MCB 110. Midterm 1. 2006ANSWER KEY1. (20 pts.) Find the one letter below that best matches the following statements. You may use each letter onlyonce.A. A+G = C+T J. 0.01B. fDNA K. Topoisomerase IIC. First replication fork protein loaded L. recAD. Measure of sequence relatedness M. propeller twistE. binds pol III and helicase N. 33 degreesF. Direction of hydrolysis for proofreading O. substrate for DNA polymerasesG. xDNA P. single-stranded DNAH. A+T=G+C Q. Substrate for DnaB helicaseI. autosomal dominant R. X-linked recessivei) D E-valueii) C DnaB helicaseiii) A follows from Chargaff’s rulesiv) P floppiest form of DNAv) M angle between bases within a base pairvi) K makes and repairs staggered, double-strand breaks in DNAvii) N average helical twist for a RNA stemviii) E clamp loaderix) O deoxyriboATPx) I phenotype does not skip generations2. The restriction endonuclease NciI recognizes and cuts the five-base-pair sequence 5’-CC(G/C)GG-3’ (G/Cmeans either G or C will work in that position).2A. (6 pts) How often, on average, would this sequence occur in random DNA? Assume the DNA is 25% A, G,T & C.Once every 512 bases:(1/4)4 x 1/2 = 1/512 bases2B. (4 pts.) Nci1 leaves a one=base 5’ overhang. Schematically draw the products of a Nci1 digestion,including the location of the restriction-site bases.5’ C C G / C G G 3’G G C / G C C2C. (6 pts.) Assuming that the genomes of different individuals are 99.9% identical, approximately how manyRFLPs would you expect for the Nci1 fragments of the human genome (3 x 109 bp).3 X 109 bp X 10–3 differences X 1 site =512 bp5.86 X 10–3 X 109 X 10–3 = 5.86 X 103 RFLPs= 5860 RFLPs2D. (10 pts.) Your friend, Gene Watson, is starting a company to test for the presence of known human diseasegenes in patient DNA. Sequencing each sample is too expensive, so he needs to figure out which probes to usein Southern blots. He asks you for advice. In this case, you know the sequence of each disease gene of interestand the sequence of the human genome. Briefly outline an experimental procedure you would use to determinewhich, if any, of the Nci1 RFLPs could provide a diagnostic marker for a specific human disease gene. Whatresults would the disease marker give in your experiment?1. Pick probes at increasing distances from gene X.2. Southern blot Nci1 digested DNA from normal and affected individualsusing each probe.3. A probe covering a linked RFLP will produce a different pattern of bandsin normal and affected DNA samples.3. The Global Ocean Survey is a project to sample the DNA sequence diversity of the oceans. Craig Venter andhis crew sailed around the world, collected water samples from various locations in the ocean, and sent them totheir lab in Maryland for sequencing.3A. (8 pts.) Suppose you’re running the lab in Maryland. Briefly list the steps of the procedure you would useto process the sea-water samples to create templates suitable for DNA sequencing.1. Extract DNA.2. Cut with a restriction enzyme.3. Clone into a plasmid that contains sites to bind sequencing primers(make a library)4. Isolate individual clones from the library.5. Purify plasmids from the individual clones.3B. (6 pts.) DNA sequencing utilizes dideoxynucleoside triphosphates. Draw the chemical structure of adideoxynucleoside triphosphate, including the sugar and phosphate groups. You can represent the base with theletter N. Indicate the two “deoxy” positions.O O O|| || ||O – P – O – P – O – P – O –| | |O–O–O–deoxy positions3C. (6 pts.) Why do the dideoxy-NTPs terminate DNA strand synthesis?They lack a 3’ –OH nucleophile.3D. (6 pts.) Why do sequencing reactions require a DNA polymerase that lacks 5’ → 3’ exonuclease activity?The 5’ → 3’ exonuclease would chew away the primer to different extents.This heterogeneity would eliminate the common start point of all labeled products.3E. (8 pts.) You run a sequencing reaction using the following dideoxynucleotides, each with its ownfluorescent marker: ddGTP-red, ddaATP-green, ddTTP-yellow, ddCTP-blue. You run the products of thereaction on a gel and the fluorescence reader scans the colors as the DNA fragments run off the bottom. Theorder of colors detected at the bottom of the gel (the positive pole) is:5’1. Red G2. Yellow T3. Yellow T4. Green A5. Blue C6. Blue C7. Red G8. Blue C3’What is the sequence of the TEMPLATE strand for these products?5’ GCGGTAAC 3’3F. (10 pts.) You discover using BLAST that one of the sequences from a sample taken at a depth of 26,000feet in the Marianas Trench off the Philippines matches the sequence of the human protein kinase, PKA. The E-value of the match is 0.05. List two different ways (experimental and/or computational) you could further test tosee if the protein encoded in the deep-sea organism is homologous to PKA.1. BLAST against a larger sequence library and find sequences homologous to bothproteins.2. Make a multiple sequence alignment and see if functional sites are conserved.3. Express and purify the protein and see if it has kinase activity.4. Solve the structure and see if it has a kinase fold.OHHHHN4. (10 pts.) Suppose you wanted to make a strain of E. coli with a high mutation rate. List two genes you wouldmutate in order to reduce the fidelity of DNA replication. Explain why you would pick those genes and whatkinds of mutations (substitution, insertion or deletion) you would make in them. Be careful not to make lethalmutations!• Dna pol I or pol III – point mutation that eliminates 3’ → 5’ proofreading exonuclease.• Delete or inactivate any mismatch repair protein – MutS, MutH…• Delete pol I5. (8 pts.) Please mark the following statements true or false.T In the absence of dNTPs, the Klenow fragment of DNA pol I will degrade the primer.F DNA pol I is a great antibiotic target, because it is required for E. coli growth.F The helicase loader, DnaC, uses deoxyATP in the loading reaction.T The clamp loader links together the two pol III molecules at the replication fork.6. (10 pts.) Experiments suggest that the 42-nucleotide RNA sequence below forms a pseudoknot. Drawschematically a possible pseudoknot structure of this RNA molecule. Indicate which bases form base pairs.Partial credit will be awarded for diagramming a pseudoknot without showing the specific base pairs.5’-G U G A C A U U G G G G A A A C G U G G G A A U G U C C A G C G G G G U C G U U U G G-3’(5 pts)or7. Briefly contrast (explain