We will proceed using the third primer pair Primer-BLAST suggests. You can visualize the PCR product (and additional annotation) in the UCSC Genome Browser using UCSC’s In Silico PCR tool. Select the most recent version of the human genome and paste the sequences of forward and reverse primers in their respective boxes. Click
submit. Normally, this returns the location and the sequence of the PCR product but our primer pair doesn’t return a match. When you think about this was to be expected since we are working with exon-exon junction spanning primers that are not able to match the genome sequence. So checking SNPs is not so straight-forward in the case of exon-exon junction spanning primers. We will repeat the primer search now searching for intron-spanning primers to show you how to use the in silico PCR tool. Taking into account the fact that the results for the exon-exon junction spanning primers were so messy we will make the search more stringent this time:
|Find intron spanning primers that fulfill the above defined criteria
|Go back to the Primer-BLAST and fill in the form like in the previous exercise except that they should span an intron:
Primer-BLAST returns 10 primer pairs. Again the seventh primer pair is the specific one.
|Take the seventh suggested primer pair and check for SNPs in the UCSC Browser
|Go to PrimerBLAST and paste the sequences of forward and reverse primers in their respective boxes. This time the search finds a PCR product:
Clicking the location visualizes the PCR product in the UCSC genome browser. Remove unnecessary trancks by right clicking the box in front of them and selecting
Add tracks showing relevant annotation like position of SNPs…
Setting the SNPs track from
full shows the SNPs in the browser. Center the forward primer by grabbing and dragging it to the center.
Zoom in to
base display to see if the forward primer is matching any SNPs.
As you can see the forward primer does match two SNPs but none of them are located near the 3’end of the primer.