F22_DS10_Mutation and Molecular Methods_KEY

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Jan 9, 2024

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Discussion 10 Bio 305 – Genetics Mutations and Molecular Methods_ KEY 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 individuals: 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? KpnI will distinguish the largest number of samples. It will allow you to identify the deletion allele (xpd b) , and the xpd c allele, although it will not allow you to identify the xpd a allele.

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? It will not allow you to identify the xpd a allele, to identify the point mutation in the xpd a allele, you will need to sequence that region of exon 1. 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? Northern Blot 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. Answers may vary. Some options are shown below, but the complementary region must be in an exon (probes only complementary to intronic regions will not anneal to RNA) Option 3 may not bind as strongly to inviduals with the deletion mutation, so may not be as robust as options 1 and 2. 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) Northern blot: Pros: You already have a probe and reagents for the experiment (since you just did northern blots to determine the best tissue type) making it a relatively quick and inexpensive procedure. Cons: quantitation may be difficult if the difference is small. Technical issues like saturation or inefficient transfer from the gel to the blot may give you an incorrect answer. Q-RT-PCR : Pros: Much more sensitive than a northern blot. Although you will need to design RT-PCR primers for both xpd and a control (housekeeping) gene, Q-PCR is probably the best answer. Q-PCR is not terribly expensive, assuming you already have access to the equipment.

Cons: A) primers may not be specific to xpd or may be located in a variably spliced exon, B) your control gene is regulated (not uniformly expressed), C) your machine is not correctly calibrated. High-throughput sequencing: Pros: The most sensitive method, will also give you information about any alternative splicing events, and coupled with pulse-chase techniques can also give you information about transcript stability after transcription. Cons: Very expensive, requires significant computational resources and time to analyze the data, might be overkill in this situation. 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. Blot B is the correct choice, as the thinner, lighter band in Javier’s lane indicates a lower relative amount of mRNA, although the RNA remaining is the same size(length) as the wt alelle (note that because Aditi is heterozygous for the b allele, she will have two bands, each of which will be lighter than the bands in homozygous lanes, because each band is half the volume of the homozygous bands) 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. xpd a had decreased mRNA levels, resulting in low protein levels – this is a hypomorphic (loss of function) alelle. xpd b is transcribed at normal levels, since the RNA transcript is smaller, but the protein appears larger, this indicateds that the 400 bp deletion created a frameshift and novel exon 3 sequence, ending in a different stop codon much later than the normal one. This could cause either LOF or GOF depending on the properties of the new AA sequence after the deletion. xpd c transcripts and protein are normal in size, and at normal levels/amounts. A point mutation which disrupts normal xpd function (LOF) is the most likely scenario. 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? Southern Blot

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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. Answers may vary, and can include introninc or exonic sequences: but if the complementary region is in an exon you will have a better chance of success (less false negatives). This is because protein coding regions are more evolutionarily constrained (and therefore have higher sequence similarity) than intronic regions. 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? cDNA should be for your PCR amplification since the prokaryotic bacteria will not be able to splice out eukaryotic introns, and will incorrectly translate a eukaryotic genome sequence. 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 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? Read depth is normalized to “1” for homozygous regions. Read depth for someone homozygous for the deletion would be represented by the line dropping to “0” on the Y axis, while an indivdual homozygous for a duplication would have a region at “2” on the Y axis – see the example read depth graph from Hill and Unkless, A Deep Learning Approach for Detecting Copy Number Variation in Next-Generation Sequencing Data , G3, Nov, 2019. Since Aditi is heterozygous for the deletion, the line should start on the left of the graph at “1” on the Y

axis, drop to “0.5” where the deletion is, and then return back up to “1” at the end of the deletion and until the right side of the graph is reached. Extra exam style question 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 The synthesized strand is 5’AGATATCCGG…..3’ Therefore, the reverse complementary template strand read 3’>5’ would be TCTATAGGCC……. Therefore, the 5 nucleotides at the 3’ end, read 5’>3’ would be TATCT

F22_DS10_Mutation and Molecular Methods_KEY

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