Please read through carefully BEFORE starting.
In this assignment, we'll design primers and compare results to various databases.
You sumbit a paper to a leading journal on human RAET1L (also known as ULBP6). This protein is a member of a family of ligands for a Natural Killer cell activating receptor, and is of great interest in the immunology world (the ligand family members are known as RAET1# or ULBP#. Unfortunately, the nomenclature is a mess). A reviewer wants to know about expression in various tissues, and you design a PCR reaction to test that. You decide to plan the primers in the protein coding area, as it is very conserved. Take the sequence of NM_130900.3 from NCBI (for a window, click here. (take the sequence with the numbers, it will make this easier) Keep this window open, you will need it later!!
Change the following parameters: Set the Mispriming library to human (on top of the input box); primer length to 18 (min), 22 (max), erase the "opt"; change the Max Tm Difference to 2.0; change the Primer GC% to Min:40.0 Opt:50.0 Max:60.0; Change the Product size ranges to 400-600 (only - erase the others). Click pick primers.
You use the primers, they work beautifully, and you resubmit the paper with the added results. The reviewer comments back that the area of the gene chosen isn't specific enough, and that you should have planned primers that are more specific. You decide to check this out at NCBI. Go back to the NCBI page for the gene that you opened before, and click on the link on the upper right hand side "Pick Primers". Copy and paste the top set of primers you got before in Primer3. Click "Get Primers".
OOPS. The reviewer was right, and we didn't do our homework before planning the primers. We'll take the sequence and compare it to the human genome at the UCSC genome browser (link here). Blat the sequence of our gene and answer the following:
Now we have to design new primers. Go back to the Primer3 page. Change the Product size ranges to 50-200. Put < after bp 150, and > at the end of the sequence
Now we'll check for specificity. We'll use both In-Silico PCR from UCSC this time. Go to your genome browser page and choose In-Silico PCR from the Tools menu in the blue menu bar at the top of the page. Paste in your new primers, make sure your Target is Genecode Genes and click submit.
We know that the in-silico PCR tool can be too specific, so we'd like to double check with another tool. Go back to the NCBI primer blast page you had before, and go back to the input. Paste in the new primers, and answer:
We ran the primer blast looking for mismatches to our sequence in particular. Go back to the primer blast input page, erase our gene accesion number from the input box on the top, and change the database (under Primer Pair Specificity Checking Parameters) to nr.
Congratulations! The paper was finally accepted! Now you just have to hand in this assignment.....