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Molecular Biology > Gene Cloning 6 > Flashcards

Gene Cloning 6 Flashcards

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1
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What did Mendel and johannsen do

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2
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What did Fredrich miescher and boveri - Sutton do

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What did albrecht Kossel and Phoebus Levene do

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What did lock and Thomas hunt Morgan do

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5
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How did Griffith discover bacterial transformation

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6
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What did Avery, MacLeod & McCarty do

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What did Hershey and Chase do

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What did Sven Furberg do

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What did Maurice Wilkins & Raymond Gosling do

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10
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What did Rosalind Franklin & Raymond Gosling do

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11
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Explain the milestones in molecular genetics from 1953 to 2003

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12
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What is molecular cloning

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13
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Why do we clone DNA

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14
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What other types of clones are there

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15
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give an overview of “traditional” gene cloning

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16
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What is sub cloning

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17
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Give an overview of “contemporary ” gene cloning

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18
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Give some applications of gene cloning

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19
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Give an overview of the process of classical DNA cloning

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20
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In classical DNA cloning what is done after the recombinant DNA is produced

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21
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In more detail, explain the DNA isolations step of classical DNA cloning (stepwise)

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22
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What is the second step of classical cloning (stepwise)

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23
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What is the basic structure of a plasmid cloning vector

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24
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What do the different labels on this plasmid mean

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25
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Explain the third step of classical DNA cloning (stepwise)

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26
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How does DNA ligase work

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27
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Name the 3 strategies of classical cloning

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Single restriction enzyme

Directional cloning – two different restriction enzymes

Blunt end ligation

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28
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What is Single restriction enzyme DNA cloning

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29
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What is directional DNA cloning

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30
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What is blunt end ligation + adv and disadvantage

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31
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What is PCR based cloning

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32
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What is T/A cloning

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Exploiting the “Terminal transferase” activity of Taq Polymerase (adds and A i.e. non-template polymerase activity

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33
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How does T/A topo cloning work

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34
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What are the different ways to introduce vectors into the organism in DNA cloning

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Electroporation

CaCl2/Heat Shock

Transformation into non-bacterial cells

Replication in bacteria

Selection in bacteria – exploit antibiotic resistance

Selection in bacteria – screening for antibiotic resistance

Screening for recombinant clones: lacZ and “Blu/White

Selection recombinant clones: use of suicide genes

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35
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How can use of suicidal genes be used as a way to introduce the vector into organisms in DNA cloning

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36
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How can lacZ and Blu/White be used as a way to introduce the vector into organisms in DNA cloning

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37
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Where does blue and white colour come from in Blu/white selection

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Blue colonies - empty vectors
White colonies - LacZ disrupted

38
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How can screening for antibiotic resistance be used as a way to introduce the vector into organisms in DNA cloning

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39
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How can exploiting antibiotic resistance (with the example of kanamycin) be used as a way to introduce the vector into organisms in DNA cloning (how does kanamycin work )

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40
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How can exploiting antibiotic resistance (with the example of ampicillin resistance) be used as a way to introduce the vector into organisms in DNA cloning (how does ampicillin work)

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41
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How can replication in bacteria be used as a way to introduce the vector into organisms in DNA cloning

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42
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How can transformation into non bacterial cells be used as a way to introduce the vector into organisms in DNA cloning (name ways vector can be incorporated)

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43
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How can heat shock be used as a way to introduce the vector into organisms in DNA cloning

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44
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How can electroporation be used as a way to introduce the vector into organisms in DNA cloning

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45
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What are some Alternatives and developments of molecular cloning

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46
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Give a limitation of the use of restriction enzymes

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47
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Explain Sequence and Ligation Independent Cloning (SLIC)

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48
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Explain Gibson assembly cloning

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The Gibson Assembly® method is a cloning procedure that allows the cloning of two or more fragments without the need for restriction enzyme digestion or compatible restriction sites. Instead, user-defined overlapping ends are incorporated into the fragments to allow the seamless joining of adjacent fragments.

