Restriction Endonucleases in Molecular Biology

Characteristics of Restriction Enzymes

• Most restriction enzymes recognize a single restriction site.
• The restriction site is recognized without regard to the source of DNA.

DNA Ligase

• DNA ligase is an enzyme that covalently links adjacent 3'-oH and 5'-po4 groups.
• The joining of DNA fragments requires energy, which in humans is provided by ATP.

Applications of Gene Cloning

• Medical applications: gene cloning plays a crucial role in synthesizing vitamins, hormones, and antibiotics.
• Medical applications: produces recombinant human insulin (Humulin) in E. coli for diabetic patients.
• Agriculture applications: cloning is used to create pest-resistant plants.
• Gene therapy: involves replacing defective genes with new healthy ones, e.g., treating leukemia and sickle cell anemia.

Steps in Gene Cloning

• Isolation of DNA fragments to be cloned.
• Insertion of isolated DNA into a suitable vector to form recombinant DNA.
• Introduction of recombinant DNA into a suitable host organism.
• Selection of transformed host cells and identification of the clone containing the gene of interest.
• Multiplication/expression of the introduced gene in the host.
• Isolation of multiple gene copies/protein expressed by the gene.
• Purification of the isolated gene copy/protein.

Enzymes Used in Gene Technology

• Restriction endonuclease enzyme (cut): cuts DNA at specific sites forming sticky ends.
• DNA ligase (paste): joins the gene of interest with the vector.

Types of Restriction Endonucleases

• Type 1: cleaves DNA at random sites, often >1000 bp from the recognition sequence.
• Type 2: cleaves DNA within the recognition sequence itself and requires no ATP.
• Type 3: cleaves DNA about 25 bp from the recognition sequence and requires ATP.

Characteristics of Cloning Vectors

• Self-replicating inside host cells.
• Possess a unique restriction site for RE enzymes.
• Introduction of donor DNA fragment must not interfere with replication property of the vector.
• Possess a marker gene for later identification of recombinant cells (usually an antibiotic resistance gene).
• Easily isolated from host cells.

Naming Restriction Enzymes

• Most restriction enzymes are named after the species in which they were found.
• The first three letters in the name of each restriction enzyme stand for the bacterial species of origin.

This quiz covers the characteristics of type I, II, and III restriction endonucleases, including their mechanisms, energy requirements, and recognition sequences. Understand the differences between these types and how they cleave DNA.