Recording-2024-12-14T15_29_47.888Z

Recognition and Cleavage by Restriction Enzymes

• Restriction enzymes, also known as endonucleases, are proteins that cut DNA at specific sequences called recognition sites.

• These recognition sites are often palindromic sequences, meaning they read the same forward and backward.

Sticky Ends

• When restriction enzymes cut DNA, they can create overhangs or sticky ends where some bases are left unpaired.

  • These overhangs are crucial for the recombination process.

  • Sticky ends allow for the binding of complementary sequences, which is significant in genetic engineering.

• The same enzyme should be used to cut both the vector DNA and the foreign DNA to ensure complementary sticky ends.

Importance of Sticky Ends

• Sticky ends facilitate the ligation process, where the foreign DNA can be inserted into the plasmid or vector DNA.

• DNA ligase is the enzyme that helps in sealing the nicks in DNA after the insertion of the foreign DNA.

• Successful ligation depends on the presence of compatible sticky ends.

Naming of Restriction Enzymes

• There are over 900 restriction enzymes identified, and they are named using a specific nomenclature:

  • The first letter represents the genus of the organism.

  • The next two letters indicate the species.

  • The fourth letter represents the strain.

  • A Roman numeral denotes the order of isolation.

• Example: EcoRI is derived from E. coli, where "Eco" stands for the genus and species, and "R" indicates the strain and "I" shows it was the first enzyme discovered in that bacteria.

Gel Electrophoresis

• Gel electrophoresis is a technique used to separate DNA fragments based on their size:

  • An electric field is applied to the gel matrix, causing DNA fragments to migrate toward the anode.

  • Smaller fragments move faster and farther than larger ones.

• Ethidium bromide dye is commonly used to visualize DNA, as it fluoresces under UV light after binding to the DNA.

• After separation, specific DNA fragments can be cut and retrieved from the gel for further analysis or use.

Genetic Engineering Process

1. Isolation of DNA: DNA is extracted from an organism that contains the gene of interest.

2. Digestion with Restriction Enzymes: Both the vector and the foreign DNA are cut with the same restriction enzyme to ensure compatibility.

3. Ligation: The sticky ends of the vector and foreign DNA facilitate their binding, aided by DNA ligase.

4. Transformation: The recombinant DNA is introduced into a competent host cell that can take up the DNA for replication.

Selectable Markers

• Selectable markers allow for the identification of successfully transformed host cells:

  • Antibiotic resistance genes are commonly used, permitting only those cells that have taken up the recombinant DNA to survive.

• Other types of selectable markers may include genes that produce color changes in colonies to indicate successful transformation.

Cloning Vectors

• Different types of vectors are used for cloning in various organisms:

  • Plasmids: Small circular DNA molecules used primarily in bacteria.

  • Bacteriophages: Viruses that infect bacteria can be used as vectors as well.

  • Ti Plasmids: Used in plants, derived from Agrobacterium tumefaciens, which induce tumors in plants.

  • Retroviruses: Can be used for cloning in animal cells, typically modified to remove harmful elements.

Competent Host Cells

• A competent host is a bacterial cell that can take up foreign DNA:

  • Methods to induce competence include chemical treatment or electroporation.

• Competent cells must be capable of allowing the vector to enter and replicate the foreign DNA.

Summary of Steps in Recombinant DNA Technology

1. DNA Isolation: Obtain pure DNA containing the gene of interest.

2. Restriction Enzyme Digestion: Cut both target and vector DNA to create compatible ends.

3. Ligation: Merge the DNA segments.

4. Transformation: Introduce the recombinant DNA into a host cell.

5. Selection: Use selective markers to identify successful transformants.