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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. 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






2. 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






3. 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






4. 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






5. 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






6. 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.






7. 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.






8. Strong positive reaction with moderate nucleic acid






9. Strong positive reactions with abundant nucleic acid






10. 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






11. 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.






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






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






14. 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






15. 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






16. 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






17. 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).






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






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. 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






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






22. 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.






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






24. 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)






25. Assist recombination between homologous DNA sequences.






26. 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.






27. 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






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






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






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






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






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






33. Use virus/bacteria phase to infect cell






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






35. 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






36. 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)






37. 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






38. 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.






39. 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.






40. 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.






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






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






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






44. 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






45. 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






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






47. 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






48. 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






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






50. 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