Molecular biology & immunology Flashcards
How does DNA and RNA composition differ between cell lineages
DNA is generally the same (assuming diploidy). mRNA patterns vary wildly.
Why is RNA less stable than DNA for testing?
RNAses are ubiquitous in cells and the environment and RNA has the ability to autodegrade.
What plasma proteins can inhibit PCR?
Heparin, hemoglobin, lactoferrin
How can amplicon contaminants be minimized in a PCR lab?
Use one-directional flow of lab layout. Use uracil and uracil glycosylases. Run water (negative) controls.
What are TMA and NASBA?
ISOTHERMAL methods of DNA amplification that use reverse transcriptase and RNA. In TMA the RNA is degraded by the reverse transcriptase, while in NASBA it is degraded by RNAse H.
How is the generation of amplicons detected in PCR?
Usually by fluorophores including those in Taqman assay, FRET, or SYBR (which only binds dsDNA)
What are the advantages and disadvantages of DNA arrays?
Quick and specific detection of sequences, but non-sensitize (only catches predefined polymorphisms)
What are the prozone and postzone effects?
Prozone: Excessive antibody overly coats antigens without agglutination.
Postzone: Excessive antigen neutralizes antibodies, reducing agglutination reactions.
What are some different ELISA assays?
Indirect: Well coated with antigen, add sample, then AHG
Sandwich: For soluble Ag detection, add capture Ab and AHG.
Competitive: Add test Ab/Ag and measure decrement of reagent Ab/Ag reaction.
How does suspension array tech work?
MIcrospheres are coated with capture antibody or antigen, washed with patient sample and secondary fluorophore, then read by flow cytometry.
Describe the structure of an antibody
Tetramers of 2 heavy and 2 light (kappa OR lambda, not both), with Fab regions (contributed to by light and heavy chains) and Fc regions (heavy chain only).
What is the difference between affinity and avidity?
Affinity refers to a specific antigen-antibody binding interaction, avidity refers to the whole antibody’s ability to bind. (see; IgM vs IgG)
Describe the structure of each isotype of antibody.
IgM: Pentamer held together with a J chain and disulfide bridges.
IgA: Dimer also held together by J chain.
IgG, IgE, IgD: All monomeric.
Compare and contrast the four subclasses of IgG
IgG1: Potently binds complement and macrophage FcR
IgG2: Weak complement binding, no FcR binding.
IgG3: Potently binds complement and macrophage FcR
IgG4: No complement binding, some FcR binding.
How is the classical complement pathway activated?
By C1 affixed to IgM or IgG which attracts C3. Results in production of C3a and C3b (opson via CR1, CRIg), and terminates in MAC lysis.
