K exam review

  • Recombinant DNA Technologies
      - Recombinant DNA:
        - Definition: A technique that isolates or joins genes from different sources to create composite DNA.
      - Homologous Recombination:
        - Definition: The exchange of genetic information between two DNA molecules. Important for repairing certain mutations by “shuffling” DNA between two separate molecules/chromosomes during reproduction.
      - Restriction Enzymes:
        - Definition: Enzymes that cleave DNA at specific sites, typically palindromic sequences of 4-8 base pairs but can be longer, up to over 20 base pairs.
      - DNA Ligase:
        - Definition: An enzyme that joins DNA strands together to rebuild double-stranded DNA.
      - Plasmids as Vectors:
        - Definition: Plasmids are circular DNA molecules that allow recombinant DNA insertion for replication in host cells. They contain:
          - Origin of Replication (ori): The start point for DNA replication.
          - Antibiotic Resistance Genes: Example: Ampicillin resistance (amp), allowing for selection of modified bacteria.
          - lacZ Gene: A gene used for positive selection (intermediary steps in experimentation/selection).
      - Palindromic Sequence:
        - Definition: A sequence that reads the same on both strands in the 5' to 3' direction. Example provided:
          - 5' GAATTC 3' and 3' CTTAAG 5'.
      - Use of Restriction Enzymes in Recombinant DNA Production:
        - Mechanism: Cutting DNA strands enables the exchange of DNA segments, facilitating the creation of recombinant DNA.
      - Plasmid Insertion Techniques:
        - Transformation: The process of inserting a foreign plasmid into bacteria or yeast.
        - Electroporation: An alternative method that applies an electrical pulse to create temporary holes in cell membranes for plasmid entry.
      - General Applications of Recombinant DNA Technology:
        - Capable of producing various protein products from microbes such as foods, vaccines, antibiotics, vitamins, hormones, and potentially extracting metals from ore.

  • CRISPR Technology
      - Definition of CRISPR:
        - Clustered Regularly Interspaced Short Palindromic Repeats, involving palindromic repeats situated with spacer DNA from previous viral infections.
      - Cas9 Protein:
        - CRISPR associated protein 9. Integral to CRISPR-Cas9 system:
          - Components: The system involves Cas9, crRNA (which encodes guide RNAs), and tracrRNA (which aids in crRNA processing).
      - Functionality of CRISPR-Cas9:
        - Cas9 is a nuclease that cleaves DNA at targeted sites, guided by crRNA.
        - PAMs (Protospacer Adjacent Motifs): Short sequences required for targeted cleavage by Cas9 protein.
          - Guide RNA Composition: Each crRNA contains a 20 nucleotide (nt) guide sequence facilitating base pairing with a target sequence.
          - Chimeric single-guide RNA (sgRNA): Lab-created fusion of crRNA and tracrRNA for directed targeting.
      - Cas9 Functions:
        - Nuclease activity: Cleaves DNA and unwinds double strands. The guide RNA binds to the cleaved DNA, leading to release if mismatched.
      - Repair Mechanisms:
        - Non-homologous End Joining (NHEJ): Direct repair without template, potentially resulting in spliced DNA regions.
        - Homology Directed Repair (HDR): Involves a template strand and polymerase for precise gene transfer.
      - FDA Approved Applications for CRISPR:
        - Treatments for sickle cell disease and transfusion-dependent beta-thalassemia.

  • Pedigrees and Inheritable Genetic Diseases
      - Definition of Pedigree:
        - A family diagram tracking the inheritance of traits or genes.
      - Symbols in Pedigrees:
        - Circle: female
        - Square: male
        - Fully Shaded: trait expressed
        - No Shade: trait absent
        - Half-Shaded: carrier of gene
      - Definitions of Inheritance Patterns:
        - Autosomal Dominant: Only one copy of the gene required to express trait. Dominates over recessive genes.
        - Autosomal Recessive: Requires both gene copies to express the trait.
        - X Linked Dominant: Present on the X chromosome, expressed in both affected males and females.
        - X Linked Recessive: Always expressed in males and expressed in females only if both X chromosomes carry the mutation.
      - Restriction Length Polymorphisms (RFLP):
        - A molecular technique that involves cleaving DNA at specific sites and using electrophoresis to analyze mutation patterns related to inheritable genetic diseases.
      - Variable Number Tandem Repeats (VNTRs):
        - Locations in genomic DNA with short nucleotide sequences organized as tandem repeats.
      - Short Tandem Repeats (STRs):
        - Short (2-7 bp) sequences repetitively occurring in non-coding regions of the genome.
      - Examples of Inherited Diseases:
        - Cystic Fibrosis: Autosomal recessive disorder linked to the CFTR gene, which regulates epithelial fluid transport. Detection through genotyping and sequencing.
        - Duchenne Muscular Dystrophy: X-linked recessive disorder involving the DMD gene, crucial for muscle fiber integrity. Common mutations include deletions and point mutations.
        - Sickle Cell Anemia: Mutation in hemoglobin's beta chain causes the replacement of glutamic acid with valine, encountered on chromosome 11.
        - Huntington’s Disease: Dominant mutation in the huntingtin gene (chromosome 4) characterized by CAG repeat expansion leading to neuronal degeneration.

  • Probe-Based Techniques
      - Definition of a Probe:
        - A nucleic acid sequence/protein that binds specifically to its target in a sample, equipped with detection labels.
        - Possible Labels: Radioactive labels (e.g., Phosphorus-32), fluorescent labels.
      - Southern Blotting:
        - Method for detecting specific DNA sequences.
        - Steps:
          1. Cleavage: Sample DNA fragmented by restriction enzymes.
          2. Electrophoresis: Separates DNA fragments by size and charge.
          3. Denaturing: DNA treated with a denaturing solution (e.g., NaOH) to prepare for transfer.
          4. Capillary Transfer: The membrane is sandwich-layered with the gel for DNA transfer.
          5. Washes and Blocking: Non-specific binding sites filled; high stringency washes are performed to ensure proper probe binding.
          6. Hybridization: Probes added to bind with targets.
          7. Visualization: The membrane is analyzed for hybridized probes.
      - Northern Blotting:
        - Similar to Southern blotting but used to analyze RNA molecules.
      - FISH (Fluorescence In Situ Hybridization):
        - Detects genetic mutations; fluorophore-labeled probes hybridize with cellular DNA viewed under a fluorescent microscope.
        - Processing Blood Samples for FISH: Colcemid halts cell division, enabling observation of cells in metaphase.
      - G-Banding and Karyotyping:
        - G-banding uses stains to visualize chromosomal patterns for abnormalities. Photographs are analyzed to form karyotypes.
      - Aneuploidy:
        - The presence of an abnormal number of chromosomes, e.g., Trisomy 21 (Down syndrome).
      - DNA Microarray Techniques:
        - cDNA Microarray: mRNA is reverse-transcribed into cDNA for gene expression analysis.
        - Comparative Genomic Hybridization: Analyzes DNA sample amplifications, deletions using different fluorophore labels (Cy3, Cy5).
      - Applications of Microarray Technology: Enables genome-wide analysis, distinguishes cancer cells from normal tissue, identifies genetic mutations for targeted therapies.

  • PCR and qPCR
      - qPCR Principles:
        - Real-time PCR allows for fluorescence monitoring during amplification.
        - TaqMan Probes: Utilize a fluorophore and quencher; separation during PCR allows fluorescence detection.
      - Threshold Cycle:
        - Minimum thermal cycles needed for detectable signal levels.