F22_DS10_Mutation and Molecular Methods

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Jan 9, 2024

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Discussion 10 Bio 305 – Genetics Mutations and Molecular Methods Consider the following hypothetical genomic locus for the gene xpd , with 3 exons and 2 introns: *Although only a fragment of a much larger chromosome is shown, for the purposes of this exercise, ignore the DNA flanking the outer KpnI restriction enzyme cut sites. KpnI and XbaI restriction enzyme cut sites are shown by the vertical lines. The numbers below each line indicate the number of nucleotides from the first KpnI site (at position 0) in the wild type allele, and the exon/intron boundarys are also listed in reference to the first cut site (so, for example, the first exon begins 400 bp after the first KpnI cut site, and is 900 bp long (1300-400=900). UTR regions of exons are shown by diagonal stripes, while the coding regions are solid color. You are studying Garnet-Feist syndrome (GFS), a novel neurodegenerative and muscle wasting disease . Using a GWAS (Genome wide association study) you have identified three mutations in the xpd gene which associate with the disease. The first, called the xpd a allele, is a point mutation in the first exon ( indicated by an asterisk in the image above ). The second, called the xpd b allele, is a much larger change, and is caused by a deletion of 400 basepairs (0.4 kb) from exon 2. (*The region deleted from exon 2 is indicated by the black double bar drawn below exon 2 in the diagram above). The third, called the xpd c allele, is a point mutation in exon 3 ( indicated by the pound sign ) and is in the middle of the KpnI recognition sequence. You will be using several of the molecular methods covered in the textbook and lecture to study the xpd DNA, RNA and proteins of four indivuals: Declan is homozgous for the wild type allele (+/+), Aditi is heterozygous for the xpd b allele ( xpd b /+), Javier is homozygous for the xpd a allele ( xpd a / xpd a ), and Heba is homozygous for the xpd c allele ( xpd c / xpd c ). You have collected skin biopsies from each individual. Using stem cell reprogramming techniques to create cell culture lines from each biopsy, you hope to test potential therapies for GFS. Unfortunately, your lab assistant labeled each culture with a unique ID, but then lost the list matching the IDs to each person. 1a. The DNA sequencing core on campus is closed for a week. Your assistant volunteers to purify DNA from each culture and perform a DNA fingerprinting assay (restriction analysis) to help identify the different samples. Should you tell your assistant to use KpnI or XbaI to do the assay? Why? 1b. Draw the expected results from the fingerprinting assay, assuming that your assistant is visualizing the DNA fragments using the DNA stain ethidium bromide (which intercalates

between DNA bases, and will allow you to visualize all fragment in the gel.) First calculate the fragment sizes for each person, then draw the expected pattern of fragments on the gel below, and indicate the sizes of each fragment on the left side of the gel. 1c. Which samples can you distinguish using this technique? Which samples can you not distinguish using this technique? For the samples you cannot definitively identify, what technique would you need to use instead? 2a. After sorting your samples, you need to determine the best cell type to test your therapies in. What technique (based on probe/sample sequence complementarity) can you use to determine which tissues express highlevels of RNA transcript expression? 2b. Design a probe that would work in tissue samples from any of your four test subjects. Draw a symbol ( ) on the diagram on the first page to indicate the region where your probe would have complementarity. 3a. You determine that WT xpd is expressed preferentially in muscle. You wonder if any of the mutations affect the stability of the RNA transcript, thereby leading to higher turnover and lower overall mRNA levels. What techniques could you use to test this hypothesis? List the pros and cons of each method. (hint: try to think of at least three methods you could use) 4a.You discover that the xpd a allele does have decreased mRNA levels, but xpd b and xpd c alleles appear to be transcribed at normal levels. Which of the following diagrams represent the blot you performed that led you to that conclusion.

4b. You perform a western blot, and find the following results. Hypothesize the possible effects of each mutation on the final protein based on both this data, and your previous results. 5a. You discover a compound, X47C, which increases xpd a transcript levels in your tissue culture experiments, and want to study the long term effects of X47C in an animal model. The spotted platypus has a muscle wasting disease phenotypically simlar to GFS, which might make a good animal model. However, the platypus genome has not been completely sequenced. What technique (based on probe complementarity) could you use to determine whether or not they have a gene (or genes) with sequence similarity to the xpd gene? 5b. Design a probe to identify any xpd homologues in the platypus. Draw a symbol in a second color ( ) on the figure on the first page to indicate the region where your probe would have complementarity. 6a. You want to amplify the xpd c allele and clone it into a bacteria to produce the protein for biochemical tests. Indicate the region complementary to your forward and reverse PCR primers on the figure on the first page using the half arrow symbols ( ). 6b. Should you use genomic DNA or cDNA reverse transcribed from mRNA to amplify the gene? 7a. You perform next-gen (high throughput) DNA sequencing on the genomic DNA from Aditi. Indicate the position and read depth of a set of mapped reads below the genome diagram. (Hint: indicate each fragment as a single straight line, and remember that the breakpoints of

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each fragment are random How many sequenced fragments would match the area of the deletion vs the areas flanking the deletion?) 7b. Can you create a line graph representing this data with average read depth on the Y axis, and nucleotide position on the X axis? Extra exam style questions 8. You amplify and sequence a small (22 bp) fragment of a viral dsDNA genome. Use the Sanger sequencing gel shown to deduce the sequence of the 5 nucleotides at the 3’ end of the template strand that was used for sequencing. A. 5’ CGAGA B. 5’ TCTCG C. 5’ AGATA D. 5’ TATCT E. 5’ AGAGC

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