DNA cloning

Summary

DNA 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.