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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. 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
Pyrosequencing Step 3
Plasmids
Pyrosequencing Step 4
Rules for primer
2. 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
Pyrosequencing Step 4
Ct = 38-40 (Cycle threshold)
FLP Recombinase System (Flippase)
Automated DNA sequencing
3. 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
Transforming and Maintaining Plasmid
Problems with Sanger method
Recognition sites of restriction endonucleases
Polymerase Chain Reaction
4. 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
Taq polymerase
Reverse Transcription PCR
Uses of Homologous recombination
Colony hybridization
5. An identical copy. This term was originally applied to individual cells that were isolated and allowed to grow to create the same cell.
Clone
Avian myelobastosis virus (AMV) reverse transcriptase
Pyrosequencing Step 2
Cycle threshold
6. 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
Markers
Touchdown PCR
Isolation of Plasmid DNA from e. coli
Chromosome walking
7. 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
Colony hybridization
Automated DNA sequencing
Applications of PCR
8. 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
Clone
Restriction endonucleases
Replication of plasmids
Uses of Homologous recombination
9. Cell lysis --> new phages. In nonrestrictive bacteria - there is more chance lysis. Plaques appear where cells have lysed.
Colony hybridization
Problems with Sanger method
Replication of plasmids
Lytic
10. Need primers - dNTP - template - thermostable polymerase - buffer - primer overhangs introduce nonnative sequences - primer mismatches introduce mutations - stops because taqP denatures after awhile
Bacteriophage Lambda
Rules for primer
Shotgun sequencing
PCR
11. Strong positive reactions with abundant nucleic acid
Ct < 29 (Cycle threshold)
3 Types of Restriction Endonuclease
Ct = 30-37 (Cycle threshold)
Quantitative Real-Time PCR
12. A viral polymerase that converts sticky ends to blunt ends. Has polymerase activity and nuclease activity.
Transforming and Maintaining Plasmid
T4 DNA Polymerase
Key Features of PCR
cDNA library
13. 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
Check PCR Product
Replication of plasmids
Moloney murine leukemia virus (MMLV) RTase
Isolation of Plasmid DNA from e. coli
14. 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
Pyrosequencing Step 4
Automated DNA sequencing
Chromosome walking
Autoradiogram
15. 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
Pyrosequencing Step 1
Reverse Transcription PCR
Homologous Recombination
Recognition sites of restriction endonucleases
16. 3' to 5' exonuclease - more expensive - yields less product - but has less error than TaqP
Why clone genes
Molecular cloning
Restriction endonucleases
Pfu Polymerase
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).
Transforming and Maintaining Plasmid
Features of cloning vector
Check PCR Product
Taq polymerase
18. Restriction nucleases - electrophoresis - vector - ligase - bacterial host - identifying the cloned gene
Problems with Sanger method
Transforming and Maintaining Plasmid
Features of cloning vector
Toolset for cloning
19. 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.
Edman degradation
Isolation of Plasmid DNA from e. coli
Features of cloning vector
Pyrosequencing Step 2
20. Introduce DNA into bacteria. Transformation efficiency can be increased by making cells competent (treating with cold CaCl2 and heat shock at 42C).
Gilbert method
Pyrosequencing Step 5
Rules for primer
Transformation
21. 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
Steps to Finding desired gene
Primer
Problems with Sanger method
Cloning Vector
22. 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
Rules for primer
Colony hybridization
Homologous Recombination
Taq polymerase
23. 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.
Primer
Ct = 38-40 (Cycle threshold)
Lytic
Pyrosequencing Step 1
24. Use virus/bacteria phase to infect cell
Transduction
Applications of PCR
Restriction Digest
Recombination enzymes
25. Primers anneal to complementary sequences on DNA template and determine the boundaries of the amplified product.
Primer
Pfu Polymerase
E. coli
Oligo(dT) affinity chromatography
26. Directional cloning of a DNA fragment - single site cloning - blunt end cloning - polylinker - creating new restriction sites
Toolset for cloning
Cloning examples
Transgenic genes
Transforming and Maintaining Plasmid
27. 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.
Features of cloning vector
Pyrosequencing Step 4
T4 DNA Polymerase
Colony hybridization
28. 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
FLP Recombinase System (Flippase)
Red recombinase enzymes
Uses of Homologous recombination
Restriction endonucleases
29. 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
Sanger method
Pyrosequencing Step 3
Cloning examples
Uses of Homologous recombination
30. 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.
Plasmids
Lysogenic
Pyrosequencing Step 3
Pyrosequencing Step 4
31. 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.
Toolset for cloning
Homologous Recombination
Ct < 29 (Cycle threshold)
Probe...
32. Two components to perform the traceless recombination on chromosomes: 1. FLP recognition target (FRT): inverted repeat 2. FLP recombinase
T4 DNA Polymerase
Bacteriophage Lambda
FLP Recombinase System (Flippase)
Pyrosequencing Step 2
33. 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
Moloney murine leukemia virus (MMLV) RTase
Cycle threshold
Restriction endonucleases
Why clone genes
34. Used to remove selection marker after Red- mediated recombination.
Chromosome walking
Ct = 30-37 (Cycle threshold)
Markers
FLP recombinase
35. 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.
Chromosome walking
Avian myelobastosis virus (AMV) reverse transcriptase
Recognition sites of restriction endonucleases
Uses of Homologous recombination
36. 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
PCR
Markers
Problems with Sanger method
Autoradiogram
37. 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
Ct = 30-37 (Cycle threshold)
Pyrosequencing Step 5
Autoradiogram
38. 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
Replication of plasmids
Avian myelobastosis virus (AMV) reverse transcriptase
Applications of PCR
Gilbert method
39. Genes that are put into a new host so that the new host can gain new/correct function
Transgenic genes
FLP Recombinase System (Flippase)
Recognition sites of restriction endonucleases
3 Types of Restriction Endonuclease
40. 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
Polymerase Chain Reaction
T4 DNA Polymerase
FLP Recombinase System (Flippase)
Oligo(dT) affinity chromatography
41. 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
Edman degradation
FLP recombinase
cDNA library
Cloning examples
42. 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.
Transduction
Colony hybridization
E. coli
Oligo(dT) affinity chromatography
43. 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)
Ct < 29 (Cycle threshold)
Recombination enzymes
Moloney murine leukemia virus (MMLV) RTase
Features of cloning vector
44. 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.
Red recombinase and FLP recombinase
Pyrosequencing Step 1
Single Recombination
Recognition sites of restriction endonucleases
45. 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.
Gilbert method
Pyrosequencing Step 1
Cloning examples
Problems with Sanger method
46. 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
Moloney murine leukemia virus (MMLV) RTase
Edman degradation
Recognition sites of restriction endonucleases
Shotgun sequencing
47. Integrate into cellular chromosome.
Plasmids
Lysogenic
Problems with Sanger method
Oligo(dT) affinity chromatography
48. (1) Gene is separated from chromosome - (2) gene is put into a vector - (3) vector replicates to produce multiple copies of the gene.
Red recombinase enzymes
Check PCR Product
Transforming and Maintaining Plasmid
Molecular cloning
49. Apyrase - a nucleotide degrading enzyme continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete - another dNTP is added.
Red recombinase enzymes
Pyrosequencing Step 4
Shotgun sequencing
Oligo(dT) affinity chromatography
50. 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
Lytic
Restriction endonucleases
T4 DNA Polymerase
Key Features of PCR