Technique
Here is the technique write-up, slides, and question, made and presented by my group.
The technique chosen was 3C: Chromosome Conformation Capture
Technique reflection
I have learned many new techniques in this course. Before taking this course, given my background in biochemistry, I knew many techniques that can be applied to examine proteins and protein-interactions. I did not know many techniques that can be applied to investigate interactions between nucleic acids and proteins, and I had never heard of a technique that could be applied to examine chromatin conformations. Such a technique is 3C: Chromatin Conformation Capture, which we chose for our presentation.
We also went through some techniques that examine chromatin conformation in class, such as 4C, and I was amazed to learn how much information you can extract from a simple 2D data plot of 3C and 4C.
Before reading up on 3C, I had a solid understanding of some of the reactions involved in this technique: cross-linking with a fixative reagent, digestion and restriction enzymes, and qPCR. Since I have never done a 3C experiment, the procedure seemed straightforward and not too difficult to me. It was also doing the work of this technique I got increased knowledge about the β-globin locus and chromatin looping, which I have written about in my final project.
The part to me that was the hardest to grasp at first was the qPCR step and the controls. The data plot of the resulting 3C experiment is illustrating the frequency of how many ligation events occurred between the examined genes and the LCR of the β-globin locus. To prove that these ligations occur more often when the gene is active, compared to when it is not, background ligation frequencies need to be obtained, and this is an important control. Therefore, in the qPCR reaction of the technique, one needs control templates that contain all possible ligation products in equimolar amounts in order to calculate the actual ligation frequency between a non-transcribed gene and the LCR, and a transcribed gene and the LCR.
Especially this control stands out to me as a very good example of why control experiments are just as important as the actual experiments, since we choose to believe that an increased ligation frequency is the consequence of close spatial interaction in the nucleus between cis-acting elements. Nevertheless, this increase could be of many reasons that would be unknown if no controls were conducted, such as variation in primer efficiency or simply non-equimolar start concentrations that would increase the possibility of some ligations over others.
It would be cool one day to conduct such a chromatin conformation experiment, and I would probably realize that it can be quite difficult to conduct.
Top 4 assignments
I was lucky to be able to make these assignments working with two different groups, which definitely can be seen in the assignments. It was fun to work with different people since different dynamics and ideas occur within different groups. Both groups worked really well and I really enjoyed the interesting debates when working out the answers.
Here is the links to my assignments and comments are written below each:
eRNA_Plan_Dean_Assignment_Sep_21
The paper, and the material covered about eRNA in class, really got me interested in eRNA. Since this was the first assignment, I remember that it was very stressful due to the time permitted, and we had to discuss our different understandings of the concepts presented in the review before writing down an answer. However, we quickly improved, and it became less stressful (once we realized that we had enough time!). This assignment, and the paper itself of course, is about some of the known concepts of enhancers and the functions hereof, and this assignment is a great introduction to these concepts. I think we did well, and I see we were not under as great time pressure as I remember, since we have answered both of the time permitting questions.
This assignment is about some of the phenotypes derived from mutations in HOXD13. One of the things the entire group was surprised about was that bone malformations due to the examined mutations in HOXD13 were not more severe. We all expected mutations in genes that are active during early development would result in much more severe phenotypes. An interesting point we also mentioned about why only 3 mutations are reported to Exon 1 is, that it could be because it has been less studied possibly because mutations in Exon 1 is lethal. The simple fact that only a small number of mutations have been reported outside Exon 2 does not tell us that mutations does not occur there.
One of the results I for some reason did not pay much attention to, or maybe misunderstood, really surprised me while discussing this assignment in the group. It was the fact that imprinting of the transgene occurs depending on the locus position, and the position-dependent effect seems to be more important when the transgene is inherited maternally but not paternally. Further, it is the actual lacZ transgene sequence that introduces this position-dependency. Even if I did understand this fact while reading the article, I did not believe that it was the actual case until we discussed it in the group. I think this fact definitely contributes to the perception of how complex epigenetics actually is, and this example could be a good candidate to further examine the sequence-dependency to imprinting.
(This is the assignment done on November 9 – the name of the file is wrong)
This was a difficult assignment, and I think this was my first encounter with medical genetics. But it was also VERY interesting. This assignment provides insight into why some mutations cause diseases depending on whether the mutated-locus is imprinted or not. With this assignment we also started to discuss the importance of dosage, and how mutations can influence e.g. dosages of transcripts. For example, SRS patients have a duplication and inversion that causes a double dosage of the maternal genes, and therefore cancels the silencing effect of the paternal chromosome. Whenever we discussed models afterwards, we always considered if a change in dosage was the outcome of e.g. a disease model.