Gene cloning and Manipulation AI Deck Flashcards
(124 cards)
1
Q
What is the definition of genetically engineered organisms?
A
An organism modified at the molecular or cellular level by means not possible under natural conditions, including recombinant DNA techniques, cell fusion, and introducing a foreign gene.
Such organisms are often referred to as genetically modified organisms (GMOs) or transgenic organisms.
2
Q
What does genetic engineering not include?
A
Modification consisting exclusively of breeding, conjugation, fermentation, hybridization, in vitro fertilization, or tissue culture.
3
Q
Who invented the Polymerase Chain Reaction (PCR)?
A
Kary Mullis in 1983.
4
Q
What is a key feature of PCR?
A
It can be very specific, readily automated, and can work with small quantities of DNA.
5
Q
What is the typical temperature for denaturation in a PCR cycle?
A
94 °C.
6
Q
What is the typical temperature range for annealing in a PCR cycle?
A
40–70 °C, often around 50–55 °C.
7
Q
What is the optimal temperature for DNA synthesis in PCR?
A
72 °C.
8
Q
What is Taq polymerase?
A
A thermostable polymerase from Thermus aquaticus used in PCR.
It has a temperature optimum of 72–75 °C and lacks 3’–5’ proofreading activity.
9
Q
What is the maximum size of DNA that PCR typically amplifies efficiently?
A
Up to 2-3 kb.
10
Q
What is one method to improve PCR specificity?
A
Touch-down PCR or Hot start PCR.
11
Q
What is a common problem with PCR related to polymerase errors?
A
Taq polymerase has a significant error rate (~1 in 10,000 nucleotides).
12
Q
What should the ideal length of PCR primers be?
A
20–30 bp in length.
13
Q
Fill in the blank: The basic reaction in PCR involves _______ to melt the DNA and then _______ to allow primers to anneal.
A
heating; cooling.
14
Q
What is the role of primers in PCR?
A
To direct DNA synthesis to the desired regions of the DNA template.
15
Q
How can you obtain sequence information for primer design?
A
Using genomic data, online resources, or cDNA from commercial sources.
16
Q
What are degenerate primers?
A
Primers that are a mixture of sequences to account for the degeneracy of the genetic code.
17
Q
What can cause non-specific priming in PCR?
A
Primers annealing to similar sequences elsewhere in the genome.
18
Q
What is the purpose of using additives like glycerol in PCR?
A
To lower strand-separation and primer-annealing temperatures, alleviating depurination effects.
19
Q
True or False: PCR can amplify DNA sequences larger than 5 kb efficiently.
A
False.
20
Q
What can be done to reduce the accumulation of truncated products in PCR?
A
Using longer extension times and high-quality templates.
21
Q
What is touch-down PCR?
A
A technique starting with high annealing temperatures and reducing in subsequent cycles to improve specificity
It ensures that the first successful reactions occur under stringent conditions.
22
Q
What is the purpose of hot start PCR?
A
To prevent non-specific priming by keeping polymerase inactive until the denaturation temperature is reached
This can be achieved by adding polymerase manually or using a specially designed polymerase.
23
Q
What is nested PCR?
A
A method involving two consecutive rounds of PCR with new primers designed to anneal within the correct product from the first round
This reduces the chance of amplifying incorrect products.
24
Q
What is a typical feature of a cloning vector?
A
Contains a Multiple Cloning Site (MCS) for restriction enzyme recognition
Example: pBluescript II SK (+/–) vector.
25
What does the lacZ' gene encode?
β-galactosidase, enabling blue-white selection in cloning
## Footnote
A functional lacZ' gene results in blue colonies, while disruption leads to white colonies.
26
What role does antibiotic resistance play in cloning vectors?
The bla gene confers resistance to ampicillin, allowing selection of transformed cells
## Footnote
It encodes β-lactamase, which breaks down ampicillin.
27
What are the steps in the basic cloning procedure?
1. Take amplified PCR product
2. Have a linearized vector
3. Mix and ligate
4. Transform E. coli host
5. Plate on selective medium
## Footnote
Selective media contain ampicillin, IPTG, and X-gal.
28
What is one characteristic of an ideal E. coli host for transformation?
High efficiency of transformation, including the ability to take up DNA and lack of degrading enzymes
## Footnote
For example, E. coli strains can be treated with CaCl2 or electroporated.
