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Molecular Biotechnology 2

Subject : engineering
Instructions:
  • Answer 50 questions in 15 minutes.
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  • Match each statement with the correct term.
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This is a study tool. The 3 wrong answers for each question are randomly chosen from answers to other questions. So, you might find at times the answers obvious, but you will see it re-enforces your understanding as you take the test each time.
1. Cell lysis --> new phages. In nonrestrictive bacteria - there is more chance lysis. Plaques appear where cells have lysed.






2. Sequencing primer is hybridized to a single stranded DNA and incubated with enzymes - DNAP - ATP sulfurylase - luciferase - and apyrase. Adenosine 5' phosphosulfate (APS) and luciferin are added.






3. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP






4. Apyrase - a nucleotide degrading enzyme continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete - another dNTP is added.






5. 20-25 nt oligonucleotide that will hybridize to DNA of interest. It can be radiolabeled with kinase and 32P-ATP or fluorescently labeled.






6. This uses a suicide plasmid (no ori) to do single crossover recombination because you want to force the plasmid to integrate its gene into the chromosome. Maintenance on chromosome allows plasmid to survive.






7. Genes that are put into a new host so that the new host can gain new/correct function






8. Restriction nucleases - electrophoresis - vector - ligase - bacterial host - identifying the cloned gene






9. Used to remove selection marker after Red- mediated recombination.






10. Each clone on the plate has the gene of interest - but there are only a few colonies that have the gene. Once do a filter paper - you need to do it again around the area where colonies popped up first until finally know where the colony is.






11. 1. Antibiotic Resistance: gene that degrades toxic compounds 2. Auxotrophic Marker: host is missing some essential amino acid/nucleotide and cell needs it to grow (eg. uracil) - nutritional markers






12. Strong positive reactions with abundant nucleic acid






13. A host for recombinant DNA because it can grow fast and to a high cell density. It can also transcribe most foreign genes efficiently and there are many strains that facilitate genetic manipulations.






14. Integrate into cellular chromosome.






15. Move plasmid into cell. In cancer biology - this means converting non - carcinoma cell to carcinoma cell.






16. Type I and III: cut and modify DNA by methylation - binding and cutting sites differ - requires ATP to move along DNA - and not efficient for DNA manipulation Type II: has only restriction activity - no modification; cutting sites are adjacent or wit






17. Plasmids have an ori sequence for replication. The sequence of ori and plasmid encoded proteins determine the 'copy- number' of plasmids. Stringent control of replication (1 copy per cell division - low cell copy number plasmid); relaxed control of r






18. The host's immune system that protects against foreign DNA (DNA binding proteins). It protects the hosts DNA through methylation and digests DNA that isn't methylated. Hydrolyze phosophodiester bond at specific sequences. Binding/cutting sites can be






19. Use polyT to 'trap' the mRNA and leave tRNA and rRNA behind.






20. DNA sequencing - Understand biological processes - Study the function of encoded protein - Introduce a mutation into the gene - Evolve a protein towards desirable functions - Obtain large amounts of a protein






21. A DNA which is complementary to an RNA (a complementary DNA); Generally made by reverse transcription of mRNA. (1) purification of mRNA with polyT because mRNA has lots of polyA on 3' end - (2) first strand DNA synthesis using RTase - (3) second stra






22. Each cell can maintain different plasmids with different selection markers. If the plasmid has the same selection marker - one will be lost. Transformation is very inefficient (<1% of the cell can be transformed).






