The relationship I have with my project is comparable to the Little Mix song. The difficulties that the girls faced with their exes has made them a much stronger person. The same applies to my feelings towards HEX cen1. I will introduce you to this project hurdle later on, but I shall start by giving you some context.
A large proportion of my project requires typing the copy number of the DEFA1A3 locus (the position of the genes) of DNA samples. The DEFA1A3 genes encode peptides found in neutrophils, which are components of the immune system that help destroy harmful microbes. Determining the copy number in as many people as possible is important because it can help with research that has shown that lower copy numbers have been associated with diseases such as IgA nephropathy and Periodontitis.
We use something called the Paralogue Ratio Test (PRT) to deduce the DEFA1A3 copy number. For a PRT, I need to amplify and then purify the DNA samples. Afterwards, my samples undergo an ABI run; this involves electrophoresis, a process that separates the DNA molecules based on their size. I wait for the all-important email containing the results of this analysis, and then use a program called PeakScanner which allows me to predict the likely copy number of DEFA1A3.
There are quite a few steps, which I will help you to get your head around. but unfortunately, this many steps means that it is more likely that something will go wrong. What is even worse is that you can rarely pinpoint the exact stage that it went wrong, so you often have to start from scratch!
My DNA samples come from UK individuals from the Human Random Controls (HRC). I add their DNA to a mix of other components for the Polymerase Chain Reaction (PCR), which increases the amount of the specific DNA sequence ‘snippet’ that I want to look at.
A PCR requires primers, which ‘clamp’ at the end of the specific DNA sequence that needs amplifying. During replication, these bind to the DNA as they have a complementary sequence. The DNA polymerase (an enzyme which helps with the reaction) adds complementary nucleotides (the building blocks of DNA) to the DNA strands. After many rounds of denaturing (separating the double-stranded DNA), annealing (when the primers attach to the single-stranded DNA) and extension (when nucleotides are added), there is now more DNA than to begin with.
Phew, that is a lot of science in one go! Not surprisingly though, there is more to come…
Finally, I can introduce HEX cen1. The cen1 primer is complementary to a sequence at the centromeric end (closest to the middle of the chromosome), and the tel2 primer is complementary to the end that is telomeric (the tips of the chromosomes). In my PRT I use two of each of these primers. They are exactly the same in their sequence but they have different fluorescent tags; FAM and HEX.
The next stage is the purification, which is important to avoid contamination and to ensure there is enough DNA for analysis. This is all I really care to share on the purification process, because it involves 20 minutes of work and far more sitting around waiting. I am better saving my words for what is to come!
Skipping past the purification stage, my samples are sent off for an ABI scan. At long last, I finally get some results to look at. Here is a ‘successful’ result;
You can see two parallel peaks in green (representing HEX products) and blue (representing FAM products) at the telomeric end. You would also expect two peaks of each colour at the centromeric end, but more spaced-out. It is important to have all the results (centromeric and telomeric in both FAM and HEX) as it is more reliable when working out the ratio of genes at the telomeric end to those at the centromeric end, which allows you to work out the total copy number! However, HEX cen1 doesn’t always cooperate;
Where are the two peaks?! FAM cen1 has done exactly what it needs to do, but why hasn’t HEX cen1? You may remember me mentioning earlier that FAM and HEX are EXACTLY THE SAME PRIMERS, only with different fluorescent tags. So why hasn’t HEX cen1 turned up? When my supervisor mentioned that previous PhD students had struggled with HEX cen1 as well, I can’t say that I was filled with optimism. Guess this means I will just have to keep trying!
I think back to when my project began and remember feeling confident that I would have everything completed with time to spare. What I didn’t realise is that EVERYTHING requires repeats. We could be making exciting scientific discoveries all the time, but we don’t, because it isn’t science unless it initially goes wrong!
BUT SCIENCE WORKS, SOMETIMES!
And when it does work, science is great!
I have valid results for 73 of my 96 samples, so I am close enough to being able to progress onto the next stage of my project. Having more PRT results is massively beneficial as it will make my results more reliable when I compare them to previously-published work. My project has taught me a lot about patience; it’s okay to be frustrated at times, but you have to accept that something has gone wrong and keep repeating it until it goes right, as a good result is definitely worth the wait.