Assay
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A quick PPT about assays for Chempetitive Group internal PR meeting. DRAFT
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Assay
- 1. Part 1: You Say, Assay Part 2: PCR- Poly What? Company Confidential
- 3. Company Confidential Did you say “essay?” Definition: Analysis (n), analyze (v)
- 4. Company Confidential More precision please. • Determine presence of sample • E.g. does the plasmid contain my gene? • Determine quantity of sample • E.g. how much protein do I have? • Determine activity of drug or biochemical in a sample • E.g. what’s the activity of my protein?
- 5. Company Confidential Assays @ Work: Protein Purification Protocol • Transfect bacteria with plasmid containing your DNA, plate on to agar • Determine optical density of cells • Pick bacteria colony from agar plate expressing your protein • Confirm bacteria contains your sequence • Grow bacteria expressing your protein in broth overnight • Determine optical density of cells • Centrifuge cells, remove broth, add buffer • Break open cells • Determine protein concentration • Determine protein activity • Determine protein purity • Fractionate protein with a salt • Determine protein concentration • Determine protein activity • Determine protein purity • Chromatography steps to enrich your protein • Determine protein concentration • Determine protein activity • Determine protein purity • Confirm protein identity
- 6. Company Confidential Wide world of assays What assay am I?
- 7. Company Confidential Does it work? • Accuracy - does it give the right answer? • Precision - does it give a consistent answer? • Reliability – does it work every time? • Sensitivity -how little analyte can you detect? • Dynamic range – can you measure your analyte over a wide concentration range? • Robustness – will the assay work under non-optimal conditions?
- 8. Company Confidential Practically speaking…. • Speed (“time-to-result”) • Throughput • Automation • Ease-of-use • Workflow • Cost • Size (“footprint”)
- 9. Company Confidential Last word: Does it smell?
- 11. PCR- Poly What? Company Confidential
- 12. Company Confidential Polymerase Chain Reaction • In short: it is a method used to analyze a short sequence of DNA or RNA • PCR is used to reproduce (amplify) selected sections of DNA or RNA • The basics of what’s needed: • Thermal cycler • Two primers • An enzyme called polymerase • A pile of DNA building blocks that the polymerase needs to make that copy
- 13. Company Confidential Some of the Applications of PCR • Diagnostics • Forensic analysis • Detection of infectious agents • Rare mutation detection • Gene expression • DNA sequencing • Copy number variation • SNP genotyping
- 15. Company Confidential 3 Steps to PCR • Denaturation • The double strand melts open to single stranded DNA • Annealing • The primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) • On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template. • Extension • The polymerase works synthesizing making a new DNA strand RINSE. LATHER. REPEAT. http://bit.ly/xGvqec
- 16. Company Confidential Real-Time PCR • Real-Time PCR (qPCR) • Enables both detection and quantification • Measures PCR amplification as it occurs– hence the name “real-time” • Uses fluorescent probes • Requires the comparison of an unknown to a standard to obtain quantitative information • Analogue measurement based on measuring amplification after each cycle of PCR
- 17. Company Confidential It’s time to digitize • Digital PCR • Works like real-time PCR, except it first partitions the sample into hundreds or even thousands of reaction chambers (chips, plates, droplets) • Some may have template, others may not • Then amplification occurs with qPCR • Reactions with target are read as a “1” and those without as a “0” • Together with the volume of each reaction and the total number of reactions analyzed, an estimate of the absolute target DNA concentration is calculated • Droplet Digital PCR • Water-in-oil droplets (approximately 20,000) • Gives off fluorescent + or – signals • Software calculates the concentration of target DNA
- 18. Company Confidential Real-time vs. Digital
Editor's Notes
- Clients run assays to make their products.Clients sell assays, e.g. Singulex sells cytokine assaysClients sell equipment to run assays, e.g. Bio-Rad sells Bio-Plex instruments to run immunoassaysClients perform assays on behalf of others, e.g. Precision Biomarker runs microarrays for its clients interested in identifying biomarkersKena cares about assays, nuf said.
- Bradford, 1976
- ProteinIs that my protein?What is the concentration of my protein?What is the activity of my enzyme?What is the weight of my protein?What is the 3-d structure of my protein?You can measure anything: cell mojo, cell death, cell poop etc.