BI441 Exam Answers PDF

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BI441 Exam Questions Lecture 6 – DNA Libraries 1. What are some advantages of being able to clone large pieces of DNA? (List 3) o Increases the amount of complete genes or set of genes o Fewer clones to screen for gene of interest o Takes less time to make 2. Define a DNA library o Population of organisms (bacteria) each of which carries a DNA molecule that was inserted into a cloning vector 3. In the “library” expression, what does the library represent, and what does each book represent? o The library is the whole genome and each vector is like a book in the library 4. What is the appropriate size of genomic DNA fragments for a library? o 12-20 kilobases 5. What conditions is a partial digest done in? Why? o Limit amount of enzyme or length of incubation 6. What are the uses of a genomic DNA library? o Cloning a gene o Sequencing a genome 7. How big is the human genome? What percent of that is non-coding DNA? o ~3.2 billion bp o ~95% 8. What does the “c” in cDNA stand for? o Complementary 9. What are the two nucleic acid polymerases needed to make a cDNA library? Describe each. o Reverse transcriptase: catalyzes formation of complementary strands of DNA using mRNA as template o DNA polymerase: catalyzes formation of complementary strands of DNA using DNA as template 10. How is mRNA different from tRNA and rRNA? o Two regognizable handles: 5’ G cap and 3’ poly A tail 11. What are the four ways to improve the efficiency of blunt end ligation? o Linker cloning: short ds oligos with RE site, blunt end ligated to ends of cDNA then cut with RE’s o Adapter cloning: variation of linker cloning, artificially made sticky ends, 2 oligos are synthesized to be partially complementary so the overhangs are the same as sticky ends produces by a known RE  DNA ligase attatches adapters then polynucleotide kinase adds phosphates o Terminal transferase  TdT, cDNA, and dCTP mixed  Tdt, vector, and dGTP mixed o High-efficiency blunt end ligation  Topoisomerase makes transient break in vector (remains attached) leaving 3’P (not 5’P)  Vector treated with AP  The two are mixed and topoisomase anneals them (ligase activity) forming two phosphodiester bonds 12. List and describe the ways we can enrich for full-length transcripts of cDNA

o 1. Trehalose: sugar that stabilizes reverse transcriptase so can work at higher temps  High temp disrupts secondary structure of mRNA by breaking H-bonds o 2. RNaseI: cuts ssmRNA, removes biotin from ssRNA and one off complete RNA/DNA molecule o 3. Biotin/streptavidin: biotin attaches to ends of mRNA, streptavidin-coated beads bind to biotin 13. List and describe the ways to screen DNA libraries o DNA (colony) hybridization:  Grow DNA library on agar  Transfer to filter  Treat with detergent and NaOH to lyse cells  Denature DNA  Fix ssDNA to filter with UV light or heat  Incubate filter with DNA probe  Probe = ssDNA, 100-1000 bases in size, 80% match to gene of interest, has radioactive or fluorescent label o Immunological assays:  Once DNA is fixed to the filter, add primary then secondary antibodies which bind to gene of interest and has a signal for detection  Block with BSA before adding antibodies o Protein/enzyme activity  Determine if your gene encode for an enzyme that the host doesn’t have  Add substrate to the media that will turn a colour when the enzyme is made o Functional complementation  Mutate the host cell so they cant survive on the media without your gene of interest 14. Name three ways to make a DNA probe for screening DNA libraries o Cloned from a related species o Degenerate probe if you know the aa sequence o Random primer 15. cDNA synthesis o 1. Add oligo(dT) to bind to poly-A tail of mRNA and provide 3’OH o 2. Reverse trascriptase makes 1sst strand of DNA using mRNA as template o 3. RNaseH nicks mRNA o 4. E. coli DNA pol synthesizes second strand of DNA and has exonuclease activity so degrades remaining mRNA o 5. T4 DNA pol cleans up ends (ligase activity) 16. Modified cDNA synthesis o 1. Add oligo(dT) with RE site on 5’ end and trehalose o 2. Reverse transcriptase uses methylated “C’s” o 3. Biotin, RNaseI, Streptavidin coated beads o 4. RNaseH degrades mRNA o 5. Poly(dG) tail added to 1st strand on 3’ end o 6. Poly(dC) with RE site (5’ end) attaches to G tail o 7. Heat resistant DNA pol synthesiszes second strand (+RNase H + DNA ligase) make 2nd strand o 8. REs added  hemimethylated DNA

