Hi everyone!

This week I’ve really started getting some great results to report! On Monday, I was able to order the first qPCR assay for L363, the cell line I had sequenced last week.

The qPCR setup I’m using is a lot like normal PCR in that I ordered two primers (one forward and one reverse) that span about 150 base pairs across the VJ breakpoint. I’m even using basically the same Taq polymerase in my reactions, which luckily makes it very easy for me to optimize my qPCR reactions first using regular PCR + gel electrophoresis. The only new ingredient I added to the qPCR is an intercalating dye called SYBR green. Intercalating basically means that it binds to double-stranded DNA, and when it does bind DNA it fluoresces green when exposed to blue light. The intensity of the green light from the SYBR green is actually directly proportional to the quantity of DNA currently in each tube. With PCR, we start with a very low quantity of the template DNA, containing anywhere from a few hundred thousand to a few copies of the IgL breakpoint we’re interested in. Each subsequent cycle the qPCR goes through essentially doubles the number of the target sequence without amplifying the rest of the template. The overall effect of this is that when you plot the fluorescence levels vs cycle number, you basically find out that every subsequent fluorescence level is roughly 2x the intensity in the last cycle.

Using a few samples of known dilutions, we can prepare what’s called a standard curve for this particular qPCR assay, essentially telling us the relationship between fluorescence levels and initial template DNA concentration. I’m probably making this sound a lot more difficult than it really is, because all the calculations are basically done automatically by the software we use.  We can work backwards from this to figure out how many copies of the IgL VJ breakpoint of L363 were present in each sample reaction we are interested in running.

Designing and testing these qPCR assays is definitely my favorite aspect of the research process so far.   is probably my favorite part of my project so far. However, I’m going to have to figure out the best conditions pretty quickly before I move onto real patient samples. Now, because I’m working with a cell line, I had nearly complete control over how much DNA I had put into each reaction, and a basically unlimited supply of DNA to work with. With patient samples, though, I’ll have only a few tries to get it right before I use them all up—so right now I’m going to focus pretty heavily on optimizing as fast as I can.

Thanks for reading!