Week 2

Hi everyone!

Today, I'm going to explain what I've done so far on project in a little bit more detail. The current goal of my project is to develop a test that can detect a certain genetic change common in multiple myeloma cells. It'll be hard to talk about the work I'm doing without first discussing the basics of the genetic change I'm studying, so we'll start with a brief summary.

Multiple myeloma arises when the growth of plasma cells--the cells which produce antibodies--becomes poorly regulated. One of the processes important to the development of plasma cells is called VJ (variable-joining) rearrangement. In the gene I'm studying, which encodes part of the antibody called the lambda light chain, VJ rearrangement randomly selects one of many variable (V) regions and brings it next to a joining (J) region later in the genome. VJ rearrangement allows for an enormous variety of antibodies to be produced from a limited number of genes, and it plays an incredibly important in maintaining our adaptive immunity.

The goal of my project is to develop an assay using qPCR (a technique that allows for the amplification and quantification of DNA) to identify which V and J regions are rearranged in any given myeloma cell line or patient sample. Identifying these normal rearrangements can allow researchers and doctors to better identify and understand associations between genetic characteristics of myeloma cells and prognosis. The project might sound simple enough, but there are 33 different V regions I’ll have to identify along with 4 distinct J regions. And I’ll also have to distinguish between functional and non-functional rearrangements, but that’s a issue I’ll tackle once I can identify all rearrangements.

In the last two weeks, I’ve been focusing on a kind of initial small-scale test for my project. Instead of starting out with the goal of identifying all rearrangements in all myeloma samples, I’m starting with a single cell line with a known rearrangement so I can be sure I have the right idea and approach.

First, I mapped out the region I’m interested in for a cell line that was already known to have a lambda rearrangement. Next, we designed primers to amplify that known rearrangement. Primers are small pieces of DNA that bind to the larger template DNA molecule on either side of the region we want to amplify, allowing a special heat-resistant enzyme to amplify that piece of DNA exponentially in a process called polymerase chain reaction (PCR). The primer design process was simpler than I expected—all I needed to do was paste the sequence I wanted to amplify into a program called Primer 3Plus (see below).  

We ordered the primers from a company in San Diego, and within a day they’d already arrived in the lab! It’s absolutely amazing that molecular biology is so advanced that the synthesis of DNA molecules with whatever sequence we want is now trivial.

Unfortunately, the PCR itself isn’t always as simple as the primer design process. I’m facing a lot of problems with the primers not amplifying the VJ rearrangement like they’re supposed to and amplifying other regions that they shouldn’t be. I've only really had one clean result out of about 40 reactions, so I’m sending it in for sequencing first thing next week so I can confirm it's correct.

I’ve tried just about every variation on the PCR reaction, so it’s probably time to move on and design primers to amplify all the possible VJ rearrangements. But that’s an issue for next week’s post. See you then, and thanks for reading!