Worksheet on FISH (Fluorescence in situ Hybridization) and ..., Lecture notes of Molecular biology

Download Worksheet on FISH (Fluorescence in situ Hybridization) and ... and more Lecture notes Molecular biology in PDF only on Docsity! Life Sciences Outreach Faculty Speaker Series for High School Biology Teachers How Biologists View Structure and Function Fall 2018 Worksheet on FISH (Fluorescence in situ Hybridization) and Chromosome Conformation Capture (e.g. 3C and Hi-C) Methods Background: FISH and 3C-based technologies are used to study 3D genome organization. FISH enables the visualization of where specific regions of the genome are located within the nucleus through the use of fluorescent probes that hybridize to a DNA sequence of interest. 3C- based technologies provide pairwise cross-linking frequencies detailing how often there are interactions between two disparate regions of DNA. Case study: After performing gene expression profiling of patient A who has an extremely rare and aggressive form of melanoma, clinicians at Boston Research Hospital have found that a key gene GROWTH1 involved in cell proliferation is significantly upregulated. Researchers hypothesize that GROWTH1 might be driving tumor expansion and decide to investigate the molecular mechanism that leads to the upregulation of that gene. The clinicians perform chromosome conformation capture (Hi-C) analysis and suspect that an enhancer E, which is known to drive the expression of very active genes, interacts with the promoter of GROWTH1 in the cancer cells but not the healthy cells. 1) Using the two Hi-C maps below, let’s hypothesize how the folding of DNA near GROWTH1 is different in healthy individuals and individuals with this particular cancer. Draw one hypothetical cancer-causing DNA conformation, and label Enhancer E and GROWTH1 as was done for the healthy conformation. Do the drawings for the healthy cancer samples support the hypothesis of the clinicians above? Bonev, Boyan, and Giacomo Cavalli. "Organization and function of the 3D genome." Nature Reviews Genetics 17.11 (2016): 661. Life Sciences Outreach Faculty Speaker Series for High School Biology Teachers How Biologists View Structure and Function Fall 2018 2) Look at the sequence below. The proposed regions of interaction are ENHANCER E and GROWTH1 and are each highlighted in their respective colors. If one designed probes to image these regions through FISH, what would a plausible imaging result look like for the Hi-C maps in question 1? Draw a diagram of the nucleus and the resulting FISH image. AGGAGTCCGAAACTTTTATTAAAGGAAAGGAGAGAAAGACTTACCAGAGACGCCGGGAAGGG GGCCAGGAAGAAGATGCCTGCCACTTACCCCAGAACCAGACGGATGGGGGTGAGGTGGTCCA GGATGTCAACAGCAGTGTACAGATGGTGATGATGGAACAGCTGGACCCCACCCTTCTTCAGAT GAAGACTGAAGTAATGGAGGGCACAGTGGCTCCAGAAGCAGAGGCTGCTGTGGACGATACCC AGATTATAACTTTACAGGTTGTAAATATGGAGGAACAGCCCATAAACATAGGAGAACTTCAGCT TGTTCAAGTACCTGTTCCTGTGACTGTACCTGTTGCTACCACTTCAGTAGAAGAACTTCAGGGGG CTTATGAAAATGAAGTGTCTAAAGAGGGCCTTGCGGAAAGTGAACCCATGATATGCCACACCC TACCTTTGCCTGAAGGGTTTCAGGTGGTTAAAGTGGGGGCCAATGGAGAGGTGGAGACACTAG AACAAGGGGAACTTCCACCCCAGGAAGATCCTAGTTGGCAAAAAGACCCAGACTATCAGCCAC CAGCCAAAAAAACAAAGAAAACCAAAAAGAGCAAACTGCGTTATACAGAGGA 3) Refer back to your hypothetical structure in Q1. If you designed a third FISH probe, MIDDLE, (in addition to the ones labeling Enhancer E and GROWTH1), in between Enhancer E and GROWTH1, what would a plausible imaging result look like for the Hi-C maps in question 1. Add an orange dot to the diagram of the nucleus from Q2. How do the relative distances between the 3 probes vary in the healthy vs cancer conditions?