Assignment 8: Primer Design

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!!

  1. Look at the DNA sequence, and answer: How long is the sequence? From where to where is the coding region?
Now open a window for Primer3: here

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.

  1. From where to where are the top set of primers? Copy here the first pair with their #s
  2. Is this in the region that I want?
  3. What are the top three reasons (give them in order) that primers failed (from the statistics, right and left separately)?
  4. What is the top reason the primer pairs failed?
  5. How many pairs were accepted at the end?

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".

  1. Which other genes might our primers pick up (according to the program)?
  2. Which ones do you think it will actually pick up? Explain why or why not.
  3. From where (nucleotide coordinates) are the primers located in the second sequence (in other words the first wrong sequence)?

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:

  1. How many good hits (that cover at least half of our sequence) do you get?
  2. Using the browser links, what are we picking up (make sure you have the RefSeq track open to pack, it will help you answer this)?
  3. Using the details links, what is the one area of the gene that is safe to use for primer design? (Give coordinates)

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

  1. How many primer pairs were found?
  2. Give them here (with coordinates)

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.

  1. Which genes did you get?
  2. Go back and change the Target to the genome assembly. How many genomic regions do you get (give coordinates)?
  3. How does this compare (on both gene and genomic level) to the previous set (take the primers from the top of the page and run the In-silico PCR with them)?

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:

  1. What other genes are found?
  2. Do we have to worry about this? Explain why or why not?

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.

  1. Describe your results. Which hits actually represent our gene? What are the others? Do we have to worry about them?

Congratulations! The paper was finally accepted! Now you just have to hand in this assignment.....


Hand in the report with all the answers as Assignment #8 (this should be a page with answers, NOT a printout of the assignment with answers on it.)