49
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Explain gateway cloning

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Prepare Your DNA: First, you need to isolate the DNA fragment (the gene you want to move) from its original organism. You also need a plasmid, which is a small, circular piece of DNA found in bacteria.
Cut the DNA: Enzymes called restriction enzymes are used to cut both the DNA fragment and the plasmid at specific points. This creates “sticky ends” on the DNA and plasmid, which will help them stick together later.
Insert the Gene: Now, you mix the DNA fragment and the plasmid together. The sticky ends of the DNA fragment will bind with the complementary sticky ends of the plasmid, effectively inserting the gene into the plasmid.
Joining: Enzymes called DNA ligases are then used to permanently join the DNA fragment to the plasmid. This creates a recombinant plasmid, which now contains the gene you want to clone.
Transformation: The recombinant plasmid is introduced into bacteria through a process called transformation. The bacteria will take up the plasmid and begin to replicate it along with their own DNA.
Selection: To make sure the bacteria have taken up the recombinant plasmid, you typically include a selectable marker in the plasmid. This marker allows only the bacteria that have successfully taken up the plasmid to survive under specific conditions (like the presence of a certain antibiotic).
Identify Clones: After selection, you can identify the bacteria that have successfully cloned your gene by examining their characteristics or by using specific tests.

50
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How does gateway cloning exploit sequence specific recombination systems of E. coli and λ bacteriophage ?

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BP Reaction (Entry Cloning):
In the BP reaction, the DNA fragment (containing attB sites) to be cloned is mixed with a donor vector containing attP sites.
The Integrase enzyme catalyzes the recombination between attB and attP sites, resulting in the insertion of the DNA fragment into the donor vector. This creates a recombinant donor vector with attL and attR sites.
LR Reaction (Destination Cloning):
In the LR reaction, the DNA fragment flanked by attL sites (from the donor vector) is transferred to a destination vector containing attR sites.
The Integrase enzyme catalyzes the recombination between attL and attR sites, transferring the DNA fragment from the donor vector to the destination vector.

51
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What are att sites in gateway cloning

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Att sites: These are short DNA sequences that serve as recognition sites for the recombinase enzymes. In Gateway cloning, there are four types of att sites: attB, attP, attL, and attR.

52
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What is the role of recombinase enzymes in gateway cloning

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Recombinase enzymes: The key enzyme involved in Gateway cloning is Integrase, which catalyzes the site-specific recombination reactions between att sites. In E. coli, the Integrase enzyme used is often called Int, while in the λ bacteriophage, it’s the Integrase enzyme.

53
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What is golden gate assembly cloning

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Golden Gate Assembly is a molecular cloning technique used to seamlessly and efficiently assemble multiple DNA fragments into a single plasmid vector. It utilizes Type IIS restriction enzymes, which cut outside their recognition sequences, enabling precise assembly of DNA fragments without leaving any scar sequences behind

54
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What is a scar sequence

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leftover DNA sequence that remains after two DNA fragments have been ligated together. Scar sequences are undesirable because they can potentially interfere with the function of the resulting DNA construct

55
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What are the pros and cons of golden gate assembly

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56
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How is RNA cloned

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Isolation of RNA: The first step in RNA cloning is to isolate the RNA of interest from cells or tissues. This can be achieved using various methods, such as phenol-chloroform extraction, column-based purification kits, or magnetic bead-based purification systems. The choice of method depends on factors like sample type, RNA yield, and downstream applications.
Reverse Transcription (RT): Since conventional cloning methods work with DNA, the isolated RNA needs to be converted into complementary DNA (cDNA) through a process called reverse transcription. Reverse transcriptase enzymes are used to synthesize cDNA from RNA templates. Typically, a mixture of random primers or gene-specific primers is used to initiate cDNA synthesis.
PCR Amplification: The cDNA obtained from reverse transcription can be further amplified using polymerase chain reaction (PCR). PCR allows for the specific amplification of the desired cDNA sequences. If the RNA of interest is rare or present in low abundance, PCR amplification can increase the yield of cDNA for downstream cloning steps.
Insertion into Cloning Vector: The amplified cDNA fragments are then ligated into a suitable cloning vector. The choice of vector depends on the downstream applications. Common vectors used for RNA cloning include plasmids, viral vectors, or expression vectors designed for RNA-based applications. The ligation reaction typically involves restriction enzyme digestion of the vector and cDNA fragments, followed by ligation using DNA ligase.
Transformation: The ligated vector containing the cDNA inserts is introduced into host cells, usually bacterial cells such as Escherichia coli, through a process called transformation. The transformed cells are then grown on selective media containing antibiotics to select for cells that have taken up the recombinant vector.
Screening and Verification: Bacterial colonies containing the recombinant vectors are screened to identify clones carrying the desired cDNA inserts. This screening can be done through colony PCR, restriction enzyme digestion, or sequencing of the inserts.

