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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. 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
Automated DNA sequencing
cDNA library
Reverse Transcription PCR
Red recombinase enzymes
2. Has been cloned and re- engineered to have negligible levels of RNase H activity - without compromising its first strand cDNA polymerizing function
Moloney murine leukemia virus (MMLV) RTase
Cloning Vector
Automated DNA sequencing
Clone
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
Taq polymerase
Check PCR Product
Shotgun sequencing
Recombination enzymes
4. Strong positive reactions with abundant nucleic acid
Ct < 29 (Cycle threshold)
Colony hybridization
Rules for primer
Sanger method
5. Assist recombination between homologous DNA sequences.
Homologous Recombination
Pfu Polymerase
Recombination enzymes
Red recombinase enzymes
6. 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
Shotgun sequencing
Quantitative Real-Time PCR
Key Features of PCR
T4 DNA Polymerase
7. 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.
Quantitative Real-Time PCR
Lysogenic
Oligo(dT) affinity chromatography
Touchdown PCR
8. Two components to perform the traceless recombination on chromosomes: 1. FLP recognition target (FRT): inverted repeat 2. FLP recombinase
FLP Recombinase System (Flippase)
Homologous Recombination
3 Types of Restriction Endonuclease
Pfu Polymerase
9. Introduce DNA into bacteria. Transformation efficiency can be increased by making cells competent (treating with cold CaCl2 and heat shock at 42C).
E. coli
Transformation
Polymerase Chain Reaction
Avian myelobastosis virus (AMV) reverse transcriptase
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
Why clone genes
Red recombinase and FLP recombinase
Edman degradation
11. 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
Transforming and Maintaining Plasmid
Reverse Transcription PCR
Bacteriophage Lambda
Restriction Digest
12. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP
Transformation
Taq polymerase
Pfu Polymerase
Red recombinase and FLP recombinase
13. Used to remove selection marker after Red- mediated recombination.
Pyrosequencing Step 3
FLP recombinase
Cloning Vector
Transformation
14. 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.
Pyrosequencing Step 2
Lytic
Automated DNA sequencing
Red recombinase enzymes
15. 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
Avian myelobastosis virus (AMV) reverse transcriptase
Homologous Recombination
Key Features of PCR
Uses of Homologous recombination
16. 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
FLP recombinase
Colony hybridization
Pyrosequencing Step 3
Quantitative Real-Time PCR
17. 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.
Shotgun sequencing
Plasmids
Cycle threshold
Quantitative Real-Time PCR
18. An identical copy. This term was originally applied to individual cells that were isolated and allowed to grow to create the same cell.
Homologous Recombination
Clone
Red recombinase enzymes
Primer
19. 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
Why clone genes
Quantitative Real-Time PCR
Red recombinase enzymes
Key Features of PCR
20. Use polyT to 'trap' the mRNA and leave tRNA and rRNA behind.
Features of cloning vector
Recognition sites of restriction endonucleases
3 Types of Restriction Endonuclease
Oligo(dT) affinity chromatography
21. Integrate into cellular chromosome.
FLP Recombinase System (Flippase)
Clone
Lysogenic
Primer
22. Primers anneal to complementary sequences on DNA template and determine the boundaries of the amplified product.
Why clone genes
Sanger method
3 Types of Restriction Endonuclease
Primer
23. 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
Isolation of Plasmid DNA from e. coli
Check PCR Product
Shotgun sequencing
24. 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
Markers
3 Types of Restriction Endonuclease
Check PCR Product
Pfu Polymerase
25. 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
Shotgun sequencing
Uses of Homologous recombination
Restriction Digest
PCR
26. Strong positive reaction with moderate nucleic acid
Molecular cloning
Autoradiogram
Plasmids
Ct = 30-37 (Cycle threshold)
27. 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)
Lysogenic
Edman degradation
Restriction Digest
Pyrosequencing Step 3
28. 20-25 nt oligonucleotide that will hybridize to DNA of interest. It can be radiolabeled with kinase and 32P-ATP or fluorescently labeled.
Recombination enzymes
Probe...
FLP Recombinase System (Flippase)
Ct < 29 (Cycle threshold)
29. Directional cloning of a DNA fragment - single site cloning - blunt end cloning - polylinker - creating new restriction sites
Automated DNA sequencing
Pyrosequencing Step 5
Clone
Cloning examples
30. 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
PCR
Cloning Vector
Sanger method
Problems with Sanger method
31. 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
Transgenic genes
E. coli
32. 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
cDNA library
Lysogenic
PCR
Touchdown PCR
33. 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.
Transduction
Transgenic genes
Moloney murine leukemia virus (MMLV) RTase
Gilbert method
34. 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).
Ct = 30-37 (Cycle threshold)
Applications of PCR
E. coli
Transforming and Maintaining Plasmid
35. 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
Taq polymerase
Isolation of Plasmid DNA from e. coli
Rules for primer
Homologous Recombination
36. 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
Pyrosequencing Step 1
Cloning examples
Taq polymerase
Transform
37. 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
Gilbert method
Cloning examples
Key Features of PCR
38. 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.
Transform
Bacteriophage Lambda
Pyrosequencing Step 3
Single Recombination
39. Cell lysis --> new phages. In nonrestrictive bacteria - there is more chance lysis. Plaques appear where cells have lysed.
Lytic
Cycle threshold
Polymerase Chain Reaction
Cloning examples
40. Restriction nucleases - electrophoresis - vector - ligase - bacterial host - identifying the cloned gene
Toolset for cloning
Gilbert method
Molecular cloning
Markers
41. 1. Detecting pathogens using genome- specific primer pairs 2. Screening specific genes for unknown mutations 3. Genotyping using known STS (sequence tagged sites) markers
Pyrosequencing Step 5
Plasmids
Applications of PCR
Transforming and Maintaining Plasmid
42. 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.
Pfu Polymerase
Pyrosequencing Step 3
Homologous Recombination
Recognition sites of restriction endonucleases
43. 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.
Key Features of PCR
Plasmids
cDNA library
Lysogenic
44. 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
Reverse Transcription PCR
Transgenic genes
Recognition sites of restriction endonucleases
Problems with Sanger method
45. Apyrase - a nucleotide degrading enzyme continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete - another dNTP is added.
Oligo(dT) affinity chromatography
E. coli
PCR
Pyrosequencing Step 4
46. 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
Transform
Why clone genes
Pyrosequencing Step 4
Red recombinase enzymes
47. 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
Moloney murine leukemia virus (MMLV) RTase
Cycle threshold
Automated DNA sequencing
48. As the process continues - the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peaks in the pyrogram.
Touchdown PCR
Pyrosequencing Step 5
Lytic
3 Types of Restriction Endonuclease
49. 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
Transforming and Maintaining Plasmid
Restriction endonucleases
Automated DNA sequencing
Colony hybridization
50. (1) Gene is separated from chromosome - (2) gene is put into a vector - (3) vector replicates to produce multiple copies of the gene.
Molecular cloning
Quantitative Real-Time PCR
Replication of plasmids
Applications of PCR