DNA cloning

Shomu's Biology
26 Dec 201204:27

Summary

TLDRDNA cloning involves extracting a specific DNA sequence and inserting it into a vector, such as a plasmid, for replication in a host cell. The process uses restriction enzymes like Eco R1 to create compatible ends, which are then ligated to form a recombinant plasmid. This plasmid, containing the foreign DNA, is introduced into E. coli bacteria via transformation. Only cells with the plasmid can grow on ampicillin-containing agar, leading to the formation of colonies. These colonies are then screened to identify the desired DNA sequence, showcasing the amplification and isolation of specific genetic material.

Takeaways

  • 🔬 DNA cloning is a technique used to isolate a specific DNA sequence from a mixture of different DNA sequences.
  • 🧬 A vector, typically a modified phage or plasmid, is necessary to replicate the foreign DNA fragment in a host cell.
  • 🔄 The vector contains a cloning site for the DNA insert, a drug resistance gene for selection, and a replication origin.
  • ✂️ Restriction enzymes like Eco R1 are used to cleave the vector at the cloning site, creating sticky ends.
  • 🔌 Sticky ends of the vector and foreign DNA hybridize and are sealed by DNA ligase to form a recombinant plasmid.
  • 🌿 E. coli bacteria are used as the host cells, made permeable to DNA through calcium chloride treatment for transformation.
  • 💊 Ampicillin resistance is used to select for bacteria that have taken up the recombinant plasmid, allowing them to grow on agar plates.
  • 📈 The replication origin on the plasmid allows for its replication within the host cell, independent of cell division.
  • 🌐 As host cells divide, the plasmids are distributed to daughter cells, amplifying the number of recombinant plasmids.
  • 🔎 Assay methods can be applied to bacterial colonies to identify the specific DNA sequence of interest.

Q & A

  • What is DNA cloning?

    -DNA cloning is a method used to isolate a particular sequence of DNA from a complex mixture of different DNA sequences.

  • What is a vector in the context of DNA cloning?

    -A vector is a highly modified phage or plasmid that can replicate in a host cell and is used to insert foreign DNA.

  • What are the three essential elements of a plasmid vector?

    -A plasmid vector typically contains a cloning site, a drug resistance gene, and a replication origin.

  • Why is a drug resistance gene important in a plasmid vector?

    -The drug resistance gene allows for selective growth of the host cell by destroying antibiotics, such as ampicillin, which helps in identifying successful transformations.

  • How does the restriction enzyme Eco R1 cleave DNA?

    -Eco R1 cleaves the palindromic sequence GAATTC to produce single-stranded ends known as sticky ends.

  • What is the purpose of sticky ends in DNA cloning?

    -Sticky ends can hybridize with any piece of DNA that has also been cut with Eco R1, facilitating the ligation of foreign DNA to the vector.

  • What enzyme is used to seal the hybridized sticky ends in DNA cloning?

    -DNA ligase is used to seal the hybridized sticky ends, creating phosphodiester linkages and forming a recombinant plasmid.

  • How is a library of recombinant plasmids created?

    -A library of recombinant plasmids is created by inserting various foreign DNA fragments into a pool of cleaved vectors, each carrying a unique fragment.

  • What is transformation in the context of bacterial cells?

    -Transformation is a process where bacterial cells, treated with calcium chloride to increase permeability, take up recombinant plasmids.

  • How are cells with recombinant plasmids selected on an agar plate?

    -Cells with recombinant plasmids are selected on an agar plate containing the antibiotic ampicillin, as only cells with the ampicillin resistance gene can grow.

  • Why are the replicated plasmids and host cells referred to as a colony or clone?

    -The replicated plasmids and host cells are referred to as a colony or clone because all cells in a colony are derived from a single cell and contain copies of the same recombinant plasmid.

  • What is the final step in isolating a specific DNA sequence using cloning?

    -The final step is to use various assay methods on the bacterial colonies to determine which contains the particular DNA sequence of interest.

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関連タグ
DNA CloningMolecular BiologyGenetic EngineeringRecombinant DNAEcoR1 EnzymePlasmid VectorAmpicillin ResistanceBacterial TransformationSticky EndsScientific Research
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