assay
TRANSCRIPT
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Company Confidential
Part 1: You Say, Assay
Part 2: PCR- Poly What?
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Company Confidential
Who cares?
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Company Confidential
Did you say “essay?”
Definition: Analysis (n), analyze (v)
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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?
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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
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Company Confidential
Wide world of assays
What assay am I?
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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?
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Company Confidential
Practically speaking….
• Speed (“time-to-result”)
• Throughput
• Automation
• Ease-of-use
• Workflow
• Cost
• Size (“footprint”)
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Company Confidential
Last word: Does it smell?
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Questions?
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Company Confidential
PCR- Poly What?
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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
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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
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So Fancy
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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
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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
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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
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Real-time vs. Digital