57
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Explain cDNA directional cloning

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Generation of cDNA: The first step in cDNA directional cloning is the generation of cDNA from RNA molecules using reverse transcription. This process produces single-stranded cDNA molecules that are complementary to the mRNA templates.
Introduction of Specific Ends: In directional cloning, the cDNA molecules are modified to have specific ends that facilitate directional insertion into the vector. This is typically achieved by using primers during reverse transcription that contain specific sequences at their ends. These sequences serve as recognition sites for restriction enzymes used in subsequent steps.
Digestion of Vector and cDNA: The destination vector (usually a plasmid) and the cDNA molecules are digested with restriction enzymes that recognize the specific sequences introduced at the ends of the cDNA. Importantly, the restriction enzyme sites are chosen such that they produce compatible cohesive ends (sticky ends) when digested.
Ligation: The digested cDNA molecules are ligated into the digested vector using DNA ligase. The cohesive ends of the cDNA and vector anneal together, allowing them to be joined in a specific orientation. Since the cohesive ends are complementary, the cDNA can only be ligated into the vector in one direction, ensuring directional cloning.
Transformation: The ligated vector containing the cDNA insert is introduced into host cells (usually bacteria) through a process called transformation. The transformed cells are then plated onto selective media containing antibiotics to select for cells that have taken up the recombinant vector.
Screening and Verification: Bacterial colonies containing the recombinant vectors are screened to identify clones carrying the cDNA insert in the correct orientation. This can be achieved through colony PCR, restriction enzyme digestion, or sequencing of the insert.

58
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Give some applications of gene cloning

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59
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How can gene cloning be used as a research tool

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60
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What is transgenic mice and how can they be used to model polio

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Transgenic mice are mouse models that have had their genomes altered for the purpose of studying gene functions.

61
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Give some uses of transgenic mice

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62
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Give the steps for producing a transgenic mouse

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The first step is to design the DNA sequence that will be inserted into the mouse genome. This transgene typically contains the gene of interest
clone the gene of interest into a vector
The transgene is then introduced into embryonic stem cells, which are derived from early-stage embryos. This can be achieved through techniques like microinjection or electroporation.
the cells are screened to identify those that have successfully incorporated the transgene into their genome. This is typically done using selectable markers

ES cells that have successfully incorporated the transgene are injected into early-stage mouse embryos. These embryos are then implanted into a surrogate mother mouse.
Breeding Transgenic Mice: The embryos implanted into surrogate mothers develop into chimeric mice, which contain a mixture of cells derived from both the host embryo and the injected ES cells. Chimeric mice are bred with normal mice to produce offspring that carry the transgene in all of their cells.
Screening Offspring: Offspring are screened to identify those that have inherited the transgene. This typically involves PCR-based methods to detect the presence of the transgene in genomic DNA extracted from tail biopsies or other tissues.

