Week 4

Hi everyone! I know my last posts have been gone into the background and initial results of my project in some detail, so this week I want to give you a better idea of what my typical day at Mayo looks like.

On Monday, I double-checked the primers I had designed earlier and mapped out where they are predicted to bind to the target strand. This helps me know how long the products I’m expecting should be, which lets me evaluate the success of my PCR reactions.

After a final screening for any obvious mistakes in my primer design, I ordered the VJ rearrangement primers on Tuesday. It takes the company about one day to synthesize them and one day to ship them, so they were delivered Thursday morning. In retrospect, I probably should’ve just ordered the primers as soon as I’d designed them because they only cost ~$5 each. I did appreciate having plenty of time to  organize my lab book and the files on my computer so the experiments I’ve planned will be well-documented even after I leave. My notes never turn out quite as neat as I’d like, but I’ve slowly gotten better at remembering to write down all the relevant details. Some advice for Honors/AP Chemistry students: recording procedures, data, and observations in lab notebooks aren’t skills you’ll need only in high school. If you plan to do any research in science, you won’t get far without keeping a diligent record of what you’re doing—and it’s always easier to maintain a good habit than to change a bad one.

While waiting for the primers, I attended two incredibly interesting seminars (along with one lab group presentation). In the lab group presentation, a professor from another lab on our floor that studies chronic myeloid leukemia. Even though there were plenty of gene, protein, and cell line names that I did not recognize, I always enjoy the opportunity to hear different approaches to answering scientific questions.

The first seminar I attended focused on viroimmunotherapy. I had no idea this existed, but apparently a new therapeutic approach is using genetic engineering to create “oncolytic” viruses, which directly attack and kill cancer cells while leaving healthy cells unaffected. And an even more promising treatment strategy is using viruses that help the immune system target cancer cells. If anyone’s interested, here’s a few papers on viroimmunotherapy by the Mayo Clinic doctor who presented at the seminar.

The second seminar I attended focused on dietary and behavioral modifications to avoid cancer, diabetes, and other obesity-related diseases. I honestly expected the seminary to be just a boring reminder to eat healthy foods and to get plenty of exercise. I couldn’t have been more wrong. Dr. Ruth Patterson, a UCSD professor, began by showing us her finding were nearly entirely independent of obesity, caloric intake, and exercise. Her research provided some strong evidence that the incidence of cancer, diabetes, and other obesity-related illnesses is instead correlated to mean sitting time and temporal patterns of food consumption. At this point, her research is too preliminary for her to make any lifestyle recommendations, but she noticed the biggest drops in insulin resistance among patients who stood in place for 2 minutes out of every hour along with patients who fasted for at least 12 hours after their last meal of the day. These seminars were a nice reminder of the many fields of research that intersect with oncology.

On Thursday, my primers finally arrived. This week, I only had time to set up one PCR reaction and gel electrophoresis. I'm still interpreting the results, but I think they are very promising so far. I can't wait to mess around with the reaction conditions next week, but for now here are some photos of the process! 

The bench I work at.

The PCR I set up Thursday afternoon. Everything is kept on ice to protect the enzyme used to amplify DNA. Just behind the ice bucket you can see four pipettes, which are used to move small volumes of liquid.

The thermal cycler I used to run my PCRs.

My gel just before I turned on the power supply. Each well is filled with a different sample from my PCR mixed with a blue loading dye that makes it easier to load the wells and visually check how quickly the gel is running. 

The same gel after 30 minutes at 100V. The loading dye has separated into blue and purple bands, indicating the gel is almost done.

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!

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! 

Week 1

Hi everyone!

I started working in the Bergsagel lab at the Mayo Clinic this Monday, and I couldn't be more excited! I’ve already been able to start analyzing cell line and patient DNA sequences, and I’ve also had the opportunity to start practicing with the programs and protocols I’ll be using.  

This lab is the first one I've worked in focusing on eukaryotic organisms and cell lines; in my previous lab experience, I had only worked with E. coli. So far, I've noticed a surprising amount of similarities between the two. The overall concepts behind growing human cell cultures are very nearly identical to those governing bacterial growth, even if the exact details of the protocol might differ slightly. For example, E. coli is usually incubated at 37°C in atmospheric air, and mammalian cell lines are usually grown at 37°C except in a slightly more controlled incubator, with 5% carbon dioxide in the air to more closely match physiological conditions. And from what I've seen, this pattern holds true for lots of the techniques I'd learned in the microbiology lab compared to the techniques I'm learning right now. As a result, most of the protocols I’ve practiced so far at Mayo are nearly identical to how I had performed them before.

           

That being said, some of them differed drastically from what I’d seen earlier, and some were entirely new. I’d never needed to work under a laminar flow hood (which prevents contamination of the cell cultures) or counted cells with a hemocytometer before. I’ll need to be careful to remember exactly how the methods used in mammalian cell labs differ from those in bacteria-focused labs so I don’t get the two mixed up.

Right now, the direction my project will eventually take isn't set in stone, but it will begin with the same initial steps regardless. The primary initial goal of my project is to develop a qPCR assay that can detect different rearrangements of the genes that encode antibodies. These rearrangements can occasionally make a mistake by swapping with the wrong DNA sequence. And if certain oncogenes have their expression increased by this inappropriate rearrangement, multiple myeloma can result. But before we can easily identify these inappropriate rearrangements, we need to be able to clearly analyze normal rearrangements. Creating this qPCR assay for normal rearrangements will be the first step to developing another assay to identify faulty rearrangements.

I’ll be posting again later this weekend to provide a short summary of multiple myeloma and the IgL genes I am focusing on in my project, so don’t forget to subscribe for email notifications if  you’re interested!

Grady Day