Tuesday, June 9, 2015

6 9 2015 Reverse Engineering a Flanking Primer

Today I needed to develop a way to reverse engineer a flanking primer from a primer that someone else designed. For my qPCR runs, I have an HSP70 and Actin primers which were developed previously by someone else with little information on how they were developed. The primers work great but to determine if they are reaching the same efficiency in every sample or cover the same isoform of the gene in every population I need to develop flanking primers. Luckily NCBI has some advance functions in the Primer Blast program which allowed me to find the template region in my transcriptome to develop flanking primers.

First I needed to take the sequences of the primers I had and find where they existed in the Transcriptome I'm using.

Under primer parameters I put in the forward and reverse primer sequences. Then under Primer Pair Specificity Checking Parameters I changed the Database type to custom and uploaded the Oly Transcriptome V3. Then for added effect I changed the organism to the Ostrea taxa. I don't think this really did much but it didn't detract either.  Then I hit get primers and waited for the results.

You can see what the input looks like above. Below is the output for the primers. 

This out put tells me the reference name of the template from the Transcriptome on the left, the size of the primer product, and where the primer lays down on the sequence. Under each primer sequence there is a line of ...................... these indicate that the complimentary strand is a 100% match. If any of these dots are replaced with a letter it tells you whats different and that its not a 100% match. You can also see that the top 3 template matches have reference names that are very similar. This is because they are overlapping reads of the same region. If the names were different then we could assume it was a different region. 

Once you've got the reference name, then you can follow the same method I used to develop the other flanking primers here

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