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






2. Integrate into cellular chromosome.






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






4. 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.






5. 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






6. Introduced on plasmids sensitive to temperature






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






8. 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.






9. 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.






10. Strong positive reaction with moderate nucleic acid






11. 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).






12. 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






13. 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.






14. 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






15. Use virus/bacteria phase to infect cell






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






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






18. 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






19. 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






20. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP






21. 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






22. 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






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






24. 20-25 nt oligonucleotide that will hybridize to DNA of interest. It can be radiolabeled with kinase and 32P-ATP or fluorescently labeled.






25. 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






26. 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.






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






28. 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)






29. 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






30. 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






31. 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






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






33. 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.






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






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






36. 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






37. 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






38. 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.






39. 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






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






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






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






43. 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






44. 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






45. 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)






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






47. 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.






48. 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






49. 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






50. 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