Recombinant DNA Technology
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Questions and Answers

Why is it crucial that plasmids used as cloning vectors have a specific sequence that exists only once?

  • To provide a binding site for RNA polymerase, ensuring efficient transcription of the inserted gene.
  • To ensure the plasmid can replicate independently within the host bacterium.
  • To ensure the plasmid is cut at only one specific location by the restriction endonuclease, allowing for precise insertion of foreign DNA. (correct)
  • To prevent the plasmid from being recognized and degraded by the host's restriction enzymes.

DNA ligase, used in creating recombinant DNA, is an enzyme exclusively used for recombinant DNA technology and not involved in natural DNA replication processes.

False (B)

Describe a potential consequence if the foreign DNA and plasmid DNA fragments lacked complementary ends during the creation of recombinant DNA.

Without complementary ends the DNA fragments would not be able to anneal, thereby preventing the creation of recombinant DNA.

After the recombinant plasmid is introduced into a bacterial host, the bacteria are cultured on a nutrient medium containing ______, which allows for the selection of bacteria that have successfully taken up the plasmid.

<p>ampicillin</p> Signup and view all the answers

Match the steps of recombinant DNA technology with their descriptions:

<p>Isolation of DNA = Extraction of DNA from both a bacterial plasmid and a eukaryotic cell. Restriction Digestion = Cutting DNA at specific sequences using restriction endonucleases. Ligation = Joining foreign DNA and plasmid DNA fragments using DNA ligase. Transformation = Introducing recombinant plasmid into a host bacterium.</p> Signup and view all the answers

What is the primary method for selecting bacteria that contain recombinant plasmids?

<p>Selecting bacteria based on their resistance to a specific antibiotic conferred by a gene within the recombinant plasmid. (D)</p> Signup and view all the answers

The process of transformation involves permanently altering the bacterial cell wall to allow plasmid entry.

<p>False (B)</p> Signup and view all the answers

What is the name given to the collective DNA fragments originating from an organism that are stored to form multitude of recombinant clones?

<p>genomic library</p> Signup and view all the answers

The restriction endonuclease EcoRI recognizes and cuts the DNA sequence ______.

<p>GAATTC</p> Signup and view all the answers

During the construction of recombinant plasmids, what outcome is possible that does not result in the creation of recombinant DNA?

<p>The plasmid reverts to its original circular form without incorporating any foreign DNA. (C)</p> Signup and view all the answers

Match the steps involved in constructing a genomic library using bacteriophages with their descriptions:

<p>Cut Bacteriophages with Restriction Endonuclease = Generating fragments of phage DNA suitable for recombination. Isolate DNA from Phages = Extracting the DNA from viral particles to prepare for subsequent steps. Mix the DNA = Combining phage DNA fragments to promote random associations. Combine Recombinant DNA with Phage Proteins = Packaging recombinant DNA molecules into new infectious phage particles resulting to new phages.</p> Signup and view all the answers

What is the most critical reason for using host cells that are not susceptible to antibiotics in recombinant DNA experiments?

<p>To allow for effective selection of recombinant bacteria, as only those with the plasmid will grow in the presence of the antibiotic. (B)</p> Signup and view all the answers

What specific process does the diagram in Figure 4.3 illustrate?

<p>Construction of Recombinant DNA Molecules</p> Signup and view all the answers

Which of the following best describes the primary role of restriction endonucleases in bacteria?

<p>To protect against invading foreign DNA. (B)</p> Signup and view all the answers

The term 'clone' exclusively refers to identical organisms produced through artificial means.

<p>False (B)</p> Signup and view all the answers

What is the term for the process of introducing recombinant DNA into a bacterial cell?

<p>transformation</p> Signup and view all the answers

A __________ library contains the entire genome of an organism, fragmented and cloned into vectors.

<p>genomic</p> Signup and view all the answers

Match each step in recombinant DNA technology with its correct description:

<p>Cutting DNA = Using restriction endonucleases to create fragments. Vector Selection = Choosing a suitable carrier for DNA transfer. Transformation = Introducing recombinant DNA into a host cell. Clone Selection = Identifying cells with the desired DNA insert.</p> Signup and view all the answers

Why is the discovery of restriction endonucleases considered a fundamental step in the development of genomic libraries?

<p>They allow precise DNA cutting and controlled fragmentation. (C)</p> Signup and view all the answers

Recombinant DNA technology is limited to applications in medicine and has no relevance in agriculture or other scientific disciplines.

<p>False (B)</p> Signup and view all the answers

What is a cloning vector, and what are its main roles in recombinant DNA technology?

<p>A DNA molecule that can carry foreign DNA and replicate inside a host cell. Its primary role is to transfer and propagate the foreign DNA.</p> Signup and view all the answers

The enzyme __________ recognizes and cuts DNA at specific sequences, creating fragments with either sticky or blunt ends.

<p>EcoRI</p> Signup and view all the answers

Match each enzyme with its specific function in recombinant DNA technology:

<p>Restriction Endonuclease = Cuts DNA at specific sequences DNA Ligase = Joins DNA fragments together Reverse Transcriptase = Synthesizes DNA from RNA template DNA Polymerase = Replicates DNA</p> Signup and view all the answers

Which characteristic of restriction endonucleases is most crucial for creating recombinant DNA molecules?