29
What is the significance of the JM109 genotype in E. coli?
It has mutations that provide endonuclease deficiency and reduced recombination, beneficial for cloning
## Footnote
Traits include hsdR17, recA1, and ∆(lacZYA-proAB).
30
What does phosphatase treatment of plasmids do?
Reduces background ligation by removing terminal phosphates from the vector
## Footnote
This prevents self-ligation but allows ligation with inserts.
31
What is blunt cloning?
A method where PCR products with blunt ends are cloned into a vector with blunt ends
## Footnote
It is less efficient than sticky end cloning and is non-directional.
32
What is TA cloning?
A method utilizing Taq DNA polymerase's terminal transferase activity to add an A residue to PCR products' 3' ends
## Footnote
This allows efficient cloning into vectors with complementary T-overhangs.
33
What are restriction enzymes?
Proteins that cut DNA at specific sequences, protecting bacterial DNA from phage infection
## Footnote
E. coli methylates its own DNA to prevent degradation by these enzymes.
34
What are isoschizomers?
Different restriction enzymes that can produce the same sticky ends
## Footnote
Useful for cloning when specific recognition sites are needed.
35
What is Gibson's assembly method?
A technique that allows joining multiple overlapping DNA fragments in a single isothermal reaction
## Footnote
It does not require specific restriction sites and is efficient.
36
What is Gibson’s assembly method?
A method to join almost any two DNA fragments regardless of sequence, allowing multiple overlapping fragments to be joined in a single isothermal reaction
37
What are the advantages of Gibson’s assembly method?
* No need for specific restriction sites
* No 'scar' between joined fragments
* Cloning is directional and efficient
38
What is required for the fragments in Gibson’s assembly?
The fragments must have overlapping ends for directional assembly
39
What is the role of T5 exonuclease in Gibson’s assembly?
Creates single-stranded 3' overhangs that facilitate the annealing of overlapping fragments
40
What does DNA polymerase do in Gibson’s assembly?
Fills in gaps within each annealed fragment
41
What is the function of DNA ligase in Gibson’s assembly?
Seals nicks in the assembled DNA
42
What does Golden Gate Cloning allow?
Simultaneous and directional assembly of multiple DNA fragments into a single piece
43
What enzymes are used in Golden Gate Cloning?
Type IIS restriction enzymes and T4 DNA ligase
44
How does Golden Gate Assembly exploit Type IIS restriction endonucleases?
They cleave DNA outside of the recognition sequence, allowing for the removal of the recognition sequence from the assembly
45
What is sub-cloning?
Using restriction enzymes to clone sequences from an existing clone without PCR amplification
46
What are synthetic genes?
Open reading frame sequences generated synthetically from publicly annotated genes
47
What are some advantages of using synthetic genes?
* Availability of hard-to-clone genes
* Increased expression yields due to sequence optimization
* Custom sequence design
48
What is Reverse Transcription and Quantitative PCR (RT/qPCR)?
A method for quantifying mRNA levels by amplifying mRNA sequences as DNA copies
49
What is the role of reverse transcriptase in RT/qPCR?
Converts mRNA into complementary DNA (cDNA) for amplification
50
What is the significance of the CT value in qPCR?
It reflects the relative amount of newly generated double-stranded DNA strands
51
What is SYBR green used for in qPCR?
A dye that binds to double-stranded DNA and fluoresces, allowing for quantification of DNA
52
What is one method to prepare in vitro transcribed RNA?
Cloning the DNA sequence into a vector with phage promoters recognized by specific RNA polymerases
53
What is required for producing recombinant proteins in E. coli?
* Promoter
* Prokaryotic ribosome binding site
* AUG initiation codon
54
What is the purpose of fusion proteins in E. coli expression systems?
To confer useful properties to the protein of interest
55
What is the role of proteases in fusion protein systems?
To cleave the fusion protein at the junction with a specific protease
56
What is a fusion protein?
A fusion protein is a protein created by joining two or more genes that originally coded for separate proteins.
57
What signals are typically included in a vector designed to produce a fusion protein?
Translation initiation signals.
58
In scenario A, how is a fusion protein generated?
Translation starts at the ATG in the vector, and the insert must be in frame with the vector's reading frame.
