1. Assays Molecular Diagnostics CSULA

2. What’s a molecular diagnostic assay? n A laboratory test for the presence or absence of a particular type of molecule u Qualitative -- is it there or not? u Quantitative -- how much is present?

3. Why perform an assay? n To diagnose an illness n To determine tissue compatibility n To identify an individual n To determine susceptibility or likelihood of drug responsiveness n To test a hypothesis of interest to you u “Experiment” is pretty much another word for “assay”

4. Essential components of a good assay n Sample - what you’re going to do the test on n Detection scheme with specificity - u To find what you’re looking for u To discriminate it from everything else n Visualization system - to see the results n Sensitivity - to minimize the amount of sample required n Controls - to aid in interpretation

5. Samples - Examples n Human cells n Simulated human blood plasma n Chicken red blood cell DNA n Patient tumor cell DNA n Patient tumor cell proteins n Sea water n Soil

6. Detection Scheme n Probe - a molecule that u Recognizes what you are trying to detect u Provides specificity by F base pairing of nucleic acid sequence to its complement F binding of antibody to its specific target n Standards of comparison

7. Vizualization - making a visible signal n Color reaction u naked eye, spectrophotometry n Light emission u film, luminometer, phosphorimager n Fluorescence emission u naked eye, fluorescence scanning instrumentation n Staining u naked eye n Radioactive emission u film, phosphorimager, counting instrument

8. Sensitivity - minimizing the sample needed n Maximize the amount of signal u Amplify the starting sample u Amplify the signal produced n Minimize the noise (background) n Bottom line - high signal to noise ratio

9. Controls n For technical success u Did the procedure work correctly? n For interpretation of results u Is the positive real or is it a false positive? u Is the negative real or is it a false negative?

10. Always ask n Does my assay have all the essential components?