Flashcards in Exam #5: Molecular Medicine I Deck (47)
How are antibodies used in western blotting?
Technique that uses specific antibodies to indicate the molecular weight of a protein
-Tells if the protein is present or not
- Used to quantify the amount present
- Shift in banding can identify a modification of the protein in question
How are antibodies used to treat cancer?
- Herceptin was the first monoclonal antibody approved for treatment of metastatic breast cancer
-inhibits ErbB2/ HER2/Neu receptor tyrosine kinase located on cell surface
What will DNA sequencing show & not show?
- Will show if the person is homozygous or heterozygous
- Will NOT show which chromosome a mutation is on
- Will NOT show a deletion or duplication
Comparative Genome Hybridization (CGH)
1) Obtain ss-DNA from patient & control
2) Label both with different dye
3) Mix & hybridize to metaphase chromosomes
4) Uneven labeling (color) indicates an insertion or deletion (variation in copy number)
What are the steps in PCR?
1) Denature DNA (want ss-DNA)
2) Synthesize oligonucleotides that are complimentary to the ends of the DNA fragment to be studied (primers)
3) Primers anneal to ss-DNA
4) Polymerase synthesizes complimentary strand
Allows for quantification of amounts of nucleic acids
- Measures amplified DNA after each cycle
- Determined how many cycles are required to pass a threshold
-Template amount can be quantified by the number of cycles required to reach threshold
- More cycles= less template & vice versa
Used for RNA analysis
- Reverse transcription of RNA to DNA by reverse transcriptase
- Makes cDNA
- cDNA serves as the template for the PCR reaction
Warfarin example of pharmacogenetics
- Oral anticoagulant that inhibits vitamin K epoxide reductase
- polymorphism in p450 (CYP2C9) causes slow metabolism & higher sensitivity
- polymorphism in vitamin K epoxide reductase (VKORC1) that leads to warfarin tolerance
What cells produce antibodies?
What is a sandwich ELISA routinely used for?
The detection of antigen or peptide in human serum
1) Parathyroid Hormone
What is an indirect ELISA routinely used for?
Diagnosis of autoimmune disease
- Also, HIV
What are the steps in a western blot?
1) Separate protein extracts by electrophoresis
2) Transfer proteins to membrane
3) Add enzyme-linked specific antibody
4) Antibodies bind antigen (proteins)
5) Detect label in band pattern
Insertion of a functional copy of a gene into mutant cells
What is the biggest obstacle to gene therapy?
What viruses are used as vectors in gene therapy?
3) Adeno-associated virus (No known disease)
What are the conflicting properties of a viral vector?
- Virus should spread aggressively through the body; however, if TOO AGGRESSIVE, can trigger sepsis
- Integration into the host genome, which can lead to cancer (leukemia in clinical trials)