Week 3

Hi everyone! I hope you’ve all had a great week! I’ll get right into what I’ve been up to this past week.

First, I’ll remind you of the main goal I’m working towards right now: developing a qPCR assay to distinguish between VJ-lambda rearranged and non-VJ-lambda rearranged plasma cells. To do this, I’ll have to design many small pieces of DNA called primers that match sequences slightly upstream of V regions and slightly downstream of J regions. Now, there are only ~70 functional V regions and 4 functional J regions, so it wouldn’t be impossible to design a single primer for each. But any reaction using that many primers would be both expensive and very inaccurate, because performing PCR with that many primers would almost certainly produce too many off-target amplicons (pieces of DNA amplified by PCR) to be an effective test for identifying VJ rearrangements.

For the next few weeks, my goal will be minimizing the number of primers I’ll need to use for my assay. I started by searching through all the published work I could find about using PCR to amplify VJ rearrangements. Unfortunately, however, the most recent list of VJ-lambda primers I found dated back to 1996—still nearly a decade before the entire human genome was sequenced! Since then, many new V regions have been discovered, and newer sequencing data has allowed for the correction of prior errors in V and J reference sequences. These factors mean I can’t necessarily trust those primers to be perfectly accurate, so I’ve decided to try designing my own primers instead.

As I mentioned last week, I’ve started with trying to amplify a known VJ rearrangement in a single cell line (a collection of genetically identical plasma cells derived from a patient). By searching through a database of previously sequenced cell lines used in our lab, we found one whose VJ rearrangement was already known. The primer design process for a single cell line was surprisingly simple, consisting only of inputting the known VJ rearrangement sequence into an online program called Primer3Plus (see below).

Although the design process was simple, the primers didn’t work exactly as intended. The first few PCR reactions that I tried appeared to produce absolutely nothing. This week, however, I was finally able to get the amplicon I expected after optimizing the temperature and template DNA concentrations for my PCR reaction (see image below).

This image shows the results of a gel electrophoresis run for the visualization of PCR product. Each bright band on the gel represents many similarly-sized pieces of DNA. Through separating these pieces of DNA by length (larger amplicons are closer to the wells at the top, while smaller amplicons travel further down the gel), we can determine the product of our PCR along with whether that product is what we expected.

This reaction was an attempt to find the optimal temperature for the cell-line specific primer pair I designed. The blue arrow represents the length of the primer pair’s expected amplicon, and the very bright bands visible in the last two wells mean the primer pair successfully amplified the VJ rearrangement in this cell line.

Unfortunately, that’s only part of the puzzle. My final qPCR assay must amplify only the VJ rearrangement region to be accurate. All the bands underneath the expected amplicon band mean that the qPCR reaction with those primers amplified several sequences other than what we expected—which is no good for a qPCR assay. 

This cell-line specific primer design was meant to be a kind of small-scale trial run for the larger qPCR assay I’ll be designing. And although I didn’t get the cleanest of results from this experiment, I’ve become much more familiar with designing and modifying PCR reaction conditions. Starting next week, I’ll be moving straight into designing primers to amplify all VJ rearrangements.

Thanks for reading!