23. Directional cloning of a DNA fragment - single site cloning - blunt end cloning - polylinker - creating new restriction sites






24. 1. Cycles of temperatures 2. 94C denatures DNA 3. Lower temperature so primers can bind to DNA at specific locations 4. Polymerase carries out templated DNA synthesis with primers at an optimal temperature (~72C) 5. Product serves as the template for






25. 1. Construct a genome library: YAC - cosmids - etc 2. If using large insert vectors - clone smaller fragments (40 kb) into overlapping cosmids 3. Fragment the cosmid into 1 kb pieces using sonication and ligate into small plasmids 4. Sequence the 1 k






26. 1. Detecting pathogens using genome- specific primer pairs 2. Screening specific genes for unknown mutations 3. Genotyping using known STS (sequence tagged sites) markers






27. Need primers - dNTP - template - thermostable polymerase - buffer - primer overhangs introduce nonnative sequences - primer mismatches introduce mutations - stops because taqP denatures after awhile






28. Increases specificity - sensitivity - and yield without redesigning primers. The initial annealing temperature is above the projected melting temperature of the primers being used. It then transitions to lower - more permissive annealing temperature






29. Introduced on plasmids sensitive to temperature






30. May get a smear - can't tell the difference between bp - and limited by # of sequence it can generate because primers may only be able to do 1000 bp






31. Can be used to linearize circular DNA - can have double digest - usually done at 37C but some done at 55C - digest time depends on the amount of enzyme






32. During meiosis - homologous recombination happens in chromosomes to generate offspring diversity. Recombination is used to repair DNA damage and can be induced by a wide array of environmental stresses.






33. The first reverse transcriptase specifically purified for use in first stand cDNA reactions






34. 1. If a product is formed: PCR can be unsuccessful if the quality of DNA is poor - one of the primers doesn't fit - too much starting template (non - specific binding) - optimization 2. Product is of the right size: primers may bind to different part






35. Know how much DNA is amplified by using Tagman which has fluorescent dye (SYBR Green) and quencher. Energy is transferred from F to Q when TaqP excises F with 5' to 3' exonuclease activity.






36. (1) Gene is separated from chromosome - (2) gene is put into a vector - (3) vector replicates to produce multiple copies of the gene.






37. From bacteriophage lambda and help in the removal of chromosomal genes in e.coli. As little as 30 nt homologous region is required - which can be introduced as overhangs in a PCR reaction using the selection marker as template 1. Gam - protects line






38. A viral polymerase that converts sticky ends to blunt ends. Has polymerase activity and nuclease activity.






39. DNA footprinting; will have an empty region if DNA has protein binding to it because that region won't be amplified.






40. 4-8 bp long (usually 6). Mostly palindromic because the nuclease is 2 enzymes coming together. There are 3 types of cleavage: (1) blunt ends - (2) 5' overhang sticky end - (3) 3' overhang sticky end.






41. Fluorescent dye is attached to 3' of each of the four bases (ddNTP) and will emit a narrow spectrum of light when struck by an argon ion laser beam. All four ddNTP can be added to the same reaction. >800 bases can be sequenced






42. Used so the cell isn't killed and can still transfer foreign DNA into a host cell. The DNA can be propagated in a host cell and hosts with the vector can be selected over hosts that don't have the vector. Plasmids - viruses - plasmids + viruses (cosm






43. Assist recombination between homologous DNA sequences.






44. 1. Label one end of DNA with radioactivity 2. Cut DNA at different places wherever A/G/C/T pop up using different chemicals 3. Line up DNA pieces by size using gel electrophoresis.






45. SDS lysis cells - potassium acetate/acetic acid is used to neutralize pH and precipitates lipids and large proteins - centrifuge to separate out plasmid DNA from precipitates






46. The number of cycles required for the fluorescent signal to pass the threshold (background level). This is inversely proportional to the amount of target nucleic acid.






47. 1. Decide the desired coverage of the genome 2. Choose an appropriate vector for making the library 3. Digest the genome pieces and clone into the vector 4. Introduce the library into e.coli host using appropriate means 5. Design probes to investiga






48. Use virus/bacteria phase to infect cell






49. ATP sulfurylase quantitatively converts PPi to ATP in the presence of APS. This ATP drives the luciferase mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are porportional to the amount of ATP and is detec






50. Primers anneal to complementary sequences on DNA template and determine the boundaries of the amplified product.