<p>Their sequence-specific cutting action. (B)</p> Signup and view all the answers

A genomic library contains only the expressed genes of an organism, representing a subset of its total DNA.

<p>False (B)</p> Signup and view all the answers

Outline the steps involved in creating a recombinant plasmid and introducing it into a bacterial cell.

<p>Digestion of plasmid and target DNA with the same restriction enzyme, ligation of the DNA fragments, and transformation of bacteria with the recombinant plasmid.</p> Signup and view all the answers

The process of selecting host cells that have taken up recombinant DNA from those that have not is called __________.

<p>screening</p> Signup and view all the answers

Match the following terms to their descriptions in the context of creating a DNA library:

<p>Genomic DNA = The whole DNA content of an organism Restriction Enzyme = Enzyme that cuts DNA at specific sites Vector = DNA molecule used as a vehicle to carry foreign genetic material DNA Ligase = Enzyme that covalently links DNA fragments together</p> Signup and view all the answers

Flashcards

Restriction Endonucleases

Enzymes that cut DNA at specific sequences, like GAATTC for EcoRI.

Plasmids

Small, circular DNA molecules in bacteria, used to carry foreign DNA.

DNA Ligase

An enzyme that joins DNA fragments with complementary ends.

Recombinant Plasmid

A plasmid that contains a foreign DNA fragment, creating a new DNA combination.

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Bacterial Culture

Growing bacteria in a controlled environment.

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Transformation (in bacteria)

The process where a plasmid enters a bacterium by transiently damaging the bacterial walls.

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Selection of recombinant bacteria

Selecting bacteria containing recombinant plasmids based on antibiotic resistance.

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Bacterial clone

A population of identical bacteria, all descended from a single ancestor, each containing a copy of the recombinant plasmid.

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Genomic library

The total DNA of an organism, fragmented and inserted into cloning vectors to create a collection of clones.

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Recombinant DNA molecules

DNA molecules created by joining DNA fragments from different sources.

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Bacteriophage

A virus that infects bacteria and is used to clone DNA segments into bacterial cells.

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Genetic engineering

Modifying an organism's genetic material.

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Recombinant DNA technology

Transferring genetic material between organisms.

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Recombinant DNA

DNA that has been formed by combining constituents from different sources.

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Cloning vector

A DNA molecule that can independently replicate inside a host cell.

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Transformation

Inserting DNA into a bacterial host cell.

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Clone

A group of identical molecules, cells, or organisms.

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Cloning

Making many identical copies of a molecule, cell or organism.

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Function of restriction endonucleases

Protecting bacteria via cutting 'foreign' DNA.

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Recognition sites

Specific DNA sequences recognized by restriction endonucleases.

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EcoRI

An example of restriction endonuclease.

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Sticky ends

Single-stranded DNA at the ends after being cut by restriction enzymes.

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Hydrogen bonds

Joining DNA fragments with complementary sticky ends.

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Selection of host cells

Selecting cells that have taken up recombinant DNA.

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Study Notes

  • Recombinant DNA technology includes all the techniques for transferring genetic material from one organism to another.

Construction of recombinant DNA

  • Total DNA from an organism is isolated which is then fragmented by special enzymes, the restriction endonucleases, and joined with a cloning vector.
  • Cloning vectors are cloning vehicles.
  • The fragments made with the restriction enzymes have unpaired bases.
  • These ends can form hydrogen bonds with the complementary bases of other DNA segments which have been cut with the same enzyme.
  • A DNA molecule, such as a plasmid or DNA of a phage, can replicate independently inside a bacterium.
  • The DNA becomes recombinant.
  • Introducing DNA into a bacterial host cell is called transformation.
  • Host cells that have taken up recombinant DNA are selected.

Selection and Isolation of host cells

  • At this stage the host cells that have been incorporated into recombinant DNA are selected from those that do not.
  • Each bacterium that has taken up recombinant DNA multiplies and produces a colony, which constitutes a bacterial clone.
  • Many thousands of clones are produced in the process, each containing a different recombinant DNA molecule that contains a distinct fragment of the donor organism’s DNA.
  • The enzyme EcoRI recognizes the GAATTC sequence in the double helix of DNA and cuts it.
  • In this way recombinant plasmids are created.
  • Some plasmids are remade into circular form, without addition of DNA from the organism.
  • Bacteria – hosts receive a small percentage of plasmids, some of which are recombinant.
  • Plasmids used as cloning vehicles usually have a specific sequence that is cut only once by the EcoRI.
  • Pieces of DNA from the same plasmid and organism are mixed.
  • The plasmids are cut apart by the EcoRI in this position, resulting in a linear DNA molecule with single-stranded ends.
  • The DNA ligase enzyme joins the two DNA pieces together.
  • The plasmids that are used as cloning vectors have a specific sequence.

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Description

Recombinant DNA technology involves transferring genetic material between organisms. Restriction enzymes fragment DNA, which is then joined with a cloning vector. The recombinant DNA is introduced into a bacterial host cell through transformation and selected.

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