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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. 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






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






3. 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






4. 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






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






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






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






8. Use virus/bacteria phase to infect cell






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. 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






11. 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






12. 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.






13. 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






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






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






16. 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






17. Strong positive reactions with abundant nucleic acid






18. 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)






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






20. 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.






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






22. 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.






23. 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






24. 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






25. 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.






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






27. 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






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






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






31. Assist recombination between homologous DNA sequences.






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






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






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






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






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






37. 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






38. 20-25 nt oligonucleotide that will hybridize to DNA of interest. It can be radiolabeled with kinase and 32P-ATP or fluorescently labeled.






39. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP






40. 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.






41. 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






42. 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






43. Strong positive reaction with moderate nucleic acid






44. 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






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






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






48. 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)






49. 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






50. Integrate into cellular chromosome.