8.4.2 Producing Fragments of DNA

Using Restriction Endonucleases to Cut DNA Fragments

• Restriction endonucleases are enzymes from bacteria that cut DNA at specific recognition sequences (often 4-6 base pairs long).
• These enzymes are highly specific, making them invaluable for precise DNA manipulation.

Types of Cuts:

1. Sticky Ends:

• Some restriction endonucleases make staggered cuts, leaving single-stranded overhangs.
• These overhangs are called sticky ends because they can easily bond with complementary sequences on other DNA fragments.
• Sticky ends enable easier recombination.

2. Blunt Ends:

• Other restriction enzymes cut straight across the DNA, leaving blunt ends with no overhangs.
• Blunt ends are less efficient for recombination as they lack complementary overhangs.

Why Sticky Ends Are Useful

• When DNA fragments are cut with the same restriction endonuclease, the sticky ends of both fragments will be complementary.
• This means the fragments can base-pair with each other.
• DNA ligase is then used to form strong covalent phosphodiester bonds between the fragments, creating a stable recombinant DNA molecule.

Example Applications

• Cutting plasmids and target DNA with the same restriction enzyme ensures compatibility for gene cloning.
• Sticky ends are critical for inserting genes into vectors like plasmids or viruses.