59
What are some applications of fusion proteins?
* Protein purification
* Protein solubility
* Tagging proteins
* Pull down assays
* Phage display
60
How can you cleave unwanted parts of a fusion protein after purification?
Using specific proteases.
61
What is a His-tag?
A His-tag is a sequence of six histidine residues used for protein purification.
62
How is a His-tagged protein purified?
Using columns containing immobilized metal cations like nickel chelated to NTA.
63
What are two methods to elute a His-tagged protein from a column?
* Using a protease if there is a cleavage site
* Using imidazole to compete for binding
64
What is the function of a Glutathione S-transferase (GST) tag?
Used for purification and pull-down assays.
65
How is a protein with a GST tag purified?
On a Glutathione sepharose column and eluted with glutathione.
66
What is the purpose of a maltose binding protein (MBP) tag?
To increase protein solubility and assist in purification.
67
How is an MBP-tagged protein purified?
On an Amylose column and eluted with maltose.
68
What is a major issue when expressing proteins in E. coli?
Formation of inclusion bodies.
69
What is the role of epitope tags?
To enable specific recognition by antibodies for various applications.
70
What are some examples of epitope tags?
* Flag
* Myc
* HA
71
What are reporter genes used for?
To analyze gene expression and promoter activity.
72
Name two popular reporter genes.
* Green fluorescent protein (GFP)
* Beta-galactosidase (X-gal staining)
73
What is the purpose of immunoprecipitation?
To determine which proteins interact with a protein of interest.
74
What is required for successful immunoprecipitation?
An antibody that specifically recognizes the protein of interest.
75
What is a pull-down assay?
A method using immobilized fusion proteins to identify interacting proteins.
76
What is the first step in a pull-down assay?
Immobilize the GST-tagged protein on glutathione sepharose.
77
What can be analyzed after performing a pull-down assay?
The protein mixture can be analyzed on SDS-PAGE and identified using mass spectrometry.
78
Why might you use lon- cells in protein expression?
To reduce proteolysis from the lon gene product.
79
Fill in the blank: A _______ gene is a gene attached to a regulatory sequence of another gene of interest.
reporter
80
True or False: The universal genetic code is not universal.
True.
81
What are some leader sequences used for protein secretion?
* OmpA
* OmpF
* PhoA
* β-lactamase
* PelB
82
What is the advantage of using epitope tags in protein studies?
They provide an easily recognizable tag for antibody-based techniques.
83
What is a potential outcome of expressing a toxic protein in E. coli?
It may kill the host, reducing yields.
84
What is the purpose of adding a complex mixture of proteins to an ion exchange column?
To purify GST-tagged protein of interest and interacting proteins
## Footnote
This process involves washing unbound proteins and eluting the target protein using glutathione.
85
What technique is used to identify interacting proteins in S. cerevisiae?
Yeast two-hybrid system
## Footnote
This method screens a library of genes for proteins that interact with a known protein.
86
What are the two domains of the GAL4 transcription factor?
* DNA binding domain
* Activation domain
87
What is the role of the GAL4 transcription factor in the yeast two-hybrid system?
To activate transcription of a gene downstream of the DNA binding site when two domains are joined
88
What is the function of the reporter gene in the yeast two-hybrid system?
It is expressed only if the bait protein interacts with a query protein, forming a functional transcription factor
89
What is mutagenesis used for in molecular biology?
To investigate the importance of specific amino acids and regulatory sequences in DNA or RNA
90
What is site-directed mutagenesis?
A method to introduce specific mutations into a DNA sequence using oligonucleotides
91
How are mutations introduced using oligonucleotides?
By annealing a mutagenic oligonucleotide to a cloned copy of the target gene
92
What is the purpose of DpnI in the mutagenesis process?
To specifically digest methylated or hemi-methylated DNA, leaving only the newly synthesized mutant DNA
93
What is RNA interference (RNAi)?
A method for specifically reducing gene expression in various cells or organisms
94
What triggers the RNAi pathway?
The presence of long double-stranded RNA (dsRNA)
95
What enzyme processes dsRNA into short interfering RNAs (siRNAs)?
Dicer
96
What are the characteristics of siRNAs?
* Double stranded
* 2 nucleotide overhangs at 3' ends
* 5' phosphate
* ~21 nt in length
97
What is the role of Argonaute 2 (Ago2) in the RNAi pathway?