Lecture 7 17. Why do we chemically synthesize DNA? o To use as primers (ex for PCR or for cDNA synthesis for reverse transcriptase) o To use as probes o Linkers/adapters 18. What is the most common method of chemical DNA synthesis? List the steps o Phosphoramidite method o 1. Link first nucleoside to solid support  First nucleoside: 5’ end blocked with DMT, bases also blocked (C, G, and A)  DMT-5’-nucleotide-3’spacer arm-CPG bead o 2. Detritylation  Remove DMT with TCA to reveal free 5’ OH o 3. Activation and coupling  Add next nucleotide with tetrazole (which activates 3’ end)  Covalent bond formed between two nucleotides o 4. Capping with acetyl o 5. Oxidaton  Phosphite triester bond between nucelotides is unstable  Oxidize to phosphate triester o Repeat 2-5, wash, remove from column, purify 19. What changes to make it biological DNA o Remove methyl groups from phosphate triester bonds o Detritylate terminal 5’ residue then phosphorylate with polynucleotide kinase o Detatch from spacer and CPG bead o Unblock C, G, and A 20. List the components of PCR (6) o DNA polymerase o Template DNA o Primers o dNTPs o Buffer o Thermal cycler 21. List the steps in PCR o Denaturation: 95C to break H bonds and make dsDNA ssDNA o Annealing: 55C lower temp so primers can anneal to ssDNA o Elongation: 70C DNA polymerase attaches to primers and synthesizes complementary strand of DNA 22. What 4 types of DNA are in a PCR reaction o Primers: remain same concentration o Template DNA: remain same concentration o Long template DNA: linear increase (2 more every cycle) made using template DNA o Short template DNA: DESIRED PRODUCT. Exponential increase, made by template DNA and other short DNA. Made in 2nd cycle, increases in 3rd 23. Explain the first 3 cycles of PCR o 1st cycle:  Primers attatch and long template DNA is made nd o 2 cycle:  Short template DNA made

 More long than short at this point o 3rd cycle:  Short template increases as each cycle increases  3rd cycle same as the rest of the cycles 24. List two applications of PCR o Reverse Transcription PCR  RNA into cDNA o Real Time PCR  Dye / reporter probe measure DNA during synthesis  Determine starting quantity of DNA Lecture 8 25. Define Pyrosequencing o Type of DNA sequencing o 1. When correct nucleotide is added, pyrophosphate is released o 2. Pyrophsphate is converted into ATP via ATP sulfurylase o 3. ATP powers oxidation of luciferin by luciferase o 4. Light signal emmited (proportional to amount of nucleotides incorporated) o 5. Apryase added to degrade remaing ATP or nucletides 26. T7 System o T7 promoter only recognized by T7 RNA polymerase o T7 RNA polymerase is under the control of lac promoter on E. coli chromosome o Target gene cloned into vector that’s under control of T7 promoter o Target gene not transcribe because LacI repressor o E. coli: lac promoter (lacI gene makes repressor), T7 gene  When not repressed, E. coli RNA pol attatched to lac promoter and makes T7 RNA pol o pET plasmid: lac promoter (lacI gene makes repressor), Target gene  When not repressed T7 RNA pol transcribes target 27. What are fusion proteins? o When two (or more) coding regions are ligated together 28. What are fusion proteins used for? o Stability  Prevent unwanted degradation (ex when in foreign host)  Partner wont be degraded o Improve solubility o Facilitate easy purification  His-tag  His-tag has metal binding properties  Ex will bind to nickel column  Then elute using imidazole 29. Give an example of a recombinantly produced protein o Insulin  used to treat diabetes mellitus Group Presentations 30. Bioremediation o Bioremediation = using biological agents to degrade toxins in the environments o Biomass: materials produced from food/agriculture industry that used to be discarded as wate but are now used to make economically important products

o Ex, bacteria with plasmid with enzyme that degrades a toxin, combine genes into one plasmid for superbug 31. Protein therapeutics o DNaseI  used to treat cystic fibrosis o Expressed DNaseI in hamster cells, purified enzyme to make aerosol sprayed over lungs on patients with cystic fibrosis o Result: decreased viscosity and adhesivity of the mucus in the lungs making it easier to breath 32. Nucleic Acid Therapeutics (Gene therapy) o Challenges  How to target specific cells?  primed with signal regognition molecules  Can they cause alternative problems? Will cells maintain the gene or are repeated treatments required?  theoretical HAC (human artificial chromosome), HAC can have regulatory elements combined into large DNA segments to limit adverse problems. HAC also provides long term stability...