Primer & Amplicon DesignOverviewThe following link is to a pdf file that provides PCR recommendations, including for amplicon and primer design. This information is also available in the Getting Started on the WAVE System manual that comes with the purchase of each WAVE System.
PCR Considerations for Successful Mutation Detection
Tools at MutationDiscovery.com®
MutationDiscovery.com® includes features for designing amplicons (or just PCR primers/products) that include specified target regions. In addition, it is possible to tell the amplicon designer to exclude specified regions when selecting primers. This feature uses the program Primer3 [(written by Helen J. Skaletsky (Howard Hughes Medical Institute, Whitehead Institute) and Steve Rozen (Whitehead Institute/MIT Center for Genome Research)]. To use this feature, you must log on to MutationDiscovery.com and your account must be set up as a premium account. To obtain a premium account, contact Transgenomic Customer Support at:
(888) 233-9283
This feature is only available to Transgenomic customers.
The following information provides instructions on how to use these features of MutationDiscovery.com
Defining PCR Target Regions
The first step in designing one or more amplicons is to define the regions of interest in a locus. These regions are called PCR target regions in MutationDiscovery.com. An example of a target region might be a complete exon, with the 5’ and 3’ splice sites.
To define a target region, position the locus display so that the desired location is contained on one screen. Then click the mouse at the first base pair of the target region and, holding the mouse button down, drag the pointer to the last base pair, then release the mouse button. The entire region will then be highlighted (shown in yellow below) in the display and the base pair location of the region will be displayed in the right margin of the display with a symbol indicating that there is a pop-up menu available (shown in the red box).
Click on the location box to access the pop-up menu, and select "PCR target" from that menu.
If your account is not set up for premium access, you will get an error message indicating that you do not have access to this feature. Please contact Customer Service for access to the premium features. A "Target Region Details" window will appear on the screen.
It is recommended that you provide a label for each target region so that they are easier to identify on later screens. Like all user-entered features, PCR target regions use the standard feature permission mechanism. By default, the new target region will be visible only to the person who created the feature. To share the target region with other users, update permissions using the permissions tab.
At this point if you click on the “Save and Design Amplicon” button, the "Amplicon Design Request" window appears.
Alternatively, you may save the target region by clicking “Save” and return to the amplicon design later. This is useful if you want to design more than one amplicon at a time.
Defining Primer Mask Regions (Optional)
Sometimes a user will want to make sure that the amplicon designer does not place primers in particular locations. They may have already tried to use primers from that location and found they failed for one reason or another. To exclude regions from consideration as PCR primers, you can define primer mask regions. To define a primer mask region, select a region of sequence just as you did for the PCR target region by mouse click and drag. Click on the location menu at the right of the display and select Primer Mask from that menu.
The primer mask region details window will then appear, where you can add a label to the region to help you identify it on later screens.
This feature is useful to mask regions close to the target sequence (e.g. 30-50bp) to ensure detection of all variants within a sequence including the splice junction. Primers too close to an exonic sequence may not allow detection of all variants.
Invoking the Designer
To access the Amplicon Designer window, click on any PCR target region.
The PCR target region detail window will appear. Click on the “Design Amplicon” button.
The Amplicon Design window will then appear on the screen.
Using the Amplicon Designer
The amplicon design window includes a list of target regions for the locus. This list contains only those target regions to which the user has access. This includes all target regions created by the user as well as any target regions created by another user to which the current user has been granted access. The label field is included in the list in parentheses to make it easier to identify each target region. One target region will be selected when you enter this window. You can select any number or target regions using shift- and control- clicks in the list. Amplicons will be designed for each selected target region.
At this point you can either simply click on “Design Amplicon” and all of the default setting will be used, or you can click on “Show Primer Design Options” to see (and/or edit) Parameters that will be used in the design process.Clicking on the "Show primer design options..." button expands the window to include options for "Product Constraints", "Masked Regions", "Primer Constraints", "PCR" and "DHPLC" as shown below.
The "Masked Regions" tab is shown below:
The “Primer Constraints” tab allows you to adjust parameters such as primer minimum/optimum/maximum lengths, melting temperatures, GC content, etc. The “MaxPolyX” field allows you to specify the maximum number of identical base pairs in a row in the primers. The "Primer Constraints" tab is shown below:
The “PCR” tab allows you to set whether the protocol should be adjusted for Optimase Polymerase. The “Adjust for Optimase” option allows the user to indicate that they would like the PCR protocol to be designed specifically for Transgenomic Optimase® Polymerase, a proofreading polymerase recommended for high-fidelity PCR. If Optimase Polymerase is not used, salt and oligo concentrations can be adjusted. In addition, either standard (3-step) or touchdown PCR can be specified.The "PCR" tab is shown below:
The “DHPLC” tab allows the user to indicate whether or not melt profile analysis should be performed (this can be turned off if the PCR product will not be used for DHPLC analysis). This tab also allows the user to specify the method that should be used to select the initial temperature(s) for the DHPLC protocol. The 75% Average Helical Fraction method is essentially the algorithm used in both Wavemaker and Navigator Software. The Delta Helicity method will use a new algorithm that predicts the impact of mutations on helicity. These two methods typically yield similar, but not identical results. While the average helical fraction method is a very strong approach to amplicon design and WAVE System users are more familiar with it, the delta helicity approach is an attempt to improve the accuracy of temperature predictions. Many WAVE System users prefer to design amplicons considering the predictions by both methods. More information on temperature prediction can be found in the “Evaluate Amplicons” section. Checking the “Save Current Settings as Default” checkbox will save any changes made in the amplicon design parameters. The “Restore Site Defaults” button will remove any customer setting the user has made and return all of the amplicon design parameters to their default state.The "DHPLC" tab is shown below:
After making any desired adjustments to the amplicon design parameters, click on the “Design Amplicons” button to initiate the design process. A pop-up window will appear indicating that the process has been initiated and that a summary of the results will be sent to the user’s email account.
Viewing the Results
If you are able to view the email message with the results summary, you will be able to click on a URL in that message for each new amplicon. Make sure that you are logged on to MutationDiscovery.com using your user name and password. Alternatively, you can simply refresh the locus display window to see the results. Amplicon design is normally completed within about one minute.
If the design process is successful, there will be a new amplicon for each selected target region in the locus display. Amplicons created by the current user (either using the "Amplicon Designer" or the "Add Amplicon" method described earlier) are shown in a different color than other amplicons.
Clicking on one of these amplicons will bring up the amplicon details window, which contains the recommended PCR primers and protocol and the selected DHPLC temperature(s) (as long as these features are enabled).
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