Name: ________KEY__________________________ Student ID #: _______________________ 1/5 MCB 110. Midterm 1. 2005 Please put your name and student ID # on the first page. 1. (20 pts.) Find the one letter below that best matches the following statements. You may use each letter only once. A. Major groove J. Number of histone chains in nucleosome core B. Minor groove K. Enzyme used in first step of cDNA synthesis C. First replication fork protein loaded L. Enzyme that extends chromosome ends D. Direction of lagging strand DNA synthesis M. Initiates nucleotide excision repair E. Number of backbone dihedral angles/base in DNA N. Number of DNA bases/nucleosome F. Direction of hydrolysis for proofreading O. Mechanism of IS1 transposition G. Percent of human genome encoding exons P. Template for telomeric repeats H. Initiates base excision repair Q. Number of human chromosomes I. Average fraction of human genome that varies between individuals R. GATC i) __D___ 5’3’ ii) __H___ Uracil DNA glycosylase iii) __E___ 6 iv) __J___ 8 v) __B___ Binding location for TBP (TATA binding protein) vi) __K___ Reverse transcriptase vii) __N___ ~150 viii) __G___ ~2 ix) __P___ RNA x) __I___ 0.001 2. Consider the following phrase: “Doc note, I dissent. A fast never prevents a fatness. I diet on cod.” 2A. (4 pts.) What feature does this phrase share with most restriction enzyme sites? Palindrome 2B. (4 pts.) If there was a restriction enzyme that could cut this phrase, would you expect it to leave a blunt end? Why or why not? No. The site has an odd number of bases, so there will be at least one single strand base after symmetrical cuts. 2C. (6 pts.) Suppose there was a restriction site with this many bases in DNA. Counting only letters (ignoring spaces and punctuation marks), what is the formula for the average number of bases between sites for this enzyme in “random sequence” DNA? Assume the DNA is 25% A, G, T & C. 451Name: ________KEY__________________________ Student ID #: _______________________ 2/5 2D. (6 pts.) If there was an enzyme that cut a site this big, how many sites for this enzyme would occur at random in the human genome? Explain your answer. 3 x 109 bases/genome x 1 site/451 bases) <<1 = 0 sites. The site would not occur at random in the human genome. 3. Detectives John and Joan are investigating a murder. They suspect a father and his son of the crime. They collected blood samples at the scene. 3A. (6 pts.) John says they should use RFLP analysis to see if either suspect left blood at the scene, but Joan says that RFLP analysis won’t conclusively distinguish father and son. Who’s right and why? John is right. Father and son share 50% of markers. 3B. (10 pts.) Briefly outline the steps in the RFLP analysis. 1. Isolate DNA (1pt) 2. Cut with restriction enzyme (2pt) 3. Run a gel (2pt) 4. Label probe with 32P (1pt) 5. Make Southern blot 6. Hybridize with probe and wash (5 and 6 got 3pts total) 7. Record autoradiogram (1pt) 3C. (6 pts.) Suppose father and son both had an inherited disease caused by an autosomal recessive gene? Would you use this gene to analyze the evidence in this case? Why or why not? No. The linked RFLP would not distinguish father and son. OR Yes. The linked RFLP would distinguish father and son from most other possible suspects.Name: ________KEY__________________________ Student ID #: _______________________ 3/5 3D. (5 pts.) John says they should look for RFLPs on the X chromosome, but Joan says that won’t work because males have just one X chromosome. Who’s right and why? John is right (2 pts.). Father and son have different X chromosomes, in general (3 pts.). 3E. (9 pts.) John and Joan consult Dr. Gene to get advice. After discussing their problem, Dr. Gene tells them his research group has just found an RFLP that has a 50% chance of being linked to an inherited susceptibility to breast cancer. Suggest three things Dr. Gene’s group should do to test if the RFLP is linked to breast cancer. (Hint: you can look up the sequence of the human genome. Hint #2: Mutations in two genes, BRCA1 and BRCA2 are already known to be linked to an inherited susceptibility to breast cancer.) 1. Repeat RFLP analysis with more patients to test for tighter linkage. 2. Pick new probes (e.g. 1Mb in both directions) and see if you measure tighter linkage. 3. See if the RFLP is linked to BRCA1 or BRCA2. 4. Find the gene and compare its location to the RFLP. 4A. (6 pts.) List three different DNA repair pathways where DNA ligase plays an essential role. 1. Base excision 2. Nucleotide excision 3. Mismatch repair 4. End joining 5. Homologous recombination 4B. (10 pts.) Briefly outline the steps by which the first Okazaki fragment to be made at an origin of replication is incorporated into the newly made chromosome as a whole. For 5’ end of the 1st Okazaki fragment: 1. 2nd Okazaki fragment runs into the RNA primer of the 1st Okazaki fragment (2pts) 2. The 5’ 3’ exo of Pol I or RNaseH removes the RNA primer (1) 3. Pol I fills in the gap (1) 4. Ligase seals the nick (1) For 3’ end of the 1st Okazaki fragment: 1. 1st Okazaki fragment runs into the RNA primer of the leading strand (2pts) 2. The 5’ 3’ exo of Pol I or RNaseH removes the RNA primer (1) 3. Pol I fills in the bases (1) 4. Ligase seals the nick (1)Name: ________KEY__________________________ Student ID #: _______________________ 4/5 4C. (8 pts.) Please mark the following statements true or false. __T__ The clamp loader complex requires ATP to load the sliding clamp. __T__ Topoisomerase II enzymes make double-strand breaks in DNA during the reaction. __F__ DNA polymerase III uses ATP during the polymerase reaction. __T__ At the replication fork, if the lagging strand polymerase stalls, the leading strand polymerase also stops. 5. You want to make the site-directed mutation shown below: 5’-GTTATTTATATGGGGATCCAATTGAACTTGACAATTAAAGCGCCC-3’ G 5A. (8 pts.) Write down the sequences of two divergent primers >16 nucleotides long that you would use. Remember to incorporate the mutation and pick primers that have approximately the same melting temperature. GATCCAATGGAACTTGAC &
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