63
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What is a knockout mouse

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A knockout mouse is a genetically modified mouse that has had one or more of its genes intentionally deactivated or “knocked out” through a process called gene targeting. This process involves the deliberate introduction of mutations into the mouse’s DNA to disrupt the function of a specific gene

64
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What is a knock in mouse

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A knock-in mouse is a genetically modified mouse that has a specific gene inserted into its genome at a particular location

65
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What is homologous recombination

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66
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How is a knockout mouse produced

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Gene Selection: Researchers choose a gene whose function they want to study. This gene may be associated with a particular disease, or its function may be of interest for understanding normal physiological processes.
Designing the Targeting Vector: A targeting vector is designed to contain the desired mutations that will disrupt the function of the target gene. This vector typically includes a selectable marker gene (such as a neomycin resistance gene) to aid in the selection of successfully modified cells.
Introduction of the Targeting Vector into Embryonic Stem (ES) Cells: The targeting vector is introduced into embryonic stem (ES) cells, which are derived from early-stage mouse embryos. This can be achieved through techniques such as electroporation or viral transduction.
Selection of Modified ES Cells: ES cells that have successfully incorporated the targeting vector are selected for using the selectable marker gene. This is typically done by exposing the cells to a selective agent, such as an antibiotic, that kills cells lacking the marker.
Verification of Gene Targeting: The targeted ES cells are screened to confirm that the desired genetic modification has been achieved. This may involve techniques such as polymerase chain reaction (PCR) or Southern blotting to verify the presence of the knockout allele.
Injection of Modified ES Cells into Mouse Embryos: The modified ES cells are injected into early-stage mouse embryos. These embryos are then implanted into surrogate female mice.
Germline Transmission: The embryos develop into chimeric mice, which contain a mixture of cells derived from the modified ES cells and cells from the host embryo. These chimeric mice are bred with wild-type mice to produce offspring carrying the modified gene in their germline (sperm or egg cells).
Breeding Homozygous Knockout Mice: Heterozygous knockout mice, which carry one copy of the disrupted gene, are bred together to produce offspring that are homozygous for the knockout allele, meaning they carry two copies of the disrupted gene. These mice are used for subsequent experiments to study the effects of the gene knockout.

67
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How does transgenesis using ES cells work

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68
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How is gene cloning used in agriculture

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69
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Why is corn genetically modified

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70
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What is used to modify corn

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71
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What is golden rice

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72
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How can gene cloning be used in medicine

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73
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How can haemophilia be treated using gene cloning

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74
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How is factor viii produced by gene cloning

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Gene Isolation: The gene encoding factor VIII is isolated from a human or animal cell. This can be done using techniques such as polymerase chain reaction (PCR) or screening gene libraries.
Construction of Recombinant DNA: Once isolated, the factor VIII gene is inserted into a vector, such as a plasmid or a viral vector. The vector acts as a carrier to deliver the gene into host cells for expression. Additionally, regulatory elements, such as promoters and enhancers, may be included in the vector to control the expression of the gene.
Transformation of Host Cells: The recombinant DNA construct is introduced into host cells, typically bacteria or mammalian cells grown in culture. This can be achieved using methods such as bacterial transformation or transfection.
Expression of Factor VIII: Within the host cells, the recombinant DNA directs the production of factor VIII protein. In the case of bacterial hosts, such as Escherichia coli, the protein may be synthesized in inclusion bodies and requires subsequent purification. In mammalian cell hosts, the protein may be secreted into the culture medium.
Purification of Factor VIII: The factor VIII protein is purified from the host cells or culture medium using a series of purification steps, such as chromatography, filtration, and precipitation. These steps isolate the protein from other cellular components and contaminants.
Quality Control and Characterization: The purified factor VIII protein undergoes extensive quality control testing to ensure its safety, potency, and purity. This includes assessing its biochemical properties, activity level, and absence of contaminants.
Formulation and Packaging: Once purified and characterized, the factor VIII protein is formulated into the desired dosage form, such as a lyophilized powder or a liquid solution. It is then packaged into vials or other appropriate containers for distribution.

75
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Why is it important to identify the gene linked to a genetic disease

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76
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What is CF

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77
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How is CF caused

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78
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What gene therapy technique was used to treat CF

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79
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How was gene therapy used to produce the Covid vaccine

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80
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How does forensic identification work?

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81
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How was DNA profiled in forensic science

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82
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What is the basis of genetics

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83
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How can the eye provide evidence for conservation of genes

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84
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What is a master gene

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85
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How is red hair produced

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86
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What is a genotype, allele, phenotype and gene

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Molecular Biology (11 decks)

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