Spring Break!

Hi everyone! I hope you’ve all enjoyed your spring breaks so far--I know I have! Last week I wrote about some of the different reactions I’ve tried with the primers I’d ordered. This week I had time to pretty much finish most of those tests. Unfortunately, I haven’t been able to get the results I’d hoped to see.

My goal was to get one single amplicon from each PCR, and you might remember from the picture I shared last week that at least four or five were consistently appearing.  I’ve tried a few different approaches, including varying the reaction temperature, primer concentrations, and template genomic DNA concentration. I saw some slight improvements, but never anything close to a single band on the gels I ran.

Now, having an initial PCR product with multiple bands isn’t an insurmountable barrier to moving forward with qPCR assay design. But having many bands in that first PCR product means it’ll be hard to tell if the correct band is present. And if the correct band isn’t present, that means when we send the PCR product in for sequencing we won’t know if we’ll get the correct result. So, while we could live with not getting a single clean result from the initial PCR, we have to be sure the correct product is in there somewhere.

I used two methods to determine whether the correct amplicon was present. The first was a restriction enzyme digest. Restriction enzymes are proteins that recognize a short sequence of DNA and cleave the DNA strand at that point. They are very useful for creating recombinant DNA by splicing together genes from different sources. In my case, though, I just needed to make a cut at a known location in the amplicon I predicted. I found a cleavage site of an enzyme called BglII that would cut the correct amplicon into two pieces of different lengths. I set up a digest on my PCR product with BglII, and then ran the digest next to the original product. If my original product was correct, then I should have seen the longer band disappear and be replaced by two shorter bands. When I actually ran the digest, though, I didn’t get a clear result.

The other method I tried is called nested PCR. Nested PCR uses the product from one PCR as the template for another PCR, this time using primers that anneal within the original product. I used two primers that would anneal inside the correct product to set up a few PCRs. The results from those were also inconclusive, unfortunately.

I haven’t come close to exhausting all the possibilities for testing my primers. I could spend months trying to get these primers to work. However, I can probably be more productive by trying a different approach. I told you before that I hadn’t found any prior research containing primers I could use, but that’s not entirely true. Originally, I was hoping to find primers that would let determine which variable and joining regions were rearranged using only a single reaction. However, there is a paper with a set of nested primers (meaning two reactions will be required) for amplifying IgL breakpoints. Dr. Riggs helped me order that paper’s primers, and they should arrive next Tuesday. That paper showed some impressive success rates for amplifying IgL rearrangements, and all the simulations I’ve ran confirm those primers should amplify all rearrangements. I’m excited to try those out! Once I can amplify these initial rearrangements, I can move towards qPCR assay design, which will let me quantify these rearrangements in patient samples.

Enjoy the rest of your break!