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Molecular Biotechnology 2

Subject : engineering
Instructions:
  • Answer 50 questions in 15 minutes.
  • If you are not ready to take this test, you can study here.
  • Match each statement with the correct term.
  • Don't refresh. All questions and answers are randomly picked and ordered every time you load a test.

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. 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.






2. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP






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






4. 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






5. Strong positive reactions with abundant nucleic acid






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






7. 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






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






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






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






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






12. 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






13. 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.






14. 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)






15. Has been cloned and re- engineered to have negligible levels of RNase H activity - without compromising its first strand cDNA polymerizing function






16. Strong positive reaction with moderate nucleic acid






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






18. An identical copy. This term was originally applied to individual cells that were isolated and allowed to grow to create the same cell.






19. 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






20. 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.






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






22. Extrachromosomal - circular DNA that has autonomous - self- replicating genetic elements. Found in bacteria - yeast. Transferred to daughter cells during cell division. Size varies from 1kb ~ 200 -000 kb.






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






24. Assist recombination between homologous DNA sequences.






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






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






27. 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






28. Need: polymerase - dNTP (one is labeled with 32P to provide signal) - ddNTP (3'H will terminate DNA synthesis; dideoxyribose; only one is put in and added in excess) - synthesizes DNA and can deduce sequence wherever DNA stops synthesizing because o






29. 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.






30. 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






31. 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






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






33. 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






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






35. Integrate into cellular chromosome.






36. 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.






37. 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






38. 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






39. 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






40. 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






41. Use virus/bacteria phase to infect cell






42. 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






43. 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.






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. 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






46. A technique that sequences the N terminus and C terminus sequence of purified proteins. These sequences can be used to design degenerate primers and probe a gene library. (1) Purify protein from cell sample - (2) break it up - (3) enzyme assay - (4)






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






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






49. 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






50. 1. Primer length is between 18-24 nucleotides long. 2. Duplex stability: both primers need to have similar Tm to have the same hybridization kinetics during the template annealing phase. Remove bases to have the same Tm 3. Non - complementary primer