Restriction Digest Activity
New England Biolabs has an excellent website that will allow you to look up a DNA sequence (by matching to a primer, for example), and visualize how that sequence will be cut with any restriction enzyme. The following description will lead you through the steps to perform this activity with the gonadotropin gene. This will not only help students understand the genotyping activity, but will serve as an introduction to Genbank, the powerful data base of all known published DNA sequences. The first few steps can be accessed by clicking on the links below:
1. Go to http://rebase.neb.com and click on the brown "Rebase Tools" icon.
2. Click on the NEBcutter icon on the bottom of the rebase tools page.
3. Click on "Browse Genbank."
4. This will bring you to the National Center for Biotechnology Information (NCBI) homepage. Click on the word "BLAST" towards the top of the page.
5. Under Nucleotide Blast, click on "Standard nucleotide-nucleotide BLAST [blastn] "
6. In the box that appears, type in the sequence of one of the PCR primers- say the GTH2B- F1 primer. Then click the "Blast" button.
7. Click "Format" and wait a few seconds for the search to complete. When the page appears, ignore the stuff at the top, and scroll down to the list of matches. The top one should be one that has the chinook salmon genus and species name (Oncorhynchus tschawytscha). Click on that reference (left of genus/species).
8. This will show you the reference and Genbank number (upper left in blue) with the published sequence that matched the primer sequence. At this point, you can do one of two things, depending on how accurate you want your restriction mapping to be. To generate a map that will be close but not exactly the same as what you expect on your gels, simply copy the Genbank number into the box back on the NEBcutter page. Then follow the next few steps to generate the map and gel picture. To get an exact picture of how the gel will look, you will need to look at the published sequence and find where the primer sequences match. This information can be found by going back to the NCBI BLAST search results list and clicking on the number (matching score) just to the right of the genus/species name. Once you find out where both forward and reverse primers match (do a separate search for each), then you can copy just the region from the beginning of the forward primer to the end of the reverse primer (remember this one will be shown reversed and opposite). Highlight this region and paste it into the box on the NEB cutter page.
9. On the NEBcutter page, after either typing in the Genbank number or pasting in the exact amplified sequence, click "Submit." You will get a map of the restriction sites for many enzymes that cut this region of the gonadotropin gene.
10. To generate a map just for the enzyme you are using (e.g. BstN1), click on "Custom Digest" button on the lower left. Scroll down the list of enzymes shown and click on the box for BstN1 (don't try to type in the enzyme name on the bottom- it doesn't always work!). Click the "Digest" button at the bottom.
11. This will show you a map of the region with the BstN1 cut sites. To see how a gel with those fragments would look, click on "View gel." The picture you will be shown is for a 0.7% gel, so go to the upper left and change the gel type to 1.4%. If you've done this with the entire sequence using the Genbank number, you'll notice that the gel picture is a little different than what you would expect with just the amplified region of the gene. Why would this be? Which allele does the Genbank sequence represent?