I. BACKGROUND Your representative has received a message from a constituent that begins:

I am one of your constituents. I've worked at a university in your district for many years, studying ways to increase agricultural productivity and thereby save the world. I've hit upon an idea I'm convinced can eliminate world hunger, particularly in sub-Saharan Africa, but have failed to gain funding from either the National Science Foundation or the US Department of Agriculture, despite many attempts. Reviewers are stuck in their old ways of thinking, ways that increase the profits of huge agribusinesses but do little for people who need help. I'm asking you to intervene.

First, let me give you an idea of how my idea works. The predominant limiting input in agriculture is nitrogen, but nitrogenous fertilizer is both ecologically devestating and beyond the reach of most farmers in sub-Saharan Africa, where the need for fertilizer is the greatest. There would be no need for nitrogenous fertilizer if crop plants could draw their nitrogen from the air. Nitrogen gas is free and plentiful, making up about 80% of the atmosphere. Certain bacteria are able to do this, but at present, the ability is beyond any crop plant. My idea is to transfer the ability to fix gaseous nitrogen from bacteria to plants.

The first step is to get a copy of a gene that encodes the bacterial enzyme that fixes nitrogen gas. I will get it from the nitrogen-fixing cyanobacterium Anabaena PCC 7120 (nicknamed A7120). This organism has a gene called ALL1440 that encodes a subunit of the enzyme. I intend to isolate the gene through polymerase chain reaction (PCR) and then...

II. YOUR TASK

Your job is to explain to your representative how PCR works. You probably don't know yourself, at least beyond generalities (if not, see Section III). We will define knowing how PCR works as the ability to amplify the gene in question, ALL1440, through PCR primers that you devise yourself. You can get the sequence of the gene within CyanoBIKE through the following command:

Alternatively, type A7120 (the name of the organism) into the entity, and execute the function. Then type all1440 into the Go to box near the top of the page and click Go. That will bring you to the location of the genome where the gene resides.

From your examination of the sequence, devise primers that amplify the entire gene, from beginning to end, and have precisely 19 nucleotides each. You will tell if the primers work by using a special BioBIKE function, which you can load by executing the following:

You should get the message:

;; BBL load of "../shared-files/run-pcr.bike" beginning...
;; BBL load of "../shared-files/run-pcr.bike" ended successfully.

Then, from the FUNCTIONS menu, click RUN-PCR, fill it in as shown below, and execute it:

where primer1 and primer2 are the 19-nucleotide primers you devised, each between "...". If you execute the function, and the primers are correct, you should get a popup window that identifies the amplified gene as A7120.all1440, of length 1539 nucleotides. If the primers are incorrect, you'll most likely get no popup window (just 'NIL' in the Results pane), or you'll get a popup window with something besides what I described above.

III. WHAT IF YOU CAN'T FIND PRIMERS THAT WORK (or are totally clueless what to do)?

• Failure is not an option.
• There are many videos on the web that want to tell you how PCR works. Try some of them.
• There are also many non-video explanations on the web of how PCR works.
• You have a textbook that probably explains PCR.
• I have a pretty fair understanding of PCR. Ask me a question, either at a staffer session or by e-mail. But see this advisory first. Yes/No questions are more apt to get definitive answers.
• It may well be that you lack concepts of DNA structure or replication on which PCR rests. You may have to educate yourself about them, in a way described above or some other way.

IV. YOUR RESPONSE

Write a brief report to your representative, explaining how PCR works, focusing on the example of all1440. Explain how you chose your primers and why they work.