. The output is also in this format. Consequently quite a few web interfaces have been developed to make Primer3 more accessible to users. MacVector also hides all this complication from you and supplies a very easy to use point and click interface to Primer3. It provides an exceedingly easy way to design a set of primers. Our aim in designing the interface was to allow a user to be able to design a set of primers to amplify a certain part of their sequence with a maximum of three mouse clicks. This was achieved and the simplest way to design primers with MacVector is:
1 – Select a particular feature or region of a sequence by highlighting it.
2 – From the menu choose
Analyze | Primers | Primer Design (Primer3)
3 – click OK.
…and that’s it! The default primer design settings will be used to produce the optimal primers to amplify that section.
The results are also presented in an easy to use way. You have a ranked list in spreadsheet form, that will allow you to quickly copy and paste your results into an email to send to your oligo synthesising service. You will also see a graphical map of the top five primer pairs along with the expected product that each primer pair would produce.
Our secondary design aim was to allow experienced users access to the powerful functionality of Primer3. For the default values we chose the settings that will give you the most sensible and useful primers in the majprity of scenarios. However, these can be changed in just a few mouse clicks. By modifying the advanced parameters it is possible to tune these values, to obtain the best primers for non-standard situations.
Design Primers also lets you have more control about where the primers are picked from. As well as selecting a single feature, you can also choose a Region to Scan . Here you can specify a region within which the amplified product should lie. Simply choose the size of product you want and Primer3 will design primers to amplify products of that size, from anywhere in that region. By default this method will design primers to produce products between 100 and 300 bases in length. However, you can change the minimum and maximum Product size values to design primers to produce shorter or longer products.
Finally, you can design primers by specifying Flanking Regions and your left and right primers will be selected from these. This is similar to the Amplify Feature/Region method but it is much more flexible, as you can specify more precisely the region that each primer is selected from.
In the next Primer Design article we’ll look at designing internal primers for realtime PCR.
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