Genetic Engineering Overview

Genetic Engineering

• Genetic engineering is the artificial manipulation of an organism's nucleic acid content, differing from genetic recombination, which is a natural process.
• Recombinant DNA (rDNA) is artificially created by combining DNA sequences from different sources.
• Genetically modified organisms (GMOs) have had their genes artificially altered.

Genetic Engineering Steps

• Cleave vector with restriction enzyme: A restriction enzyme cuts a plasmid vector at specific restriction sites.
• Cleave foreign DNA: A restriction enzyme cuts foreign DNA at matching restriction sites.
• Mix vector and DNA fragment: The vector and DNA fragment are mixed to encourage base pairing between complementary sequences.
• Treat with DNA ligase: DNA ligase covalently joins the DNA pieces.
• Recombinant vector: The resulting recombinant vector carries the gene of interest.

Recombinant DNA Technology Applications

• Studying gene arrangement, expression, and regulation.
• Modifying gene expression to enhance or suppress a specific product.
• Creating multiple copies of a nucleic acid segment.
• Creating organisms with altered characteristics (transgenic organisms).
• Using transgenic organisms as cell factories for commercial purposes.

Enzymes Used in Cloning-Restriction Endonucleases

• Restriction enzymes (REs) are endonucleases that cut DNA at specific recognition sites.
• Restriction sites are short, specific nucleotide sequences.
• REs are bacterial in origin.
• Three major classes of REs exist, based on their recognition sequences, cut type, and enzyme structure.
• Type I and III are not commonly used in cloning due to non-specific cutting sites.
• Type II enzymes are widely used for cloning because they cut precisely at recognition sites.

Restriction Endonuclease Nomenclature

• REs are named based on the organism they are isolated from (e.g., HindIII, EcoRI, BamHI).

Recognition Sequences for Type II Restriction Endonucleases

• These enzymes recognize specific palindromic sequences.
• Some create "sticky ends"—single-stranded overhangs that form hydrogen bonds with complementary sequences.
• Others create "blunt ends," producing even cuts in the DNA.

Ligases

• DNA ligases form covalent bonds between DNA fragments' 5' and 3' ends.
• T4 DNA ligase is commonly used to connect DNA with sticky or blunt ends.
• Ligation is the joining of DNA fragments using ligase.

Terminal Deoxynucleotidyl Transferase

• This enzyme adds deoxynucleotides to the 3' ends of DNA molecules.

Phosphatases

• Removal of 5' phosphate from DNA molecules, preventing self-ligation of the vector.
• Calf intestinal phosphatase is used.

Large Fragment of DNA Polymerase I (Klenow Fragment)

• Cleavage of intact DNA polymerase I produces this 76-kDa enzyme.
• This enzyme possesses 5' to 3' polymerase and 3' to 5' exonuclease activity.
• Used in DNA sequencing, filling recessed 3' termini, labeling DNA fragments.