Genetic Engineering - Class 3

Genetic Engineering - Class 3

• Outline for today: Quiz, Advanced Organizer, Basic methods and tools (Extraction of nucleic acids, Enzymes working on nucleic acids, Polymerase chain reaction (PCR)).

Advanced Organizer

• Focus Areas: Transgenic plants and animals (golden rice, GM salmon), Medicine (CAR T-cell therapy, inherited defects, CRISPR/Cas in gene therapy, replacement gene therapy), Cloning strategies (cloning vectors and plasmids, getting DNA into cells, DNA fragments into vectors, isolation of nucleic acids, PCR, host cells, selection and screening, synthesis of cDNA, separation of nucleic acids, amplification and quantification, enzymatic modifications), Bioinformatics, Databases, Alignment, Genome editing, RNA interference, Next-generation sequencing.

Basic Methods and Tools - Nucleic Acid Extraction

• General Steps: Cell disruption/lysis, Separation/purification, Concentration/precipitation.
• Cell Disruption/Lysis: Lysis buffer and/or heat are used to destroy cell structures containing genetic material. Common components include SDS (detergent), enzymes (e.g., Proteinase K), a chelating agent (EDTA), or Tris buffer.
• Separation/Purification: Lipids and proteins are eliminated from the cell lysate.
• Concentration/Precipitation: Impurities are further removed using methods like alcohol precipitation, to concentrate nucleic acids.
• Methods: Lysozyme (digests peptidoglycan in bacterial cell walls), Detergents (e.g., sodium dodecyl sulfate (SDS) to burst cell membranes), Physical force (grounding for tissue samples), Proteinase K (degrades tissue).
• Cultured Cells: Detergents alone can release intracellular components in cells without cell walls.

Basic Methods and Tools - Nucleic Acid Extraction (Continued)

• Phenol-Chloroform Extraction: Used to separate proteins from DNA. DNA and protein are mixed with phenol, the protein dissolves in the phenol layer, and nucleic acids go into the aqueous layer. The phases are separated by centrifugation, and the aqueous DNA layer is removed.
• Removal of RNA: RNA is also nucleic acid, and is not soluble in phenol. Ribonuclease, which digests RNA into ribonucleotides, is sometimes used. Then, ethanol precipitation is commonly used to further purify the DNA. The precipitated nucleic acid is isolated by centrifugation.
• Column Methods (Silica or Beads Based): Different commercial kits are available. These often involve a spin column, with stages of binding, washing, and elution steps to purify the nucleic acid.

Nucleic Acid Extraction - Guanidinium Thiocyanate Method

• Method Summary: Homogenization/lysis, Phase separation, Extraction/precipitation, and Resuspension.
• Principle: Guanidium thiocyanate is a chaotropic agent that disrupts cells, denatures proteins, and deactivates nucleases, stabilizing DNA and RNA during the extraction.
• Separation of Protein from Nucleic Acids: The extract is treated with phenol/chloroform at a low pH; RNA is precipitated using isopropanol, and often commercially available as TRIzol, TriFast, or TRI Reagent.

Enzymes Working on Nucleic Acids

• Restriction Enzymes: Four types; Type I cut away from recognition sites, Type II are commonly used, recognizing inverted palindromes, Type III cut 20 base pairs from the recognition site, and Type IV cleave modified (methylated) sites.
• Type II Restriction Enzymes: Most frequently used in genetic engineering. They cut DNA at specific sequences, often creating overhangs (sticky ends). Some common enzymes and their properties are included in a table.
• Isochizomers and Neoschizomers: Isochizomers recognize the same sequence and cut in the same way, while Neoschizomers recognize the same sequence but cut in a different way.

Polymerase Chain Reaction (PCR)

• Overview: PCR is used to make millions of copies of a specific DNA segment (usually 200-1000 base pairs).
• Components: Two primers, a thermostable DNA polymerase (e.g., Taq polymerase), DNA template, dNTPs, and buffer.
• Cycles: PCR involves repeated cycles of denaturation, annealing, and elongation, increasing the amount of the DNA target.
• Thermocycler: PCR reactions occur in a thermocycler, which is a machine that automates temperature changes for these cycles.
• Quantitative PCR: (qPCR) Monitors the PCR reaction while it's happening and measures the amount of target DNA present. Two common methods are the SYBR Green and Taqman methods.
• Digital PCR: Divides the reaction into tiny compartments. This method measures the presence of DNA target or not.

Gel Electrophoresis

• Purpose: Separates DNA fragments by size.
• Method: DNA fragments are loaded into wells in a gel matrix, and an electric field is applied. DNA migrates to the positive electrode; smaller fragments travel further than larger fragments.
• Visualization: UV light is used to visualize the DNA fragments. The pattern of bands represent fragments of a certain size.

Quantification of Nucleic Acids

• Spectrophotometry: Used to measure the absorbance of DNA and RNA solutions at 260 nm.
• Absorbance Ratios: A ratio of absorbance readings at different wavelengths (e.g., 260 nm/280 nm for DNA and RNA purity). A ratio of 1.8 is acceptable for DNA, and 2.0 for RNA. Lower ratios indicate impurities (e.g. protein).