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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. 1. Use RTase to go from RNA to DNA 2. Use RNAseH to get rid of RNA 3. Use TaqP to make top strand of DNA - can't detect quantity of RNA/DNA






2. 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.






3. 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






4. 20-25 nt oligonucleotide that will hybridize to DNA of interest. It can be radiolabeled with kinase and 32P-ATP or fluorescently labeled.






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






6. 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






7. 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






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






9. Introduced on plasmids sensitive to temperature






10. 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






11. 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






12. 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






13. Two components to perform the traceless recombination on chromosomes: 1. FLP recognition target (FRT): inverted repeat 2. FLP recombinase






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






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






16. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP






17. Strong positive reaction with moderate nucleic acid






18. E. coli polymerase denatures at 95C and new enzyme has to be added each time. TaqP is a thermal stable organism and only need to add once - but will denature after 30 min at 95C (may be able to reduce temperature after a few cycles; increase denatura






19. 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.






20. 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.






21. Use virus/bacteria phase to infect cell






22. 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.






23. Small size (between 3-50 kb) and it is more efficient to transfer into host cell. Unique restriction enzyme sites and selectable marker (antibiotic resistance genes)






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






25. dNTP is added to the reaction Each time dNTP is incorporated to DNA - pyrophosphate (PPi) is released in a quantity equimolar to the amount of incorporated nucleotide.






26. Assist recombination between homologous DNA sequences.






27. 1. Delete genetic information on the chromosomes of species of interest (knock outs) 2. Insert new genes and DNA sequences into desired positions on the chromosome (not relying on plasmids) 3. Generate genetically engineered species






28. 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






29. 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






30. 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






31. Weak reactions with minimal nucleic acid (representing an infection state or environmental contamination).






32. Introduce DNA into bacteria. Transformation efficiency can be increased by making cells competent (treating with cold CaCl2 and heat shock at 42C).






33. 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.






34. 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






35. 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.






36. 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






37. As the process continues - the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the pyrogram.






38. 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






39. 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






40. A method to assemble long sequences of chromosomal DNA. It involves hybridizing a primer of known sequence to a clone from an unordered genomic library and synthesizing a short complementary strand. The complementary strand is then sequenced and its






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






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






43. A DNA Virus that infects bacteria with its chromosomal DNA. The Phage DNA is linear (35-50 kb) but circularizes in host. It encodes virus specific enzymes and is replicated in the host. It gets integrated into bacteria genome.






44. 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






45. 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






46. 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.






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






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






49. Integrate into cellular chromosome.






50. Cell lysis --> new phages. In nonrestrictive bacteria - there is more chance lysis. Plaques appear where cells have lysed.