Determining the Reading Frame of the Gene

• Begin by saving a copy of the DNA sequence to a local drive.  Click on the following link (HSV-1 ICP34.5 gene sequence) with the right-mouse button and select the Save Link As option.  Once you have saved a copy, you can then click on the link to bring up a copy in Netscape.  Select the entire sequence (by dragging your mouse over the entire sequence or simulataneously depressing (<CTRL>A) and copy the selection onto your clipboard (<CTRL>C).

•  Link to Baylor College of Medicine Search Launcher Page (This will open a new browser window)  Select Sequence Utilities (pulldown menu).  Paste sequence into window from the clipboard (<CTRL>V) and select 6-frame translation.  Click on the Submit button to start the utility.

 
• Save page output to your folder and print a copy for examination.  Find the correct frame reference by 1) determining which sequence has a start codon (ATG) and has the best sequence not interrupted by a stop codon (*).  The correct translation will begin with the peptide sequence MARRR.

 
• Click on the Back button to return to the Sequence Utilities page.  Your original data should still be in the clipboard.   Re-paste the original sequence (<CTRL>V), if necessary, into the data window and select Reverse Compliment.  Submit the sequence, save the output page to your disk and print a copy for inspection.

 
• Using your printouts for both the coding sequence and its compliment, mark off where the frames of the gne and its reverse compliment start and stop.   (Hint 1:  Fasta formatted data contains lines of 50 characters, which should make counting residues easier.)  The 5' and 3' ends of both the coding and compliment strands will determine the starting point for you primer search. (Hint 2: Both sequences are written 5' to 3'.  The frame of the gene will be a certain number of residues from the beginning.  The frame of the reverse compliment will be that same number of residues from the end.)

 
• Look at the coding sequence.  Up the page from the gene frame lies the region containing your potential primer.  Remember: The primer for the reverse compliment should initiate the polymerization of a sequence that contains the coding sequence and the primer for the coding sequence will polymerize to incorporate the reverse compliment.  Typically, the primer will start a good distance from the ends of the actual coding sequence and will be 18-24 nucleotides in length.  Once you have a starting point, link to the NetPrimer site.  (Caution: This will load a Java applet and may take a few minutes to complete)

Primer Selection and Screening
 

• Click on the Help button for the NetPrimer window and familiarize yourself with what the program will do.

• Once you are comfortable with how the analysis program works, try a sequence.  The easiest way to do this is to copy sequences from the original text window and paste sequences into the NetPrimer Oligo Sequence window.  (Briefly: select your sequence by scrolling over with you mouse with left button depressed.  Type <CTRL>-C to copy onto clipboard.  Swith to NetPrimer window and click cursor into correct window and type <CTRL>-V.  And voila, your sequence is pasted intact.)

• Experiment with the sequence by moving the frame or extending/truncating the sequence.  With each modification, keep track of the rating posted by NetPrimer.  It gives you a sense of whether you are getting warmer or colder.(A score of 100 pretty much means you've hit the bullseye).  Once, you have a primer you are happy with, we'll then map it against the GenBank data base to make sure you don't go replicating lots of genes by accident.

• Link to NIH-Blast search engine.

• Copy and paste your primer sequence into the input window.  The label the sequence you need to type in the first line as follows:

• The angle bracket (>) denotes comment text, rather than nucleotide data.  Try to keep the name reasonably short but descriptive enough so you know what it is later.

• Click on the Search button and follow the directions as posted.  The final output will give you a lising of all sequences in the NIH databank that would match against your primer.  The fewer matches you get, the more selective you primer.  If your primer matches a lot of mammalian genes, you'll probably have to go back to the sequence and find another starting point.