Advanced DNA Technology, Genetic Modification, and Genomics Study Notes

Exam Logistics and Oral Examination Format

  • Exam Structure and Scoring:

    • The exam consists of exactly five questions.

    • Each question is worth 2020 points.

    • The professor estimates that 1010 minutes is more than enough time for students to complete these five questions.

    • The exam variety includes some problem-solving (e.g., Punnett squares, which may take time for some students) and some fact-based questions.

    • Fact-based questions offer less opportunity for partial credit unless they incorporate subsections (e.g., defining an autonomous transposon and identifying two associated enzymes).

    • Genetic problem-solving questions usually allow for more partial credit.

  • Oral Exam Procedure and Tools:

    • The exam is conducted orally with a camera required to ensure the student is not looking at notes or typing answers.

    • Questions will be displayed on the screen via PowerPoint; they will not be read aloud by the professor as many students prefer reading themselves.

    • Students are permitted to write down their thoughts or work out problems on paper or a tablet first.

    • Once ready, the student verbally provides the answer to the professor.

    • Students do not have to answer questions in order (e.g., answering question five first is acceptable).

Genetic Modification and Gene Targeting

  • Definition of Genetic Modification:

    • Moving beyond characterization to modify a gene's function, remove it entirely, or overexpress it.

    • Gene targeting specifically refers to targeting an endogenous gene within the chromosome of a living cell or organism, rather than modifying DNA in a test tube (cloning).

  • Homologous Recombination (HR):

    • A mechanism derived from meiosis (prophase I) where similar maternal and paternal chromosomal sequences cross over.

    • Scientists use this natural mechanism to manipulate chromosomes by introducing very similar DNA.

    • The Process:

    • Targeting Vector: A DNA construct designed to exchange an endogenous gene (e.g., Lamin A) with a mutated or deleted version.

    • Homology Arms: The targeting vector must contain sequences homologous to the target area in the endogenous chromosome to induce recombination.

    • Lamin A: A cytoskeletal element that forms a scaffold for the nuclear envelope; used as an example of a gene that can be targeted in a mouse model to study functional loss.

  • Selection and Efficacy in Mouse Embryonic Stem Cells (ESCs):

    • HR is inefficient, with success rates as low as 11 in 10001000.

    • Massive numbers of ESCs (hundreds of thousands) are grown in petri dishes to overcome this low efficacy.

    • Selectable Markers:

    • Neomycin Resistance: Used as a positive selectable marker. Neomycin is added to the medium; only cells that integrated the targeting vector survive.

    • Thymidine Kinase (TK): Used as a negative selectable marker. If HR occurs correctly, the TK enzyme is not integrated. If the vector integrates randomly (non-homologous), TK is present and can be detected. Researchers discard cells with TK activity.

    • Blastocyst Injection: Targeted ESCs are injected into mouse blastocysts (the stage of embryogenesis consisting of stem cells). These cells then contribute to the development of the entire mouse.

    • Model Organisms: Mice were the primary vertebrate model for this because their ESCs were uniquely easy to culture compared to other species.

    • Noggin Gene Example: Identified initially in frogs, Noggin is involved in brain formation. Removing it via gene targeting in mice results in skeletons lacking major brain parts and showing limb defects.

CRISPR/Cas9 Technology

  • GEAN (Genome Editing with Engineered Nucleases):

    • CRISPR Cas9 acts as a specific nuclease targeting one exact gene out of the entire genome, unlike restriction enzymes which cut at every occurrence of a consensus sequence.

    • Derived from the bacterial immune system used to target foreign DNA.

    • Components:

    • Cas9: The enzyme that perform the double-stranded DNA cut.

    • Guide RNA (gRNA): A 20-base pair sequence engineered to be specific to the gene of interest (e.g., Lamin A).

  • Mechanisms of Repair:

    • Non-Homologous End Joining (NHEJ):

    • The cell "chews back" the DNA ends to create blunt ends and ligates them together.

    • Usually results in random deletions or mutations of variable length.

    • Homology Directed Repair (HDR):

    • A donor template is provided along with CRISPR/Cas9.

    • The cell uses the donor template to repair the cut through strand invasion and homologous recombination.

    • Allows for precise insertion of specific mutations, such as changing a single amino acid associated with a human disease.

  • Historical Context: Emmanuelle Charpentier and Jennifer Doudna won the 2020 Nobel Prize for characterizing this system. Jennifer Doudna’s life and discovery are chronicled in the book The Code Breaker.

Transgenic Organisms

  • Definition: Living cells or organisms where a DNA construct is incorporated into the genome through random integration.

  • Contrast with Gene Targeting: Unlike gene targeting, the endogenous gene remains intact and untouched. The transgene integrates into an unknown, random location.

  • Visualizing Gene Expression:

    • Promoter Fusions: Combining the promoter/regulatory sequence of a gene with a reporter gene like GFP (Green Fluorescent Protein).

    • Transgenic Zebrafish Example: Using a heart-specific promoter to drive GFP, allowing researchers to see the heart beating and pumping in a living organism. This helps analyze the effects of drugs on organ development (e.g., identifying split heart defects).

    • LacZ/Beta-galactosidase: Another reporter gene used to look at developing nervous systems in sections of tissue.

