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Test your basic knowledge |
Molecular Biotechnology 2
Start Test
Study First
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. Assist recombination between homologous DNA sequences.
Markers
Pyrosequencing Step 5
Features of cloning vector
Recombination enzymes
2. 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
Touchdown PCR
Uses of Homologous recombination
Red recombinase and FLP recombinase
Restriction endonucleases
3. Used to remove selection marker after Red- mediated recombination.
Cycle threshold
FLP recombinase
Molecular cloning
Pyrosequencing Step 2
4. A viral polymerase that converts sticky ends to blunt ends. Has polymerase activity and nuclease activity.
T4 DNA Polymerase
Avian myelobastosis virus (AMV) reverse transcriptase
Transformation
Cloning Vector
5. Directional cloning of a DNA fragment - single site cloning - blunt end cloning - polylinker - creating new restriction sites
Pyrosequencing Step 1
PCR
Polymerase Chain Reaction
Cloning examples
6. 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.
Restriction endonucleases
Clone
Bacteriophage Lambda
Touchdown PCR
7. Move plasmid into cell. In cancer biology - this means converting non - carcinoma cell to carcinoma cell.
Restriction endonucleases
Pyrosequencing Step 3
Moloney murine leukemia virus (MMLV) RTase
Transform
8. An identical copy. This term was originally applied to individual cells that were isolated and allowed to grow to create the same cell.
Ct = 30-37 (Cycle threshold)
Shotgun sequencing
Cloning Vector
Clone
9. 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
Cycle threshold
Chromosome walking
Steps to Finding desired gene
Check PCR Product
10. 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
Touchdown PCR
E. coli
cDNA library
Markers
11. Introduced on plasmids sensitive to temperature
Check PCR Product
Automated DNA sequencing
Transgenic genes
Red recombinase and FLP recombinase
12. 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)
Pyrosequencing Step 4
Edman degradation
Ct = 30-37 (Cycle threshold)
Check PCR Product
13. 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
Pyrosequencing Step 5
Colony hybridization
Molecular cloning
Shotgun sequencing
14. 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.
Colony hybridization
Lysogenic
Transforming and Maintaining Plasmid
cDNA library
15. 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
Pfu Polymerase
Sanger method
Touchdown PCR
Red recombinase enzymes
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
Isolation of Plasmid DNA from e. coli
Restriction endonucleases
Transgenic genes
Touchdown PCR
17. The first reverse transcriptase specifically purified for use in first stand cDNA reactions
Primer
Avian myelobastosis virus (AMV) reverse transcriptase
Single Recombination
Transgenic genes
18. Two components to perform the traceless recombination on chromosomes: 1. FLP recognition target (FRT): inverted repeat 2. FLP recombinase
FLP Recombinase System (Flippase)
Lytic
Automated DNA sequencing
Isolation of Plasmid DNA from e. coli
19. 1. Detecting pathogens using genome- specific primer pairs 2. Screening specific genes for unknown mutations 3. Genotyping using known STS (sequence tagged sites) markers
Shotgun sequencing
Applications of PCR
Why clone genes
Automated DNA sequencing
20. Strong positive reaction with moderate nucleic acid
Polymerase Chain Reaction
Ct = 30-37 (Cycle threshold)
Why clone genes
Avian myelobastosis virus (AMV) reverse transcriptase
21. Use virus/bacteria phase to infect cell
Applications of PCR
Homologous Recombination
Molecular cloning
Transduction
22. 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.
E. coli
Reverse Transcription PCR
Single Recombination
Lysogenic
23. 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
Chromosome walking
Oligo(dT) affinity chromatography
Pyrosequencing Step 4
Probe...
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
Touchdown PCR
Avian myelobastosis virus (AMV) reverse transcriptase
Ct = 30-37 (Cycle threshold)
cDNA library
25. 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
Red recombinase enzymes
Recombination enzymes
Pyrosequencing Step 2
Primer
26. 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).
Cloning Vector
Problems with Sanger method
Transforming and Maintaining Plasmid
Recognition sites of restriction endonucleases
27. 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)
Features of cloning vector
Reverse Transcription PCR
Transforming and Maintaining Plasmid
Transduction
28. 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.
Lytic
cDNA library
Homologous Recombination
Cloning Vector
29. 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.
E. coli
Cloning examples
Homologous Recombination
Cloning Vector
30. 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.
Applications of PCR
Pyrosequencing Step 2
Toolset for cloning
Ct < 29 (Cycle threshold)
31. Genes that are put into a new host so that the new host can gain new/correct function
Transgenic genes
Cloning Vector
Moloney murine leukemia virus (MMLV) RTase
Cloning examples
32. 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 Types of Restriction Endonuclease
Transforming and Maintaining Plasmid
T4 DNA Polymerase
Ct = 30-37 (Cycle threshold)
33. Weak reactions with minimal nucleic acid (representing an infection state or environmental contamination).
Why clone genes
Pyrosequencing Step 4
Ct = 38-40 (Cycle threshold)
Cloning Vector
34. Primers anneal to complementary sequences on DNA template and determine the boundaries of the amplified product.
Primer
FLP Recombinase System (Flippase)
Shotgun sequencing
Transformation
35. 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
Ct = 38-40 (Cycle threshold)
Moloney murine leukemia virus (MMLV) RTase
Polymerase Chain Reaction
Steps to Finding desired gene
36. 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.
Cycle threshold
PCR
Molecular cloning
Gilbert method
37. 20-25 nt oligonucleotide that will hybridize to DNA of interest. It can be radiolabeled with kinase and 32P-ATP or fluorescently labeled.
Features of cloning vector
Probe...
Cloning examples
Markers
38. Use polyT to 'trap' the mRNA and leave tRNA and rRNA behind.
Pyrosequencing Step 1
Oligo(dT) affinity chromatography
Recombination enzymes
Taq polymerase
39. Restriction nucleases - electrophoresis - vector - ligase - bacterial host - identifying the cloned gene
Ct = 30-37 (Cycle threshold)
Red recombinase and FLP recombinase
Toolset for cloning
Homologous Recombination
40. Apyrase - a nucleotide degrading enzyme continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete - another dNTP is added.
Steps to Finding desired gene
Pyrosequencing Step 4
Pfu Polymerase
Recombination enzymes
41. 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.
Molecular cloning
Polymerase Chain Reaction
Quantitative Real-Time PCR
Isolation of Plasmid DNA from e. coli
42. 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
Red recombinase and FLP recombinase
Why clone genes
Replication of plasmids
Pfu Polymerase
43. As the process continues - the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the pyrogram.
Pyrosequencing Step 5
Homologous Recombination
Replication of plasmids
Rules for primer
44. 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
Transduction
Pfu Polymerase
Homologous Recombination
Markers
45. Cell lysis --> new phages. In nonrestrictive bacteria - there is more chance lysis. Plaques appear where cells have lysed.
Recognition sites of restriction endonucleases
Lytic
Markers
cDNA library
46. 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
Pfu Polymerase
Quantitative Real-Time PCR
Problems with Sanger method
Lytic
47. 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
FLP Recombinase System (Flippase)
Ct = 30-37 (Cycle threshold)
Transduction
Automated DNA sequencing
48. 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
Key Features of PCR
FLP Recombinase System (Flippase)
Red recombinase enzymes
Recombination enzymes
49. DNA footprinting; will have an empty region if DNA has protein binding to it because that region won't be amplified.
Autoradiogram
Molecular cloning
Oligo(dT) affinity chromatography
Pyrosequencing Step 4
50. 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.
Rules for primer
Avian myelobastosis virus (AMV) reverse transcriptase
Transduction
Gilbert method