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






2. 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






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






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






5. 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.






6. 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






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






8. 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






9. 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)






10. 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.






11. 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






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






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






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






15. 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






16. 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






17. 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






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. 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).






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. 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






22. 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






23. 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






24. 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






25. 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






26. 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






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






28. 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






29. Introduced on plasmids sensitive to temperature






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






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






32. 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






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






34. 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.






35. 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






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






37. 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.






38. 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






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






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






41. 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






42. 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






43. Assist recombination between homologous DNA sequences.






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






45. Use virus/bacteria phase to infect cell






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. 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






48. 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.






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






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