To cleave the targeted mRNA
98
How can siRNAs be introduced into mammalian cells?
By transfection using Lipofectamine®-3000 or plasmids expressing shRNAs
99
What is the CRISPR/cas9 system used for?
Genomic editing, including downregulating gene expression and making mutations
100
What are the three components of the CRISPR/Cas system used in gene editing?
* Cas9 nuclease
* Target complementary crRNA
* Auxiliary tracrRNA
101
What is the significance of designing specific crRNA using dedicated software?
To target desired sequences while minimizing off-target effects
102
What does the yeast two-hybrid system rely on to find interactions?
The function of the GAL4 transcription factor
103
What must be considered when designing primers for site-directed mutagenesis?
* Primers must contain desired mutations
* Minimum GC content of 40%
* Melting temperature (Tm) of ≥78 °C
104
What is the typical size range for primers used in mutagenesis?
25-45 nucleotides
105
What is the potential consequence of using a high-fidelity polymerase in PCR for mutagenesis?
To minimize the potential for errors
106
True or False: RNAi can only be used in model organisms like Drosophila and C. elegans.
False
## Footnote
RNAi can be used in various organisms, including plants and mammalian cells.
107
Fill in the blank: The cleavage position by Ago2 occurs between nucleotide _____ and _____ when targeting mRNA.
10 and 11
108
What must be done after mutagenesis to ensure no errors were introduced?
Sequence the amplified sequences
109
What is the purpose of using shRNAs in mammalian cells?
To induce RNA interference for gene knockdown
110
What is the purpose of crRNA in CRISPR systems?
To specifically base pair with target DNA and guide the Cas9 nuclease to the desired sequence
## Footnote
crRNA is designed using software tools that predict the best crRNA sequences for targeting.
111
What are some characteristics of CRISPR plasmids?
They produce gRNA and often encode the Cas9 nuclease, may include selectable markers, and can contain unique restriction sites for cloning
## Footnote
Selectable markers like GFP or OFP are used to identify successfully edited cells.
112
What are the two main DNA repair pathways after Cas9 cleavage?
* Non-homologous end joining (NHEJ)
* Homology directed repair (HDR)
113
What is a Cas9 variant used for?
To improve efficiency, specificity, minimize off-target effects, and deliver effector molecules
## Footnote
Examples include dead Cas9 (dCas9) which does not cut DNA but can bind and recruit other proteins.
114
True or False: CRISPR technology has been used in clinical trials for genetic diseases.
True
115
What is gene disruption?
A method to knockout a gene by replacing it with a mutant version through homologous recombination
## Footnote
This method can be applied to various organisms, including bacteria and mice.
116
What is the cre-lox system used for?
For controlled excision of genes using site-specific recombination
## Footnote
The Cre protein mediates recombination at lox sequences.
117
Fill in the blank: The process of removing sequences flanked by lox elements is called _______.
floxing
118
What are the two main methods for generating transgenic organisms?
* Microinjection of DNA into fertilized eggs
* Use of cultured embryonic stem (ES) cells
119
What happens during microinjection of DNA into fertilized eggs?
The DNA randomly integrates into the genome, potentially enabling its expression
## Footnote
This method was demonstrated with a rat growth hormone gene in mice.
120
What is the role of embryonic stem (ES) cells in creating transgenic animals?
They can differentiate into any type of cell and can be manipulated to knockout genes or introduce mutations
## Footnote
ES cells are injected into embryos to create mosaic organisms.
121
How do you ensure proper integration of DNA in ES cells?
Using positive selection markers (e.g. neomycin) and negative selection markers (e.g. Thymidine kinase)
## Footnote
These markers help to select for cells that have successfully undergone homologous recombination.
122
What is a knockout mouse?
A mouse with one or both copies of a gene disrupted or knocked out
## Footnote
These mice are used for studying gene function and disease models.
123
What is the significance of generating homozygous knockout mice?
They serve as a model for studying the complete loss of function of a gene
## Footnote
However, creating homozygous knockouts can be challenging if the gene is essential for life.
124
What is a tissue-specific knockout?
A knockout that occurs in specific tissues using the Cre-lox system
## Footnote
This allows for the study of gene function in particular contexts without affecting the entire organism.
