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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. 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






2. Assist recombination between homologous DNA sequences.






3. Integrate into cellular chromosome.






4. Strong positive reaction with moderate nucleic acid






5. 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.






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






7. 20-25 nt oligonucleotide that will hybridize to DNA of interest. It can be radiolabeled with kinase and 32P-ATP or fluorescently labeled.






8. 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






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






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. 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






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






13. 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






14. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP






15. 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.






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






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






18. 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.






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






20. 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






21. 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.






22. 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






23. Introduced on plasmids sensitive to temperature






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






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






26. 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.






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






28. 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






29. 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)






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






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






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






34. Four Components: 1. Template (Target DNA) - doesn't need to be purified and can be from anything 2. Primers (short oligonucleotides) 3. dNTP (building blocks) 4. Thermostable polymerase - no need for RNA primers like in actual DNA replication






35. 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






36. 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






37. 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






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






39. 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






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






41. 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.






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






43. 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






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






45. 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.






46. 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






47. 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).






48. 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






49. 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.






50. Use virus/bacteria phase to infect cell