Wednesday, July 18, 2012

PCR, Forrest Gump and Nomenclature


Do people really get bright ideas in the shower? That is a topic for another day, but I think I recently had a good one. It all started with a question from a post baccalaureate trainee at NIH, where I currently work. She encountered a problem when designing primers for Polymerase Chain Reaction (PCR) based on a published paper. Since I was the resident PCR specialist she came to me for advice.

I think a brief introduction to PCR will help here, though a detailed blog on PCR will follow. It is the biochemical reaction that revolutionized DNA amplification, the basis for genome sequencing and cloning. It involves choosing two short stretches of DNA (primers) from a template (genome, plasmid, etc) and exponentially amplifying the DNA between primers using an enzyme called DNA polymerase. DNA, such as our genome, consists of two complementary strands running in opposite directions, while PCR primers are single-stranded. For PCR to work, each chosen primer needs to bind one template strand and the two primers need to be directed towards each other (illustrated below).

The postbac’s problem was that of the two published primer sequences, only one could be located in the mouse genome. Furthermore, she was surprised that what was labeled as forward primer in the paper was actually a reverse primer! By convention, the primer that is identical to the sequence of your gene of interest is called forward primer and the one that resembles the complementary strand is called the reverse primer. The postbac was confused because the paper labeled the primer from the complementary strand as forward. But does that really matter? As an aside, this actually happens with surprising frequency; scientists don’t seem to have enough time to confirm many of their findings and rush their publications out the door. Naturally once a finding is out, very few try to repeat those experiments; what is the point anyway… if you confirm the results, then you would have wasted your time and resources on something that is already known!

Getting back to PCR, the primers don’t really know whether they are forward or reverse; if they see DNA sequence that they can bind to, they will. What we name the primers is up to us. They don’t care what we call them, forward/reverse, left/right, up/down, or mango/banana. Put them in the PCR reaction, they work the same whatever their names.

So, what was my shower idea? I happened to be thinking about my drive through Highway US 163, a two-lane road with one lane going in each direction, near Monument Valley in Utah/Arizona a few years ago. This is where Forrest Gump stopped running. He probably did not know which direction he was running in, but he knew how to run! Seemed like a great locale to illustrate how PCR works... Now drop a couple of drivers on that straight road and tell them whichever direction they go is the right one. The only way they see each other is if they start driving in opposing directions and are heading towards each other, assuming they drive at the same speed. It is immaterial who drives towards Utah and who drives towards Arizona. The objective is for them to meet. This is what needs to happen for PCR to work. What are the parallels to PCR? The two opposing lanes of US 163 are the two strands of DNA, the two cars are the primers and the drivers are the polymerase enzymes. So, the bottom line for my postbac friend was, not to worry about the nomenclature, but to design the primers and see if they work!



In this illustration, the thick green line corresponds to the sense strand (the gene sequence) and the thick red line corresponds to the antisense strand. Thin green line is the forward/sense primer and thin red line is the reverse/antisense primer. Blue oval is the DNA polymerase enzyme. PCR product would be a double helix formed by the two dashed lines (red and green). The DNA molecules have arrows because DNA is directional. 5 represents the end of DNA that has a Phosphate group and 3 represents the end of DNA with the sugar group. In this example, a green sequence will always bind a red sequence and vice versa.

Tuesday, July 17, 2012

What's in a name?

The question arose when I decided to start my own blog and had to come up with a name that succinctly and accurately represented my feelings about what I was going to deal with in the blog.  I knew I could find the right words, but in the process relearned a lesson that was first imparted to me when I started my Ph.D.

What is the lesson?  It is that our thoughts may be random, but they need not necessarily be unique; in other words, multiple people can have identical random thoughts.  When I started my Ph.D., I was full of these wonderful, original AND unique ideas; there was no one better qualified to do a Ph.D. than I was, or so I felt.  I would run these ideas by my thesis advisor Dr. Ren, who gave me a patient ear whenever I ran into his office.  Then one day after a discussion of another one of my fabulous ideas for half an hour, he said, “Just remember that if you can come up with a novel experiment, someone else would have already done it or would be doing that experiment right now”.  I was thoroughly offended...  What does he know?  Who does he think he is anyway?  Well, next month my idea was published in a scientific paper (proof that it indeed was a fabulous idea and that someone else thought of it before me)!  Maybe Dr. Ren could guess that my idea was an easy one for others to come up with, or maybe he reviewed the paper that was about to be published!  Either way, I learnt the lesson that coming up with unique ideas is not an easy task.

That lesson was revisited when I tried to find a name for my blog.  I first thought of calling it “Random walks of a wandering Brain”, but when I did a Google search for the phrase, I came across other, similar sounding phrases the closest one being “Random thoughts from a wandering mind”.  Upon further cogitation, I came up with the name “Brownian Brain” for my blog, based on Browning motion, which describes the random movement of particles and is named after Robert Brown who first observed it.  Quite naturally others thought of similar names or descriptions for their blogs, such as Brownian movement of my thoughts and Brownian motion of thoughts.  Hmm, what to do?  Well, if people can share a Nobel Prize for coming up with similar ideas independently, why can’t we share similar but original ideas in mundane stuff like names of blogs?  So, with a salute to all others who had thought similar thoughts, I will be calling my blog “Random Walks of a Wandering Brain”, with the link to the blog being, “brownianbrain.blogspot.com”. Thanks for visiting!