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!
