Textbook Question
How can gene knockouts, transgenic animals, and gene editing techniques be used to explore gene function?
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18. Molecular Genetic Tools
Genetic Cloning
2:51 minutesProblem 1a
Textbook Question
In a recombinant DNA cloning experiment, how can we determine whether DNA fragments of interest have been incorporated into plasmids and, once host cells are transformed, which cells contain recombinant DNA?
Verified step by step guidance1
Understand the goal of the experiment: The objective is to determine whether the DNA fragments of interest have been successfully incorporated into plasmids and to identify which host cells contain recombinant DNA after transformation.
Step 1: Use a selectable marker: Plasmids often contain antibiotic resistance genes (e.g., ampicillin resistance). After transformation, grow the host cells on a medium containing the corresponding antibiotic. Only cells that have taken up the plasmid (recombinant or non-recombinant) will survive.
Step 2: Use a screening method to identify recombinant plasmids: Incorporate a reporter gene or a multiple cloning site (MCS) into the plasmid. For example, the lacZ gene can be used for blue-white screening. If the DNA fragment of interest is inserted into the MCS, it disrupts the lacZ gene, preventing the production of β-galactosidase. Colonies with recombinant plasmids will appear white, while non-recombinant colonies will appear blue when grown on a medium containing X-gal.
Step 3: Confirm the presence of the DNA fragment: Isolate plasmid DNA from white colonies and perform a restriction enzyme digestion or PCR. Use specific primers to amplify the inserted DNA fragment. Analyze the results using gel electrophoresis to confirm the presence and size of the DNA fragment.
Step 4: Sequence the recombinant DNA: To ensure the correct DNA fragment has been incorporated, sequence the plasmid DNA using Sanger sequencing or another sequencing method. Compare the sequence to the expected DNA fragment to verify its identity.
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Key Concepts
Here are the essential concepts you must grasp in order to answer the question correctly.
Recombinant DNA Technology
Recombinant DNA technology involves combining DNA from different sources to create new genetic combinations. This process typically includes the use of plasmids as vectors to carry foreign DNA into host cells. Understanding this technology is crucial for determining how specific DNA fragments can be inserted into plasmids and subsequently expressed in host organisms.
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Selection Markers
Selection markers are genes introduced into plasmids that allow for the identification of successfully transformed cells. Common markers include antibiotic resistance genes, which enable the growth of only those cells that have taken up the recombinant DNA. By applying selective pressure, researchers can easily distinguish between cells that contain the plasmid with the DNA of interest and those that do not.
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Molecular Cloning Techniques
Molecular cloning techniques encompass various methods used to isolate and amplify specific DNA fragments. Techniques such as PCR (Polymerase Chain Reaction) and gel electrophoresis are essential for analyzing DNA fragments before and after transformation. These methods help confirm the presence of the desired DNA sequences in plasmids and facilitate the identification of recombinant DNA in transformed host cells.
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