The eight weeks of this term didn’t seem like long enough to work on my project, but it was just enough time to obtain the results that I needed. And fortunately, I didn’t lose my sanity.
My final weeks were far more chilled out than I anticipated. Part of my project involved tasks that I could complete ‘given time’, and surprisingly enough I had some time to do them. The main task out of these was genotyping the centromeric-flanking single nucleotide polymorphisms (SNPs) of some samples – I’ll break down that piece of information step-by-step!
A SNP is one single base pair within a DNA sequence that may be one allele (a form of a base pair or gene found at the same place) or another. Let’s use rs4300027 (a SNP) as an example; the ‘T’ allele is the most common, but it may be that the ‘C’ allele is present instead. The possible combinations are therefore ‘TT’, ‘TC’ or ‘CC’. Genotyping is the process where you determine which alleles they are!
There are four SNPs that I genotyped in my project; rs7825750, rs62487514, rs4300027 and rs7826487. These SNPs are in linkage disequilibrium. This is when the alleles are found in particular combinations, instead of just random combinations. Previous studies have found that there are five possible combinations, and these combinations are referred to as haplotype classes. Every person has a combination of two of these classes that make up their genotype. The names of the classes are ‘Reference Sequence’, ‘Class 1’, ‘Class 2’, ‘Exchange 1’ and ‘Exchange 2’. Below I have made a table that shows which SNP alleles are associated with each class;
Each of the haplotype classes are also associated with different DEFA1A3 copy numbers. For example, a person with the Reference Sequence class tends to have higher copy numbers (8+), whereas Class 2 individuals have lower copy numbers (under 6). If you are looking at combinations of two of these classes, there are fifteen possible combinations. What we want to know is the most likely genotypic copy number for each of these combinations. Sequencing to find the copy number takes a lot of time and effort, whereas we have the SNP data for thousands of people. If there is a way to look at someone’s SNP genotypes and determine the most likely copy number for each haplotype class combination, this would be very useful!
I am comparing my results, which are generated by a programme that calculates the most probable copy number, with that seen in real life. There are individuals with a copy number that was determined in another study, but they also need some SNP genotypes to go with it. To determine the genotypes of these individuals, there are four separate assays (experiments) that I need to do.
All these assays use restriction enzymes, which are proteins that cut the DNA at a specific base pair. Once you have cut your DNA with the restriction enzyme, you can run your product on a gel (agarose that the DNA will move through). Gel electrophoresis works by having a positive charge at one end of the gel tank, and as DNA is negatively charged, it will move through the gel towards the positive end.
A restriction enzyme cuts DNA into fragments, and the smaller fragments will be able to run more quickly through the gel in comparison to the larger fragments. You can then scan the gel using UV light to see where these fragments have ended up. Here is a picture of a good result;
You can make out some very bright bands. These are homozygotes, meaning that the allele is present twice. A heterozygote has one of each allele, and these are shown by fainter bands. You will also notice that one bright band is lower down than the rest. This assay is for Class 2 and looks at the allele at rs7825750. The product for a ‘C’ allele is smaller than it is for a ‘T’ allele. As it is smaller, it can move further down the gel. We can therefore say that this person is a Class 2 homozygote, as Class 2 is the only class with a ‘C’ allele at that SNP.
You can hopefully see that by doing an assay with a different restriction enzyme for each SNP, you eliminate the possibilities and can conclude what the genotype of that individual is! The only problem I encountered is that the DNA isn’t in the best condition; I have had to do up to three PCRs on some of the DNA to amplify enough of it!
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My perception of what labs were like completely changed throughout my fourth year. I believed that working in a laboratory was solitary and involved very, very long hours. However, it was the people involved that made my project so enjoyable and showed me that who you work with really does make a difference.
I cannot fault the support of my supervisor, who shockingly never lost his patience with me! There are very few people who would reply to your email and help you with your work at 11 at night or six in the morning, but I am thankful that he is one of them. I was also fortunate enough to work alongside fellow MSci students that I bonded with over our incompetency; never have I sent so many science-related memes! Other Masters, PhD and PostDoc students were also friendly, inviting and helpful.
As far as my science career goes, my time in the laboratory is up. My research project was much more enjoyable than I thought it would be, but I am looking forward to its completion, as it has been a stressful year! I appreciate having the opportunity, but my journey through research has reached its final destination. Although I got some good results this time round, I am not sure whether I would have the patience to wait four years until I had good results again…
I adore science, and I especially love communicating that through my blog. Thankfully enough ‘Science Communication’ is a real thing and it is a career that I have decided I want to work towards. With regards to my blog, you won’t be hearing the last of me just yet!