So far, things look great: from using the TapeStation on our DNA extracts, we know that there are small fragments of DNA (likely ancient DNA, or aDNA) preserved in the samples that we brought from our C.ohioensis specimens at JMM– Heather treated us to ice cream to celebrate! We do not yet know for sure whether the ancient DNA is endogenous giant beaver DNA (which is what we want) or whether it’s environmental DNA (we don’t want that). Ancient DNA is difficult to find in specimens that are so old, for various reasons including DNA damage due to poor preservation. At least, now we know that our samples probably contain aDNA and for now, that’s reason enough to celebrate!
For our next step on Thursday, we started a long-range PCR with the modern beaver DNA. (Just a heads up, from here onwards this post is going to have a lot of scientific details, so you can skip to the last paragraph if you’re not into all the details). The whole mitochondrial DNA of a modern beaver is about 16,000 base pairs (bp) long. So, we ordered primers that would amplify the entire mitochondrion in two large overlapping pieces that are 6,000 bp and 11,000bp. We ran a long range PCR to amplify those fragments. We were a little nervous whether the long range PCR would work at the first attempt, because Heather told us that they fail more often than not. We left the products in the thermocycler overnight because it takes ~6 hours to run a PCR that can amplify such long fragments!
Every day, we plan our work in such a way that we finish all our ancient DNA lab work first, before moving on to the modern lab, in order to reduce contamination from modern DNA and PCR products. So, the next morning (Friday), we couldn’t go in to look at our long range PCR results until after we finished any work we wanted to do in the ancient DNA lab. So, first we suited up in our smurf suits and started working on our single-stranded DNA library prep. This method has been optimized to retain very small DNA fragments, which is important for working with the small fragments of aDNA. Since the library prep is a long process, we decided to break up our work over 3 days and on Friday, we did the first three steps:
- Ancient DNA is often damaged by the Cytosine base being deaminated and converted into Uracil, artificially changing the DNA sequence from a Cytosine to a Thymine. Since we want accurate DNA sequences, we cut the damaged DNA at any Uracil site.
- We then dephosphorylated and denatured the DNA using heat to separate the two DNA strands from each other so we could work with single stranded DNA.
- We then added a short DNA sequence, called an adaptor, to each DNA strand. This adaptor has a biotin molecule on it, which will make it easier to use in later steps, which we will explain in future posts!
This is a good stopping point because ligation products can be stored for several days at 20°C.
After finishing up our work in the aDNA lab for the day, we went back to our long range PCR. Heather was very nervous about how the PCR would turn out because we had limited time on hand before she has to return to the U.S. and so we really needed the first trial to work out. I, on the other hand, was (way too) excited about the pretty purple gloves they had in the modern lab!
Luckily, our PCR did work on the first try (see the photo below)! The 6kb piece was amplified very nicely (right side of the photo on top and bottom left) but the 11kb needs to be cleaned by running a second gel because some other smaller pieces of DNA were also amplified (top left). After cleaning, we will chop the DNA to ~500bp fragments which will be used to make baits for hybridization capture (more details on this later).
Since our long range PCR worked, it was time for another celebration! We know that we are still many steps away from getting our final results which means there are many steps where things could go wrong and we could end up with no results. So far, nothing had gone wrong, and that’s what we were going to celebrate!