Podcast
Questions and Answers
During DNA cloning, what is the primary role of a cloning vector?
During DNA cloning, what is the primary role of a cloning vector?
- To cut the genomic DNA into smaller fragments.
- To provide the enzymatic machinery for DNA replication within the test tube.
- To synthesize the DNA segment to be cloned.
- To act as a carrier to deliver the DNA segment into a host organism. (correct)
Which enzyme is responsible for covalently linking the cloning vector and the DNA fragment to be cloned?
Which enzyme is responsible for covalently linking the cloning vector and the DNA fragment to be cloned?
- Restriction endonuclease
- Reverse transcriptase
- DNA ligase (correct)
- Polymerase
What is the significance of using Escherichia coli in recombinant DNA technology?
What is the significance of using Escherichia coli in recombinant DNA technology?
- Its DNA metabolism is poorly understood, making it ideal for novel experiments.
- It is the only organism with plasmids and bacteriophages.
- Its DNA metabolism is well understood, and techniques for DNA transfer are readily available. (correct)
- It lacks naturally occurring cloning vectors, ensuring no interference with the experiment.
Which of the following best describes recombinant DNA?
Which of the following best describes recombinant DNA?
What is the purpose of selecting host cells that contain recombinant DNA during cloning?
What is the purpose of selecting host cells that contain recombinant DNA during cloning?
How do restriction endonucleases contribute to the process of DNA cloning?
How do restriction endonucleases contribute to the process of DNA cloning?
What is the ultimate outcome of DNA cloning?
What is the ultimate outcome of DNA cloning?
Why is it important for cloning vectors to have features that allow host cells to survive in a selective environment?
Why is it important for cloning vectors to have features that allow host cells to survive in a selective environment?
Which of the following is NOT a general procedure involved in the classical cloning of DNA?
Which of the following is NOT a general procedure involved in the classical cloning of DNA?
How does the Polymerase Chain Reaction (PCR) contribute to obtaining DNA segments for cloning?
How does the Polymerase Chain Reaction (PCR) contribute to obtaining DNA segments for cloning?
What distinguishes a 'clone' in the context of DNA from its original definition related to cells?
What distinguishes a 'clone' in the context of DNA from its original definition related to cells?
If a researcher wants to study a particular gene and needs to create many copies of it, what is the most appropriate first step according to the text?
If a researcher wants to study a particular gene and needs to create many copies of it, what is the most appropriate first step according to the text?
In the context of DNA cloning, what is meant by the 'selective amplification' of a DNA segment?
In the context of DNA cloning, what is meant by the 'selective amplification' of a DNA segment?
When creating recombinant DNA, what best describes the order of steps?
When creating recombinant DNA, what best describes the order of steps?
What scenario would necessitate the use of PCR over restriction endonucleases for obtaining a DNA segment for cloning?
What scenario would necessitate the use of PCR over restriction endonucleases for obtaining a DNA segment for cloning?
What potential outcome might arise if DNA ligase were non-functional during recombinant DNA technology?
What potential outcome might arise if DNA ligase were non-functional during recombinant DNA technology?
In contrast to using E. coli, what might be a challenge in cloning DNA in a less-studied organism?
In contrast to using E. coli, what might be a challenge in cloning DNA in a less-studied organism?
A researcher successfully creates a recombinant DNA molecule but fails to select host cells containing the recombinant DNA. What is the most likely consequence?
A researcher successfully creates a recombinant DNA molecule but fails to select host cells containing the recombinant DNA. What is the most likely consequence?
What is the most direct consequence of a cloning vector lacking features that allow host cells to survive in a selective environment?
What is the most direct consequence of a cloning vector lacking features that allow host cells to survive in a selective environment?
How has the knowledge of genomic sequences impacted the DNA cloning process?
How has the knowledge of genomic sequences impacted the DNA cloning process?
Flashcards
Clone
Clone
An identical copy, originally referring to cells but now also to DNA segments.
DNA Cloning Definition
DNA Cloning Definition
Separating a gene, attaching it to a carrier DNA, and allowing microorganisms to make many copies.
Restriction Endonucleases
Restriction Endonucleases
Enzymes that recognize specific DNA sequences and cut genomic DNA into smaller fragments.
Cloning Vector
Cloning Vector
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DNA Ligase
DNA Ligase
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Recombinant DNA
Recombinant DNA
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Recombinant DNA Technology
Recombinant DNA Technology
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Plasmids
Plasmids
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Bacteriophages
Bacteriophages
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Study Notes
- A clone is an identical copy, originally referring to cells reproducing into a population of identical cells. When referring to DNA, a clone means many identical copies of a specific gene segment.
DNA Cloning Process
- DNA cloning involves separating a gene from a larger chromosome, attaching it to a smaller carrier DNA, and allowing microorganisms to make many copies. The process results in selective amplification of a DNA segment for study and use.
- Classically cloning DNA from any organism involves 5 general procedures.
Five General Procedures for DNA Cloning
- Obtaining the DNA segment to be cloned: Restriction endonucleases are used to cleave genomic DNA into smaller fragments. Genomic DNA can also be sheared randomly into fragments of a desired size. DNA segments to be cloned are often amplified by PCR or synthesized.
- Selecting a small molecule of DNA capable of autonomous replication: These small DNAs are called cloning vectors. Cloning vectors are modified versions of naturally occurring small DNA molecules found in bacteria or eukaryotes or viral DNAs.
- Joining two DNA fragments covalently: The enzyme DNA ligase links the cloning vector to the DNA fragment to be cloned. Composite DNA molecules, comprising covalently linked segments from two or more sources, are called recombinant DNAs.
- Moving recombinant DNA from the test tube to a host organism: The host organism provides the enzymatic machinery for DNA replication.
- Selecting or identifying host cells that contain recombinant DNA: The cloning vector generally has features that allow the host cells to survive in an environment in which cells lacking the vector would die. Cells containing the vector are thus “selectable” in that environment.
Recombinant DNA Technology
- The methods used to accomplish these and related tasks are collectively referred to as recombinant DNA technology or genetic engineering.
DNA Cloning in Escherichia Coli
- DNA cloning is often done in bacterium Escherichia coli, the first organism used for recombinant DNA work.
- E. coli advantages include well understood DNA metabolism, readily available cloning vectors like plasmids and bacteriophages, and techniques for moving DNA between bacterial cells.
- The principles discussed are broadly applicable to DNA cloning in other organisms.
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