Molecular Tools: Restriction Enzymes, Ligases, and Cloning
Building Recombinant DNA
Core toolkit
Classic cloning relies on cutting DNA, joining fragments, and propagating the construct in a host cell. Restriction enzymes recognize specific sequences, DNA ligase seals phosphodiester bonds, and plasmids serve as common vectors.
Tool summary
| Tool | Function |
|---|---|
| Restriction enzyme | Cuts DNA at or near specific recognition sites |
| DNA ligase | Joins DNA fragments by forming covalent bonds |
| Plasmid | Small circular DNA vector commonly used in bacteria |
| Competent cell | Cell prepared to take up DNA |
What is the role of DNA ligase?
Ligase seals breaks in the sugar-phosphate backbone, joining fragments.
Correct answer: To join DNA fragments
Why are plasmids useful as vectors?
Plasmids are easy to manipulate and maintain, making them convenient delivery vehicles.
Correct answer: They can replicate in cells and carry inserted DNA
Restriction enzymes are valued because they:
Their sequence specificity enables predictable DNA assembly.
Correct answer: Recognize specific DNA sequences
Sticky vs blunt ends
Some restriction enzymes create overhangs that can base-pair with complementary ends, while others create blunt ends. Sticky ends often make assembly easier.
Typical cloning workflow
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1
Step 1: Choose an insert and a vector.
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2
Step 2: Cut both DNAs with compatible enzymes.
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3
Step 3: Ligate insert into vector.
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4
Step 4: Transform cells with the recombinant plasmid.
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5
Step 5: Screen colonies for the correct construct.
What does 'competent cell' mean in cloning?
Competent cells are treated so they can efficiently receive plasmid DNA.
Correct answer: A cell prepared to take up foreign DNA