RNA Interference (RNAi)

  • Mechanism: Builds on endogenous cellular machinery for microRNAs (miRNAs).

  • Process: Single-stranded RNA is designed to bind to a specific target mRNA sequence. This either blocks translation or decreases the stability of the mRNA, causing it to break down.

  • Use: This is referred to as "knocking down" gene expression rather than "knocking out" the gene itself.

Techniques to Study Gene Expression

  • In Situ Hybridization (ISH):

    • Used to identify which cells in a tissue are transcribing a specific gene.

    • Probe Design: A nucleic acid probe is designed using base-pairing rules to be complementary to the target mRNA.

    • Sense vs. Antisense: If the mRNA sequence is 5′-UCA-3’5' \text{-UCA-3'}, the antisense probe must be 3′-AGU-5’3' \text{-AGU-5'} (written as 5′-UGA-3’5' \text{-UGA-3'}) to bind properly.

    • BlueScript Plasmid: Contains binding sites for T7 and T3 RNA polymerases, allowing for the commercial production of RNA probes.

    • Chemical Labeling: Probes often incorporate digoxigenin (DIG). An antibody against DIG, which produces a purple color when a substrate is added, is used to visualize the target RNA.

  • Genetic Labeling (GFP Fusions):

    • Promoter-GFP Fusion: The promoter of a gene drives GFP expression. This shows where the gene is transcribed. GFP typically stays in the cytoplasm (e.g., making an entire eye or heart glow green).

    • Protein-GFP Fusion: The GFP coding sequence is fused directly to the gene's coding sequence. This creates a fusion protein. Scientists can track the movement of the protein within a living cell (e.g., traveling from the cytoplasm to the nucleus).

  • Immunocytochemistry (ICC):

    • Focuses on the protein product in preserved (not living) tissue.

    • Uses a primary antibody to bind the target protein and a secondary antibody with a fluorescent tag for visualization.

  • Western Blotting:

    • An electrophoresis approach for proteins.

    • A "cellular smoothie" (protein extract) is run on a gel matrix and transferred to a membrane. Antibodies are then used to detect the presence and size of specific proteins in different tissues.

Genomics and Genome Evolution

  • History and Databases:

    • Competition between the Sanger Center (UK) and the NIH (USA) to sequence the human genome.

    • The Sanger Center initially wanted to patent sequences; the NIH pushed for public, free access, which is why sites like NCBI (National Center for Biotechnology Information) are free today.

    • The first human genome cost approximately 300,000,000300,000,000 to produce using the dideoxy method.

    • Modern Next Generation Sequencing can sequence 500,000,000500,000,000 bases per day.

  • Genome Structure Facts:

    • Approximately half (50%50\%) of the human genome consists of transposable elements.

    • Prokaryotes: Circular chromosomes, no introns, repetitive sequences, and polycistronic messages (one promoter for multiple genes).

    • Eukaryotes: Diploid (two sets of linear chromosomes), organelle DNA (circular, maternal inheritance), and extensive non-coding DNA. Yeast is an exception as it can be haploid.

    • Viruses: Non-living entities with diverse genomes: double or single-stranded DNA or RNA.

Genome Annotation and Open Reading Frames (ORFs)

  • Annotation: The process of superimposing biological information onto DNA sequences (mapping genes, identifying promoters, finding homologs).

  • ORF Identification Challenges:

    • High amounts of non-coding DNA and large introns make finding coding genes difficult.

    • In DNA, introns interrupt codons, meaning an ORF might not be a single continuous stretch of triplets.

    • There are six possible reading frames for any stretch of DNA.

  • Tools for Identification:

    • cDNA Alignment: Comparing genomic DNA to cDNA (reverse-transcribed mRNA) is the most effective way to find exons.

    • ESTs (Expressed Sequence Tags): Randomly sequenced cDNA fragments used for mapping.

    • CpG Islands: Regions of at least 200200 base pairs with high GC content, often found near promoters; used to detect methylation and epigenetic control.

Applications of Genomics

  • Functional Genomics: Establishing what genes do through experiments involving removing or overexpressing functions.

  • Clinical Genomics: Mapping mutations and alleles associated with specific diseases.

  • Forensic Genomics: Focusing on highly polymorphic non-coding DNA to distinguish individuals.

  • Evolutionary Genomics: Comparing conserved genes (like the cytochrome family) to study relationships across all organisms from bacteria to humans.

    • Example: Genomic rearrangements explain why humans see more wavelengths of light compared to dichromatic primates.

Questions & Discussion

  • Question (Gerda): Will the PowerPoint with questions be visible during the oral exam?

  • Answer: Yes, the professor will display the questions so students can read them. No reading aloud is required by the professor.

  • Question (Gerda): Can I answer questions out of order?

  • Answer: Yes, students can answer in any order (e.g., question 5 before question 1).

  • Question (Boris): Is there a submission link for the practice problem regarding the zebrafish GFP labels?

  • Answer: The professor confirmed the link is open on Brightspace; students may need to refresh their browsers to see it.

  • Practice Problem Answer: The zebrafish embryo showing general transcription in endothelial cells used a promoter-GFP fusion. The video showing protein movement between the cytoplasm and nucleus utilized a protein-GFP fusion.